KCNK5 in Physiology and Pathophysiology Kirkegaard Petersen.pdf · 2014-07-18 · Submitted to “...

F A C U L T Y O F S C I E N C E

U N I V E R S I T Y O F C O P E N H A G E N

KCNK5 in Physiology and Pathophysiology

- Focus on Cell Volume Control

PhD Thesis

Signe Skyum Kirkegaard Petersen

Academic advisors: Professor Else Kay Hoffmann & Dr.med. Steen Gammeltoft

Submitted: September 30 th 2013

FACULTY OF SCIENCE UNIVERSITY OF COPENHAGEN

KCNK5 in Physiology and Pathophysiology

- Focus on Cell Volume Control

PhD Thesis By


Section for Cell and Developmental Biology

Department of Biology

Faculty of Science

University of Copenhagen

Author: Signe Skyum Kirkegaard Petersen Thesis title: KCNK5 in Physiology and Pathophysiology – Focus on Cell Volume Control Academic advisors: Professor Else Kay Hoffmann Section for Cell and Developmental Biology Department of Biology University of Copenhagen Denmark Dr.med. Steen Gammeltoft Sleep Research Unit Diagnostic Department Glostrup Hospital Denmark Submitted: September 30th 2013

Copenhagen, sep. 2013

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Preface and acknowledgements

This PhD thesis will focus on the potassium channel KCNK5 and its role in different cell types i.e.

the cell lines Ehrlich Ascites Tumor cells and Ehrlich Lettré Ascites cells and in human primary T

cells. More precisely I have studied the involvement of protein tyrosine kinases in swelling-

induced activation of the channel, the long-term effects of hypotonicity on channel physiology

and expression pattern and last but not least I have looked into its role in T cells.

The thesis is based on the following papers:

Paper I: Activation of the TASK-2 channel after cell swelling is dependent on tyrosine phosphorylation Signe Skyum Kirkegaard, Ian Henry Lambert, Steen Gammeltoft and Else Kay Hoffmann Am J Physiol Cell Physiol 299: C844–C853, 2010

Paper II: KCNK5 is functionally down-regulated upon long-term hypotonicity in Ehrlich ascites tumor cells Signe Skyum Kirkegaard, Tune Wulff, Steen Gammeltoft and Else Kay Hoffmann

Submitted to “Cellular Physiology and Biochemistry”. Status 09-30-2013: The paper has been found in “principle acceptable for publication”.

Paper III: The potential role of KCNK5 in activated T cell physiology with specific focus on cell volume control Signe Skyum Kirkegaard, Pernille Dyhl Strøm, Anker Jon Hansen, Steen Gammeltoft and Else Kay Hoffmann

Paper in preparation.

Through this thesis I will touch upon various themes such as the activation of the KCNK5 channel

upon acute cell swelling, the physiological function and expression of the channel upon long-

term hypotonicity and on its potential role in activated T cell physiology. I thus used different

cells and cell systems in order to investigate these topics - a fact that will be reflected in this

thesis, where the introductory part of the thesis will deal with a variety of quite different topics.

I will describe how the immune system works, how a cell can regulate its volume and hereunder

various sub-topics, what is known about the KCNK5 channel and about potassium channels in

general. With relative many large topics to describe some details will not be addressed, thus the

reader might find some topics left unmentioned but hopefully this will not cloud the greater

overview, issues and facts presented here.

During my time as a PhD student at Section for Cell and Developmental Biology at the University

of Copenhagen and at the Department of Clinical Biochemistry, Glostrup Hospital, Denmark I

have had the pleasure of guidance from my two supervisors, Else Kay Hoffmann and Steen

Gammeltoft. I have had great scientific challenges and have learned a lot about research, project


Page 2 of 117

management, teaching and about myself as a person. With this in mind I cannot begin to

express my gratitude towards my two supervisors, but I will try.

First of all I would like to thank Professor Else K. Hoffmann for everything! – for giving me a

chance in her laboratory, first as a master student and later on as a PhD student, for sharing her

extensive expertise on the area of cell volume, ion flux and on physiology in general. On a more

personal level I would like to thank her for always having time to talk, listen and give advice. One

thing in particular also comes to mind when thinking of the opportunities given to me by Else - I

have attended a great deal of meetings and conferences and have met so many interesting

scientists (probably more than most other PhD student), which is due to the fact that conference

attendance and networking is highly prioritized by Else. You are indeed a very social person Else,

and that fact has always had a huge impact on the great working environment in the lab. During

the last couple of years we have visited Slovenia, USA, Canada and Japan besides the many

meetings in Denmark. I have enjoyed every chance of presenting my work and have found the

participation in all the conferences and meetings very beneficial. Thank you so much!

I am also extremely grateful to Dr. Steen Gammeltoft for giving me the opportunity to be a part

of his group, first as a master student, since as a research assistant and last but not least as a

PhD student. The project might never have been without his financial and scientific support. I

would also like to thank Dr. Ian Lambert for always having his door open for me, whenever I

needed advise or practical help and for including me in the little group of “Friends of the Coulter

counter”.

I would very much like to express my gratitude to Dr. Anker Jon Hansen from Novo Nordisk, for

giving me a chance to work in his lab, for giving me the opportunity to see how the industry

works and for challenging my knowledge and ability to explain my work and ideas. I have very

much appreciated his comments and thoughts.

A special thank should also be given to technicians Birthe Juul Hansen, Pia Birn, Dothe Nielsen

and Birte Kofoed for their most appreciated help and friendship.

Last but not least I would like to thank friends and family for their everlasting support and

patience, especially I would like to thank my husband Mikkel, for whom without I would not

have been able to do this. I would also like to thank my son Oskar for making me learn how to

get the most out of, the now more limited time, I could spend at work.



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Abbreviations

[Ca2+]i

AA

APC

AVD

B cell

BCR

cDNA

cADPr

CNS

CRAC

ChTX

DAG

EAT cells

ELA cells

ER

FAK

FLAP

fMLP

ICl, vol

IK

IK, vol

IP3

JAK

K2P

KCa3.1

KCNK5

Kv1.3

LTC4

LTD4

MHC

mRNA

NFAT

Intracellular Ca2+ concentration

Arachidonic azid

Antigen presenting cell

Apoptotic volume decrease

B lymphocyte (“B” from bursa of Fabricius or bone marrow)

B cell receptor

cyclic ADP-ribose

Complementary DNA

Central nervous system

Ca2+ release-activated Ca2+ channel

Charybdotoxin

Diacylglycerol

Ehrlich ascites tumor cells

Ehrlich lettré ascites cells

Endoplasmatic reticulum

Focal adhesion kinase

5-LOX activating protein

N-formyl-methionine-leucine-phenylalanine

Swelling activated Cl- current

Intermediate conductance

Swelling activated K+ current

1,4,5-inositol triphosphate

Janus kinase

Two-pore domain K+ channel

Ca2+-activated K+ channel 3.1

Two-pore domain channel 5.1

Voltage-gated K+ channel 1.3

Leukotriene C4

Leukotriene D4

Major histocombatibility complex

Messenger RNA

Nuclear factor of activated T cells

PCR

PIP2

PLCγ

PTK

RA

RT-PCR

RVD

RVI

STIM

T cell

TALK

TASK

TCR

TEA

THIK

TM

TRAAK

TREK

TWIK

VRAC

qPCR

Polymerase chain reaction

Phosphatidylinositol 4,5-biphosphate

Phospholipase C-γ

Protein tyrosine kinase

Rheumatoid arthritis

Reverse transcription polymerase chain reaction

Regulatory volume decrease

Regulatory volume increase

Stromal interacting molecule

T lymphocyte (“T” from thymus)

TWIK-related alkaline pH activated K+ channel

TWIK-related acid-sensitive K+ channel

T cell receptor

Tetraethylammonium

Tandem pore domain halothane-inhibited K+ channel

Transmembrane

TWIK-related arachidonic acid-stimulated K+ channel

TWIK-related K+ channel

Tandem of p domains in a weak inwardly rectifying K+ channel

Volume-regulated anion channel

Real-Time quantitative PCR


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Indhold

Preface and acknowledgements ...................................................................................................... 1

Abbreviations ................................................................................................................................... 3

Purpose ............................................................................................................................................ 6

Abstract ........................................................................................................................................... 7

Resumé ............................................................................................................................................ 8

1. Introduction ................................................................................................................................. 9

1.2 K+ channels in general ........................................................................................................... 9

1.2.1 Potassium channel grouping by topology .................................................................... 10

1.2.2 The 2TM and 6TM families........................................................................................... 10

1.2.3 The 4TM family – the two pore-domain potassium channels ..................................... 11

1.3 KCNK5 – a two-pore domain potassium channel ................................................................ 13

1.3.1 KCNK5 history and tissue distribution .......................................................................... 14

1.3.2 KCNK5 characteristics .................................................................................................. 14

1.3.3 KCNK5 activation and gating ........................................................................................ 16

1.3.4 KCNK5 in lymphocytes ................................................................................................. 16

1.4 Cellular volume .................................................................................................................... 17

1.4.1 Cell volume regulation ..................................................................................................... 17

1.4.1.1 Pathophysiological conditions .................................................................................. 18

1.4.1.2 Regulatory mechanisms in response to volume changes ......................................... 18

1.4.1.3 The volume set-point ................................................................................................ 19

1.4.1.4 RVD............................................................................................................................ 19

1.4.1.5 K+ channels in volume regulation .............................................................................. 20

1.4.1.6 Volume regulation in Ehrlich cells ............................................................................. 20

1.4.1.7 Long-term effects of anisotonicity ............................................................................ 21

1.4.1.8 Volume regulation in lymphocytes ........................................................................... 22

1.4.2 When changes in cell volume act as signals ..................................................................... 23

1.4.2.1 Programmed cell death ............................................................................................. 23

1.4.2.2 Proliferation .............................................................................................................. 24

1.4.2.3 Transepithelial transport .......................................................................................... 24

1.4.2.4 Migration and invasion ............................................................................................. 24

1.5 The immune system ............................................................................................................ 26


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1.5.1 B and T lymphocytes .................................................................................................... 26

1.5.2 T cell activation ............................................................................................................ 27

1.5.2.1 T cell subtypes ........................................................................................................... 27

1.5.3 When the immune system attacks the body ............................................................... 29

1.5.3 Ion channels in T cell activation ....................................................................................... 29

1.5.3.1 Ca2+

signaling ............................................................................................................. 29

1.5.3.2 K+ channels in Ca

2+ signaling ..................................................................................... 30

1.5.3.3 Other ion channels described in T cells .................................................................... 31

1.5.4 T cell proliferation ............................................................................................................ 32

1.5.4.1 K+ channels in proliferation ....................................................................................... 32

2 Summary and conclusions .......................................................................................................... 34

3 Experimental procedures ........................................................................................................... 35

3.1 cDNA quantification instead of reference genes ............................................................ 35

3.2 T cell subtypes ................................................................................................................. 35

References ..................................................................................................................................... 36

4 Paper I ......................................................................................................................................... 51

5 Paper II ........................................................................................................................................ 63

6 Paper III ....................................................................................................................................... 83

7 Final thesis discussion, future work and perspectives .............................................................. 103

8 Appendix ................................................................................................................................... 108


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Purpose

The purpose of this thesis can be divided into three sub-purposes all centered around the two

pore-domain potassium channel KCNK5 (or TASK-2).

1) To study the potential role of protein tyrosine kinases in the swelling-mediated KCNK5

activation in Ehrlich Ascites Tumor (EAT) and Ehrlich Lettré Ascites (ELA) cells

2) To study the long-term effect of hypotonicity on KCNK5 physiology and expression in

EAT and ELA cells

3) To study the expression pattern and potential role of KCNK5 in activated human T cell

physiology


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Abstract

The KCNK5 potassium channel (also known as TASK-2 or K2p5.1) belongs to the latest discovered

potassium channel family - the two-pore domain potassium channels. KCNK5 was first described

in 1998 by Lazdunski and co-workers and has since been given a role in e.g. volume regulation,

and recent been proposed to play a role in T cell activation and in multiple sclerosis.

When a cell experiences swelling do to osmotic changes in the extracellular or intracellular

environment most mammalian cells will regulate their volume back towards the starting point –

an important feature in keeping cellular homeostasis. This swelling-induced response is called

Regulatory Volume Decrease (RVD) and involves the efflux of KCl and organic osmolytes through

specific volume sensitive channels, with a concomitant water efflux and shrinkage back towards

starting volume as a result. KCNK5 has been shown to be an important player in RVD in different

cell types and tissue e.g. in Ehrlich cells. It has been speculated that tyrosine phosphorylation is

necessary for the swelling-activation of the channel and in “Paper I” we show how acute

hypotonic swelling induces a time-dependent tyrosine phosphorylation upon the channel itself.

Acute cell swelling and its regulatory mechanisms is by far the most studied part of the cells

response to hypotonic stress and little is known about the consequences of long-term hypotonic

stimuli on cells, thus we induced a long-term hypotonic stimulation on EAT cells and ELA cells to

study the effect on physiology and channel expression patterns (Paper II). We found that 48 h of

hypotonic stimulation reduced the maximum current through the channel and likewise

decreased the cells ability to perform RVD. This impairment was found to be likely due to

lowered KCNK5 protein expression.

In 2010 it was suggested that KCNK5 play a pivotal role in the autoimmune disease multiple

sclerosis more precise in activated T cell. Numerous studies on ion channels in T cell activation

have been published and potassium channels Kv1.3 and KCa3.1 are thought to be the two main

channels involved. A potential role for KCNK5 made us shift cell type and thus look closer into

the channel in human T cells. We found a time-dependent massive up-regulation of KCNK5 on

mRNA and protein levels in activated human T cells, but KCNK5 up-regulation did not facilitate

an increased RVD, instead we found RVD in activated T cells to be impaired in comparison with

non-activated control cells. This impairment, despite of up-regulation of KCNK5, we show to be

due to a decreased volume activated Cl- flux, thus making the volume regulated anion channel

(VRAC) the rate-limiting factor in RVD in activated human T cells (Paper III).


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Resumé

Kaliumkanalen KCNK5 (også kaldet TASK-2 eller K2P5.1) tilhører den senest opdagede familie af

K+ kanaler og tilhører således gruppen af to-pore-domæne K

+ kanaler. KCNK5 blev første gang

beskrevet af Lazdunski et al. i 1998. KCNK5 er vist at være involveret i volumenregulering i en

række celletyper og er relativt for nylig foreslået at være af vigtig betydning i T celle aktivering

og i den autoimmune sygdom multipel sklerose.

Når mammale celler oplever cellesvulmning som følge af osmotiske ændringer i det intra- eller

ekstracellulære miljø, vil de fleste være i stand til at regulere deres volumen tilbage mod

udgangspunktet – en vigtig evne i forhold til at bevare den cellulære homeostase.

Volumenreguleringen som følge af svulmning kaldes ”Regulatory Volume Decrease” (RVD) og

involverer efflux af KCl og organiske osmolytter via specifikke volumenaktiverede kanaler.

Aktiveringen af disse kanaler og den efterfølgende KCl efflux resulterer i et osmotisk vandtab og

celleskrumpning. KCNK5 er vist at være vigtig i RVD i flere celletyper og væv bl.a. i Ehrlich celler.

Det er blevet foreslået, at tyrosin-fosforylering er vigtig i den svulmningsinducerede aktivering af

KCNK5 og i ”Paper I” viser vi, hvordan akut hypoton cellesvulmning og kanalaktivering resulterer

i en tidsafhængig tyrosin-fosforylering af selve kanalen.

Akut cellesvulmning, og de regulatoriske mekanismer involveret i korrelation af dette, er uden

sammenligning den mest studerede og bedst beskrevne effekt af hypotont stress og der er

således en begrænset viden om konsekvenserne af langtids-hypoton stimuli af celler. Vi

undersøgte derfor effekterne af langtids-hypoton påvirkning af EAT og ELA celler med henblik på

KCNK5 kanalfysiologi og ekspressionsmønster (Paper II). Vi fandt at 48 timers hypoton

påvirkning reducerede den maksimale svulmningsaktiverede strøm og ligeledes sås et hæmmet

RVD. Denne fysiologiske hæmning ser ud til at skyldes en nedsat KCNK5 protein-ekspression.

Der har været utallige studier af ionkanaler i T celle aktivering og K+-kanalerne Kv1.3 og KCa3.1

menes, at være de to vigtigste kanaler i denne proces. Da det er blevet foreslået, at KCNK5

spiller en vigtig rolle i sygdommen multipel sklerose og også er involveret i T celle aktivering

besluttede vi, at kigge nærmere på en potentiel rolle for KCNK5 i aktiveringen af humane T

celler. Vi fandt, at KCNK5 blev kraftigt opreguleret i aktiverede T celler både på mRNA- og

proteinniveau. Denne opregulering medførte dog ikke et øget RVD, tværtimod fandt vi at RVD i

aktiverede T celler var hæmmet i forhold til RVD i ustimulerede kontrolceller. Vi har evidens for,

at denne hæmning af RVD skyldes en nedsat volumenaktiveret Cl- flux, hvilket gør VRAC (volume

regulated anion channel) til den begrænsende faktor for KCl efflux under RVD i aktiverede T

celler (Paper III).


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1. Introduction

Potassium channels are ubiquitously distributed in all living organisms and are thus found in

cellular membranes in such different organisms as e.g. bacteria, plants and mammals. To this

day more than 75 known mammalian potassium channel subunit coding genes are known, which

makes this family of ion channels the most diverse of all ion channel families. They are found in

almost all cell types and hold a variety of cellular functions. This thesis will focus on the

potassium channel KCNK5 and its activation in response to acute hypotonicity, its physiology

when experiencing hypotonicity over a longer period of time and its role in T cell physiology. The

following sections will thus (in broad terms) deal with potassium channels in general, KCNK5,

volume regulation and T cell physiology.

1.2 K+ channels in general

K+ channels are protein complexes made of subunits that allow the passive transport of K

+ across

the membrane which, if not for ion channels and transporters, would be functional

impermeable to ions. The passive transport follows the electrochemical gradient. K+ serves a

wide range of functions including setting the membrane potential, controlling the action

potential in excitable cells and being involved in cell proliferation, cell volume control, muscle

contraction and secretion of various hormones and transmitters. As potassium channels are

implicated in such various physiological functions, many diseases or pathological conditions can

be contributed to an altered K+ channel function. Of diseases and pathological conditions

implicating K+ channels can be mentioned arrhythmia, diabetes, neuronal diseases and renal

diseases, for further information see e.g. (60; 140). Several K+ channels are also found to be up-

regulated in cancer cells (see (125)), though one way of cancer cells to survive is to avoid

apoptosis, which can be obtained by the down-regulation of K+ channels involved in apoptotic

volume decrease (see section 1.4.2.1).

As stated above, potassium channels hold a range of different functions and so they differ both

in topology and regulation. The K+ current running through the channel can be activated in

different ways reflecting the physiology of the cell or tissue in mind. There are different ways of

grouping the potassium channels e.g. by activation and type of current or by topology (see

section 1.2.1). There are four main types of potassium channels based on activation and current;

i) voltage-gated K+ channels which are sensitive to changes in membrane potentials and opens

or closes accordingly, ii) Ca2+

-activated K+ channels which, as the name implies, has a Ca

2+

dependent activation, iii) Inwardly-rectifying K+ channels that favors K

+ fluxes in an inward


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direction and last but not least iiii) two-pore domain K+ channels which are constitutively active

and carries currents designated leak-currents see (110).

The potassium channels consist of various transmembrane (TM) spanning regions (or TM

domains) together with a highly conserved pore-forming domain, that can transport between

106

to 108 K

+ ions across the membrane pr. sec, while at the same time rejecting other cations

(110; 140).The pore region of the channel is a feature shared by all potassium channels and thus,

all potassium channel subtypes poses the very conserved amino acid sequence threonine,

valine, glycine, tyrosine and glycine (TVGYG). This amino acid sequence make up the selective

filter of the pore (60) and is called the signature sequence of potassium channels (48). To make a

functional K+ pore, four pore-domains have to come together.

Scientists are always on the look for new discoveries including new ion channel families, thus the

pore sequence was used to search DNA sequence databases for new potassium channels. This

approach resulted in the discovery of the two-pore domain potassium channel family further

described in the following sections (83).

1.2.1 Potassium channel grouping by topology One way to group the many different K

+ channels is by activation pattern and another is by

topology and thus the number of TM segments. Dividing the potassium channels by topology

gives three main groups, the 2TM family, the 4TM family and the 6TM family which also include

a 7TM spanning member (the BK family of Kca potassium channels (21; 60)) (see Figure 1).

1.2.2 The 2TM and 6TM families In the 2TM family we find the KIR channels which are inward rectifying channels that include the

ATP- sensitive channels as well as G-protein coupled channels (fig. 1). KIR channels are found in

e.g. skeletal and heart muscle and in neurons see e.g. (21). The family contains 7 members and is

found as a tetramer in the membrane, since the generation of a functional pore requires four

pore domains to come together and in the 2TM family each subunit holds one pore-forming

domain.

The voltage-gated, the Na+-activated and the Ca

2+-activated K

+ channels all belong to the 6TM

family which in spite of the name also include a 7TM channel namely the large (big) conductance

(BK) Ca2+

-activated potassium channel (60) (see fig.1). The functional channel is, as for the 2TM

family found as a tetramer with four pore domains generating the pore.


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1.2.3 The 4TM family – the two pore-domain potassium channels The 4TM family comprises the latest discovered family of K

+ channels, namely the two-pore

domain channels in which we find the channel of special interest for this thesis – the KCNK5 K+

channel (also known as TASK-2 or K2p5.1). The 4TM family has an interesting structure that

varies from the other two families since members of the 4TM family have four TM segments and

two pore-forming domains (43; 82). Because of the two pore domains in each subunit the

functional channel is found as a dimer and not as a tetramer as seen in the other two families

(84) see also (83).

The KCNK5 channel belongs to the TALK (TWIK-related alkaline pH activated K+ channel)

subfamily and besides TALK the 4TM family consists of five other subfamilies namely the TWIK

(tandem of p domains in a weak inwardly rectifying K+ channel) subfamily, the THIK (tandem

pore domain halothane-inhibited K+ channel) subfamily, the TREK (TWIK-related K

+ channel)

subfamily, the TRAAK (TWIK-related arachidonic acid-stimulated K+ channel) subfamily and the

TASK (TWIK-related acid-sensitive K+ channel) subfamily (9) (fig. 2).

Figure 1: Overview of potassium channels and potassium channel families There are three potassium channel families based on membrane topology namely the 2TM family with one pore domain and two transmembrane segments containing the inward rectifying KIR channels, the 4TM family of two-pore domain channels including KCNK5 and the 6TM family with one pore domain and 6 (or 7) transmembrane segments including the voltage–gated and the Ca2+- activated K+ channels. Own figure.


Page 12 of 117

The idea of potassium leak-currents in

the resting membrane was proposed a

long time before it was possible to

measure ion currents across the

membrane and before cloning

techniques made channel identity

possible. Thus Bernstein had in 1912

proposed that the resting membrane

was more permeable to potassium than

other ions, which was described and

further analyzed by Hodgkin and Huxley

(49; 50). Though the idea of leak-

currents came to light long ago, it was

not until 1995 that the identity of a

background channel was known by the

cloning of the TOK1 channel in

Saccharomyces cerevisicae (66). The TOK1 channel differs from the mammalian two pore-

domain channels since it has eight TM domains, whereas all other two pore-domain K+ has the

characteristic four TM segments and it further more differs in functionality. The first mammalian

two pore-domain potassium channel with the characteristic morphology of two pore-domains

and four TM domains to be cloned, was TWIK-1 which was cloned in 1996 (82).

Despite a relatively poor sequence similarity between some of the members of this relatively

newly described family, they share not only the distinct topology, but are also similar in regard

to electrophysiological properties. They are (almost) voltage independent, constitutively active

at resting membrane potentials and have “open rectification”. For more on two-pore domain

potassium channels see e.g. (32).

The background channels or potassium leak channels all share the same morphology with four

membrane spanning domains, two of them being pore forming and they are special in the way

that they are constitutively active at resting membrane potential (41). The potassium channel

signature sequence is conserved in the first pore-forming loop, but is slightly different in the

second pore-forming loop, where GYG is substituted by GFG or GLG (140). Since this group of K+

channels posses two pore domains, only two subunits are required to make a functional

channel. Thus the channel is formed by the dimerization of two subunits (83; 84).

The potassium leak-channels are defined by the lack of voltage and time-dependencies, which

means that they are open even at resting membrane potentials leaking K+ ions, and thus

Figure 2: Phylogenetic tree Phylogenetic tree for the known two-pore domain K+ channels which shows how KCNK5 is related to the other members based on sequence similarities. Model based on (41).

<zx<xc


Page 13 of 117

bringing the plasma membrane potential towards the equilibrium potential for K+. So they serve

an important function in maintaining the negative resting membrane potential by constitutively

leaking K+ ions down the concentration gradient, which in turn helps maintaining the Na

+/K

+

pump function see ref. (42).

To this day there are 15 known members of the family of human two-pore domain potassium

channels see refs. (20; 41). For more about two-pore domain potassium channels see e.g. refs.

(8; 32; 41; 42; 66; 115).

1.3 KCNK5 – a two-pore domain potassium channel

In the 4TM family one of the subfamilies was named TASK (TWIK-related acid-sensitive K+

channel) and started out containing the channels TASK-1 (which was the first TASK channel and

the third K2P channel to be cloned (31)), TASK-2, TASK-3, TASK-4 and TASK-5, but as seen from

fig. 2 the TASK-2 channel or KCNK5 together with TASK-4 (also known as TALK-2) are now found

in the TALK subfamily (fig. 2). This subfamily re-arrangement was partly due to the poor

molecular relation between TASK-2 and TASK-4/TALK-2 against TASK-1, thus the sequence

similarity between TASK-1 and TASK-2 is only about 30% (110). In 2001 TALK-1 and TALK-2 were

described (26; 39) and they were named as TWIK-related alkaline pH-activated K+ channels due

to the high extracellular pH needed for activation.

TASK-2 and TASK-4 were now regarded as TALK

channels due to their alike alkaline pH sensitivity and

because of their molecular relations mentioned above.

Furthermore TASK-channels are gated only by

extracellular pH (32) and thus not affected by

intracellular pH as seen in the TALK channels KCNK5

and TALK-2, which are indeed sensitive to intracellular

pH changes (105). Since TASK-2 is now regarded as a

TALK channel and though the name TASK-2 is still in

use, the TASK-2 channel will be designated KCNK5

throughout the rest of this thesis. I should though be

noted that the channel is called TASK-2 in “Paper I”

(69) and KCNK5 in “Paper II” and “Paper III”.

Figure 3: KCNK5 pH sensitivity Current-voltage relationship recorded from

a TASK-expressing oozyte (31).


Page 14 of 117

1.3.1 KCNK5 history and tissue distribution KCNK5 was as the first member of the TALK subfamily to be cloned, which was done from

human kidney in 1998 by Reyes and co-workers (128). KCNK5 Northern blot analysis have shown

KCNK5 to be present in human kidney, pancreas, liver, placenta, lung and small intestine (97;

128) whereas RT-PCR from on mouse tissue showed abundant KCNK5 presence in liver, kidney,

small intestine, lung and uterus (97; 128). Some studies including the KCNK5 channel is strictly

expression studies and do not deal with physiological importance and between the various

studies there is some species variation in regard to KCNK5 expression (see table 1). In that

regard some tissue expression is up for discussion, while there is a broad agreement on the

expression and importance in kidney cells where many of the functional studies on the channel

has been conducted.

Since the initial studies on KCNK5 it has (besides being described as a K+ leak-channel) been

reported to be implicated in RVD in several cell types including Ehrlich Ascites Tumor (EAT) cells

(69; 104), mouse proximal tubules (7), human and murine spermatozoa (4; 5) and murine T

lymphocytes (12) but has also been reported to hold other functions in different cell types (see

table 1).

1.3.2 KCNK5 characteristics

KCNK5 has been studied in a variety of cell types

and it has been found to be highly sensitive to

external pH, thus open probabilities increases

with alkalization and decreases with acidification

(31; 64; 104) (see fig. 3) with a 90% maximum

current measured at pH 8.8 and only 10% at pH

6.5 (31; 128). At pH 6.0 the KCNK5 activity is

almost non-existing (107). The pH sensing

mechanism has been proposed to be located in

the extracellular loop between TM1 and channel

pore domain one (99), but in 2007 Niemeyer and

co-workers showed how an arginine residue

(R224) located near the second channel pore

domain acts as the pH sensor and that it

functions by applying an electrostatic effect on

the pore domain (107).

Figure 4: KCNK5 characteristics See the text for further details. Own figure.


Page 15 of 117

Table 1

Reported mammalian KCNK5 expression and function

Species Tissue Function Reference

Mouse Ehrlich ascites tumor cells Volume sensitive (69; 104)

Mouse Proximal tubules Volume sensitive (7)

Human Spermatozoa Volume sensitive (4)

Mouse Spermatozoa Volume sensitive (5)

Mouse T lymphocytes Volume sensitive (12)

Mouse Proximal kidney cells AVD (73)

Human T lymphocytes Possible T cell activation (10)

Human T lymphocyte Volume sensitive (3)

Rat

Mouse

Human

Mouse

CNS

Proximal tubules

MCF-7 and T47D

WEHI-231 cells

Possible cell excitability & signal transduction

Stabilizing HCO3- transport

Regulating proliferation

BCR-ligation-dependent apoptosis

(37)

(151)

(1)

(100)

Mouse Retrotrapezoid nucleus neurons CO2 and O2 chemo-sensing (38)

Rat

Rat

Human

Mouse

Mouse

Rat

Rat

Rat

Human

Rat

Proximal neurons in inner retina

Neurons in hippocampus

Peripheral blood and T cells of RA patients

Gastrointestinal smooth muscles cells

Gastroenstesinal tract

Carotid body

Taste receptor cells

Cerebellar astrocytes

Cerebellar Purkinje cells

Cerebellar granule neurons

Neuronal excitability

Contribution to properties of epilepsy

Correlation between expression and disease activity

Background current

Contribution to resting potential

Regulating K+ currents and acid sensing

Setting resting potential and sour taste transduction

Not speculated on

Not speculated on

Component of standing-outward potassium conductance

(157)

(68)

(11)

(137)

(156)

(154)

(89)

(136)

(136)

(23)

Furthermore the channel has proven to be insensitive to Ca2+

and a wide range of known

potassium channel blockers e.g. tetraethylammonium (TEA) (128) and charybdotoxin (ChTX) but

can be blocked by e.g. clofilium (104) and quinine (128) (fig. 4). For a recent review on KCNK5

see ref. (20).


Page 16 of 117

1.3.3 KCNK5 activation and gating Different studies have been made to elucidate the activation mechanisms of KCNK5 and as

mentioned earlier, KCNK5 has proven to be volume sensitive and thus activated by cell swelling.

In addition we have demonstrated how protein tyrosine kinase activity is involved in RVD (Paper

1, fig. 1) and how cell swelling elicits a time-dependent tyrosine phosphorylation of the channel

itself upon activation (Paper I, fig. 5). We have also shown how inactivation of the swelling-

activated channel involves protein tyrosine phosphatases (Paper I, fig. 3).

Furthermore swelling-activation of KCNK5 has in kidney cells been proposed to be caused by an

extracellular alkalinization due to Cl-/HCO3

- exchanger mediated Cl

- influx (72). LTD4 has likewise

been implicated in the swelling-activation of KCNK5, since hypotonicity and cell swelling is

followed by LTD4 release (74; 76) and since addition of LTD4 results in activation of a K+ current

similar to the swelling-activated K+ current (57). In addition it was shown that desensitization of

the LTD4 receptor impairs RVD in EAT cells (61).

Of other gating mechanisms can be mentioned gating by intracellular pH and also extracellular

pH both working in the same pH range though they are independent of each other. A recent

study have also shown how KCNK5 is inhibited by heterotrimeric G protein subunits Gβγ an

effect that could be eliminated by mutation of lysine residues of the C terminus (2) (also see

(20)).

Despite all of these findings there are still many questions to be answered in regard to KCNK5

channel activation, thus we have yet to identify the tyrosine sites on KCNK5 being

phosphorylated upon swelling-mediated activation (see section 7 and discussion in Paper I) and

the direct connection between KCNK5 and LTD4 has still not been shown. Furthermore though

the effect of Gβγ G protein subunits has been established, the answer to how exactly it works

and through which signaling pathway also remains to be answered.

1.3.4 KCNK5 in lymphocytes KCNK5 was initially described in kidney cells and thorough studies have also been done in Ehrlich

cells. The presence of KCNK5 in lymphocytes on the contrary is a relatively new observation. The

first evidence of a background channel (TREK-2) present in lymphocytes was described in WEHI-

231 cells, which is a murine cell line equivalent to immature B cells (101; 158). Further studies

have revealed the presence of two-pore domain channels in lymphocytes e.g. TASK- 1 and TASK-

3 in human T cells (98) and in regard to lymphocytes KCNK5 has so far been described in human

T cells (10) and in WEHI-231 immature B cells (100).


Page 17 of 117

In B cells KCNK5 has been shown to be involved in apoptosis which is initiated by B cell receptor

stimulation (100). In T cells Bittner and co-workers has proposed that KCNK5 holds a role in

multiple sclerosis since an up-regulation of the channel was found in T cells purified from

multiple sclerosis patients. The study from Bittner et al. led us to look into the potential role of

KCNK5 in activated human T cells. We found that KCNK5 is indeed expressed in T cells and that

activation resulted in a time-dependent expression pattern with an initial down-regulation

followed by a strong up-regulation of the channel on protein level (Paper III, fig. 2). For more on

KCNK5 in lymphocytes see (20; 35).

1.4 Cellular volume

The permeability of water across the plasma membrane is much larger than the permeability of

potassium, sodium and chloride, thus the water permeability was measured to be 105 times

higher than sodium and potassium (51) and 106 times higher than chloride (75) in EAT cells. A

shift in intracellular and/or in extracellular osmolarity can be observed in response to e.g.

accumulation of metabolic waste products, protein synthesis and transport over epithelia. The

extracellular content has very little variation under physiological conditions, thus changes in the

intracellular osmolyte composition are the main determinant for volume changes. The cell is

very vulnerable to changes in cell volume which will affect cellular homeostasis e.g. is the actin

cytoskeleton very susceptible to volume changes and it is seen how cell swelling results in a

decrease in F-actin whereas cell shrinkage result in an increased amount of F-actin (117; 139).

For more on volume regulation see e.g. refs. (56; 78).

1.4.1 Cell volume regulation

Maintaining a steady cell volume is crucial for keeping cellular homeostasis, thus a fundamental

ability in mammalian cells, with only some exceptions, is the ability to volume regulate. Cells are

constantly osmotically challenged by extracellular and especially intracellular changes in

osmolyte content and since most mammalian plasma membranes contain aquaporins water will

rapidly move across the membrane in response to the osmotic changes. These osmotic changes

are in general met by activation of regulatory mechanisms bringing the cell volume (almost)

back to normal see e.g. refs. (56; 78). There are though exceptions where the changed volume

does not result in volume regulation (see section 1.4.2 and (56)).


Page 18 of 117

1.4.1.1 Pathophysiological conditions An altered cell volume is found in a number of pathophysiological conditions such as

ischemia/hypoxia and epilepsy where an increase in intracellular osmolarity is seen. Hepatitis

cirrhosis, nephrosis and hyponatremia are all examples of conditions that include a decreased

plasma osmolarity, whereas increased plasma osmolarity is seen in diarrhea, uremia and

diabetes mellitus/insipidus see (96; 111). Of other pathophysiological states can be mentioned

cerebral edema (see (96)).

1.4.1.2 Regulatory mechanisms in response to volume changes As stated above, osmotic challenged cells will often respond to the resulting swelling or

shrinkage by initiating a regulatory response thereby returning their volume back towards the

starting point. The cell uses different regulatory mechanisms in regard to swelling contra

shrinkage. Regulatory Volume Decrease (RVD) is the cells response to swelling and consists of

mechanisms excluding osmolytes and with concomitant water efflux as result, whereas

Regulatory Volume Increase (RVI) is seen in response to shrinkage and involves the uptake of

osmolytes and water (fig. 6).

As seen from the scanning electron images in fig. 5 EAT cells have clear invaginations of the

membrane under isotonic conditions, letting them tolerate some swelling by unfolding of the

surface thereby not disrupting the cell membrane. Shrinkage on the other hand causes more

prominent invaginations (51). This is not only true in Ehrlich cells but also in lymphocytes (18)

and certainly in a lot of other cell types. Since the mechanisms of RVI is not of direct interest to

this work I will not go more into detail on the matter, but merely refer to fig. 6 and the extensive

review by Hoffmann et al. (56).

Figure 5: scanning electron images of cells kept under normal, hypotonic and hypertonic conditions Scanning electron microscopy images of Ehrlich ascites tumor cells kept under various osmolarities. B: cells kept in isotonic media shows invaginations of the membrane which are unfolded upon cell swelling (A). Cell shrinkage will on the other hand generate even more pronounced membrane-folding as seen on image C (51).


Page 19 of 117

1.4.1.3 The volume set-point Most cells have a fine tuned volume regulation which occurs instantly upon osmotic changes

making volume changes almost undetectable and the cells are said to be isovolumetric.

Mammalian cells are very sensitive to volume changes and as little as a 3.5% (44) volume change

can result in the activation of regulatory mechanisms – the volume set-point of the cell is

reached. The volume set-point defines the boarder for cell volume regulation and volumes

higher or below this threshold will result in volume regulation. The volume set-point is not to be

viewed as a definite size, but rather a dynamic mechanism by which the cell adapts to its

environment and function (52) and it is thus seen that cells that undergo RVD or RVI often will

have an altered volume set point (53; 103). The volume set-point can also change accordingly to

cellular function, thus proliferating cells experience a shift in the volume set-point since volume

regulation in this case is not beneficial for the growing cell (see section 1.4.2.2).

1.4.1.4 RVD When subjected to cell swelling the cell will

release KCl and organic osmolytes (52)

through specific channels and co-transporters

which will then drive water to leave the cell

and subsequently lead the cell volume back

towards its starting point. Besides the volume

sensitive K+ and Cl

- channels KCl can also

leave the cell through transporters and so the

K+-Cl

- co-transporters, the H

+/HCO3

- anion

exchanger and the K+/H

+ exchanger (fig. 6)

are likewise important players in a RVD

response (see (111)).

The exact composition and influence of the

different transporters and channels involved

in RVD differs from cell type to cell type, thus

different K+ channels have been shown to be

implicated in the swelling induced K+ efflux.

Whereas the K+ channel picture is very

diverse, it is commonly agreed that the

swelling activated Cl- channel holds one

identity namely that of VRAC (volume

regulated anion channel), though it’s

Fig. 6: Ion movement during RVD and RVI Cell swelling results in KCl efflux through specific K+ (varies from cell type to cell type) and Cl- channels (VRAC), the K+-Cl- co-transporter (KCC), the H+/HCO3

- anion exchanger (AE), the K+/H+ exchanger together with an organic osmolytes efflux (not shown here). The ion efflux drives osmotic obliged water to leave the cell and let it reach a volume close to its starting point. RVI on the other hand rely on ion up-take through transporters such as AE, the Na+/H+ exchanger NHE, the Na+/K+/2Cl- co-transporter (NKCC), the Na+/Cl+ co-transporter and organic osmolytes up-take (not shown

here). Own figure.


Page 20 of 117

molecular identity for the time being remains unknown. The molecular identity of VRAC is very

much a hot topic and it has been suggested to belong to various chloride channel families

though no definite answer to the puzzle has been found. A further discussion of that subject is

outside the scope of this thesis and will not be further dealt with here.

1.4.1.5 K+ channels in volume regulation As mentioned previously the K

+ channels picture is quite diverse and so far, and to my

knowledge members of 9 different potassium channels subfamilies has been shown to be

volume sensitive (see (56)). It should be noted that the discrepancies between the number

stated here and the number of channel families listed as including volume sensitive members in

ref. (56) is due to the fact that the volume sensitive channels KCNK5 was recently moved from

the TASK subfamily to the TALK subfamily as described previously in this thesis (see section 1.3).

Volume sensitive potassium channels can e.g. be found amongst the family of Ca2+

-activated K+

channels (47) in the BK subfamily (see (56; 111)), the IK (63; 67; 150) and SK (63; 132)

subfamilies and amongst the voltage-gated K+ channels has e.g. Kv1.3 (28) and Kv1.5 (4; 34)

been shown to contribute to RVD in lymphocytes. Of special interest to this thesis the KCNK5

has been shown to be the volume regulating K+ channel in EAT cells (104; 106) and mouse

proximal tubules (7) see table 1 and (20).

1.4.1.6 Volume regulation in Ehrlich cells Volume regulation in Ehrlich cells (EAT and ELA cells) have been intensely studied over the years

and much knowledge has come from that. The main pathways for KCl efflux in Ehrlich cells are

through specific volume sensitive K+ and Cl

- channels and the currents through these channels

are designated IK,vol and ICl,vol see (55)

ICL,vol

The outward rectified chloride current activated by cell swelling runs through the volume

regulated anion channel (VRAC). It has been described as being Ca2+

independent, tamoxifen

inhibited, relative insensitive to DIDS and niflumic acid (118) and to hold a permeability

sequence of SCN->I

->NO3>Br>Cl

-F

- (see ref. (53)).

IK,vol

Initially an intermediate-conductance K+ (IK) channel was shown to be activated in cell attached

patch studies on EAT cells (19) but later studies e.g. showing how IK,vol was poorly affected by

clotrimazol, apamin, kalitoxin, margatoxin, TEA and ChTX which are all known inhibitors of K+

channels (including Ca2+

-activated K+ channels), together with the fact that IK,vol was activated

despite buffering [Ca2+]i (57) led to the conclusion that RVD in Ehrlich cells were dominated by a


Page 21 of 117

Ca2+

-independent channel (62; 129). It was further more demonstrated how IK,vol had a specific

selection profile favoring entry of Rb+ and K

+ which eliminated the stretch-activated K

+ channel

since it was proven to be non-specific (19; 108). For further information see (53). IK,vol did not

belong to the family of voltage-gated K+ channels either (108). One drug clofilium, did though

inhibit IK,vol hence evidence pointed towards the family of two-pore domain channels (106). PH

sensitivity with potentiation of the current with alkalization and inhibition at acidification

narrowed the field to – at that time - a TASK channel (see section 1.3) (54; 71). It was later

shown that KCNK5 (TASK-2) but neither TASK-1 nor TASK-3 was present in Ehrlich cells (104).

Activation of IK,vol in EAT cells

When swollen phospholipase cPLA2α is translocated to the nucleus (120) where it is

phosphorylated and activated by a G-coupled process. This activation results in a release of

arachidonic acid (AA) which is 3.3 times higher than seen in cells kept isotonic (145). AA which is

a precursor for leukotriene C4 (LTC4) requires the action of FLAP (5-LOX activating protein), 5-

LOX and LTC4 synthase to be generated. LTC4 is subsequently transported out of the cell where it

is converted to leukotriene D4 (LTD4) by γ-Glutamyl transpeptidase, here it binds to its receptor

which in turn activates IK,vol (57; 61; 74) (also see (53; 56)). The direct mechanisms between LTD4

and channel activation still remains to be studied.

Protein tyrosine kinase activation has been shown to play a role during RVD in various cell lines

(6; 22; 25; 88) and it was speculated that tyrosine phosphorylation was also of importance in the

RVD response in Ehrlich cells somewhere in the signaling pathway just described. In “Paper I” we

show how tyrosine phosphorylation indeed is vital for RVD, since protein tyrosine kinase

phosphorylation of the volume sensitive K+ channel (which in Ehrlich cells is KCNK5) is part of the

activation mechanisms upon cell swelling. Thus we found a swelling-induced and time-

dependent tyrosine phosphorylation on the channel itself in response to acute hypotonicity

(Paper I, fig. 5).

1.4.1.7 Long-term effects of anisotonicity During long-term exposure to an anisotonic environment cells respond with late-phase volume

regulatory mechanisms in contrary to the acute phase response (described in section 1.4.1.2)

which takes place within minutes of cell swelling. The late-phase mechanisms are often

connected with an altered gene expression and protein synthesis changes. Organic osmolytes

makes good late-phase regulators since accumulation of organic osmolytes compared to e.g.

electrolytes are harmless, thus an accumulation of electrolytes can upset various metabolic

processes e.g. denature macromolecules and influence membrane potential (see (142)).


Page 22 of 117

Long-term anisotonicity can be both hypo- or hypertonic and hypertonicity is by far the most

studied of the two conditions. It has been shown how long-term hypertonicity results in an

increased gene transcription of various volume regulatory genes (14) while the number of

studies on long-term hypotonicity is scarce. Thus we studied the KCNK5 function and expression

on various levels in Ehrlich cells upon long-term exposure to hypotonicity (Paper II). We found

that the maximum current through KCNK5 in ELA cells were inhibited (Paper II, fig. 1) as were

the EAT cells ability to perform RVD (Paper II, fig. 2). We further more found a decreased KCNK5

protein expression in both cell lines upon stimulation (Paper II, fig. 4) and our results thus show

how a functional down-regulation of KCNK5 upon long-term hypotonicity is probably due to a

decreased KCNK5 protein synthesis (see section 7 and the discussion in Paper II).

1.4.1.8 Volume regulation in lymphocytes Lymphocytes like most other mammalian cells are subject to changing intracellular environment

causing potential volume changes and subsequent regulation. T cells are of course not static in

the sense that they are in constant movement around the body and thereby eligible to

extracellular osmotic changes e.g. when passing around the kidney. Lymphocytes are as most

other mammalian cells capable of performing RVD in response to cell swelling (18; 29; 135) and

as seen in many cells the higher the osmotic challenge the lesser the recovery (18). T cells are

only able to perform RVI if previously experiencing hypotonically elicited RVD – also called post-

RVD RVI (29).

As seen in other volume regulating cells VRAC is responsible for the swelling activated Cl- current

in lymphocytes (17; 81; 85) (also see (16)), whereas the K+ channel picture is more diverse and

different regulatory volume pathways and K+ channels have been suggested to play a role in T

cell volume regulation including the voltage-gated K+ channel Kv1.3 (17; 28; 80), the Ca

2+-

activated KCa3.1 (138), and two-pore domain potassium channels (3; 12). Kv1.3 is involved in

RVD in mature T cells whereas KCa3.1 is so far only implicated in RVD in thymocytes (immature T

cells), thus it is seen that RVD in mature T cells is Ca2+

-independent (16). Kv1.3 is not directly

volume sensitive, but is thought to be activated by a change in membrane potential due to

activation of volume sensitive anion channels causing Cl- efflux, plasma membrane depolarizing

and subsequent Kv1.3 activation (16; 29; 87).

KCa3.1 and Kv1.3 is by far the most studied and described K+ channels in T cell physiology

including cell volume regulation and the role of other K+ channels is still object to some

discussion (see e.g. (16; 112)).

Recently members of the two-pore domain potassium channel family were proposed to play a

role in cell volume regulation in murine and human T cells (3; 12), thus both KCNK5 (in mouse


Page 23 of 117

and human T cells) (3; 12) and TRESK-2 (in human T cells) (3) were given prominent roles in RVD.

We have likewise studied the effect of KCNK5 in human T cell volume regulation and find that

the channel is expressed in these cells and that this expression is up-regulated when the T cells

are activated (Paper III, fig. 2). We find that the RVD response in activated T cells is impaired

(Paper III, fig. 3), contradicting findings by Andronic et al. (see section 7 and the discussion in

Paper III). We furthermore found that the Cl- permeability in activated and swollen T cells were

inhibited compared to non-activated control cells (Paper III, fig. 4), thus we suggest that Cl- is the

limiting factor in the RVD response in activated T cells and that the strong up-regulation of

KCNK5 has another function. We speculate whether this function could be in maintaining an

electrochemical gradient favoring a sustained Ca2+

influx, by extruding K+ and thus keeping the

plasma membrane hyperpolarized (see paper III and section 1.5.3.2).

1.4.2 When changes in cell volume act as signals

Not all cell volume changes will result in the activation of volume regulatory mechanisms and

often the change in cellular volume is an important player in cell signaling. As described

previously an altered cell volume is seen in various pathophysiological events, but changes in cell

volume without any regulatory mechanisms activating is also seen under physiological

conditions. It has for instance been shown that volume changes serves as signaling events in cell

proliferation and migration, transport across an epithelia and in apoptosis (fig. 7) which will be

further addressed below (also see ref. (56)).

1.4.2.1 Programmed cell death Different conditions can lead to

programmed cell death (or apoptosis), thus

programmed cell death is part of both cell

physiology (e.g. cells lacking growth factor

stimulation) and pathophysiology (e.g. cells

with DNA damage). If not wanted, the cells

can become suicidal by undergoing

programmed cells death a process that in

contrast to necrosis will not damage the

surrounding cells. Apoptosis is a

physiological process that is essential to

keep the balance in cell number (see (79)). A

number of characteristics can be observed

Figure 7: volume changes as a signaling mechanism There are various examples of an altered cell volume acting as a signaling event and some of these examples are depicted in this figure. See the text for more details. Model based on (56).


Page 24 of 117

in an apoptotic cell and one well described feature in apoptotic cell death is the initial loss of cell

volume. The apoptotic loss of cell volume is called Apoptotic Volume Decrease (AVD) and will

not result in RVI. The volume decrease is concomitant to a loss of K+ and Cl

- ions which initially

does not change the K+ concentration but later in the AVD it decreases significantly (123). The

decrease in K+ is followed by an increase in the Na

+ concentration. AVD was first believed to be a

secondary event in apoptosis, but have shown to be an important signal in facilitating the

further progress into programmed cell death (79; 93; 123). It has further more been shown that

the decrease in intracellular K+ concentration is vital in apoptosis and that this decrease is to

some extend due to an inhibition of Na+/K

+ pump function. The role of K

+ channels in apoptosis

is complex and so various K+ channels have been implicated depending on cell type. It is most

often seen that inhibition of K+ channel function and thus inhibited K

+ loss will protect against

apoptosis, but in some cell types the opposite has been seen. For more on ion channels in

programmed cell death see (79).

1.4.2.2 Proliferation When looking at cell volume changes and cell proliferation it is well known that the proliferating

cell undergo swelling to be able to produce two daughter cells of the same volume as the initial

parental cell, thus the dividing cell has increased in size without activating RVD mechanisms.

What is perhaps less well known and definitely less described is how this increase in volume

probably serves as a signal for the cell to progress through the cell cycle (see (56; 70; 92)). It has

thus been shown how proliferation in some cells can be stimulated in swollen cells and inhibited

in shrinking cells (30; 77; 122; 134). It is well known that cancer cells often have dysregulated

control of cell cycle progression involving changes in ion channels (119). How exactly these

volume changes influence cell cycle progression is yet to be fully elucidated.

1.4.2.3 Transepithelial transport Keeping a steady cell volume in epithelial cells constitute a challenge for the cells, since they are

subject to massive changes in osmolyte composition due to absorption and secretion of ions and

nutrients which are followed by water (see (119)). Though cell volume under normal conditions

are without large changes due to tight regulatory mechanisms there is evidence of epithelial cell

volume as a mechanism in the physiology of absorption and secretion. In that respect cell

swelling has been implicated in epithelial absorption and cell shrinkage in secretion (see (56;

78)).

1.4.2.4 Migration and invasion In cell migration there is likewise evidence of cell volume being an important signal and it has

e.g. been shown that hypertonicity inhibits neutrophile migration in the lung (131) and that cell

swelling plays a role in human polymorphonuclear leucocytes migration stimulated by fMLP


Page 25 of 117

(130). In transformed renal epithelial cells it was seen that migration could be inhibited both by

hypo- and hypertonicity, thus it seems that the type of impact cell volume changes has on

migration, can vary between cell types. For more on the subject of volume changes as a signal

(see (56)). Briefly, it is thought that a migrating cell is capable of cell swelling in the protruding

part of the cell and cell shrinkage in the back. These volume changes are facilitated by specific

shrinkage-activated transporters and swelling-activated channels resulting in cell protrusion in

the leading end and retraction in the rear end of the cell respectively (139).


Page 26 of 117

1.5 The immune system

The immune system can be divided into two parts; the innate and the acquired (or adaptive)

immune system. As the name implies the acquired immune system is under constant

development through the exposure to pathogens, the following immune response and the

subsequent activation of memory cells. The immune system is able to remember different

pathogens, an ability that is acquired throughout the numerous infections and pathogenic

influence an individual undergoes. This memory function makes a second exposure to a

pathogen elicit a far greater and faster immune response compared to the first exposure

response, even if the second exposure occurs many years after the initial one. Immunological

memory can be obtained by exposing an individual to a pathogen either by disease or by

vaccination.

The innate immune system, on the contrary, is the part of the immune system that we have with

us from birth and it constitutes the first barrier that meets an invading pathogen. Only when the

innate immune system has proven to be insufficient, the more specialized acquired immune

system is activated. Some infections can be defeated solely by an innate immune response and

those infections will hardly give any symptoms or do much harm to the body.

The innate and the adaptive immune systems, though playing very different parts in the battle

against intruders, should not be viewed as two distinct features, but rather as players on the

same team. The two system overlaps in different ways e.g. the dendritic cells of the innate

immune system plays a crucial role in the activation of the adaptive immune system since they

act as antigen presenting cells (APC’s), in fact the dendritic cells are the most important of APC’s.

Two other cell types can also act as APC’c namely the macrophages and the B cells.

Both the innate and the adaptive immune system relies on white blood cells or leucocytes, but

whereas the major players in the innate system are the macrophages, granulocytes, mast cells

and dendritic cells the highly specialized T and B lymphocytes (T and B cells) constitutes the

acquired part. A small fraction of activated T and B cells end up as memory cells making a future

response to the same pathogen more efficient (see above).

1.5.1 B and T lymphocytes When encountering an antigen, it is recognized by different mechanisms being a T cell or a B cell

and whereas T cells recognize antigens with the T cell receptor (TCR) B cells have

immunoglobulins acting as antigen receptors. Upon antigenic binding to the TCR T cells

activates, proliferate and differentiates into effector cells.


Page 27 of 117

While B cells are specialized in detecting antigens of pathogens living outside the cells, T cells

are specialized against pathogens living inside the host cell, thus they will always act on another

cell and not the pathogen itself. So to be activated T cells have to recognize a specific antigen

which can be from viruses, intracellular bacteria or from pathogens which have been

internalized, degraded by the host cell and taken up by major histocompatibility complex (MHC)

molecules to be displayed on the cell surface.

Whereas B cells recognize the antigen directly, T cells cannot recognize antigens unless

displayed by MHC molecules. B cells when activated differentiate into plasma cells which are

antibody producing (fig. 8), but since the B lymphocytes are of lesser interest in regard to this

thesis they will not be further dealt with here.

1.5.2 T cell activation T cell activation – from naïve to effector T cell – requires various signals and mechanisms. The

activation signaling can be divided into three sub-signals; signal 1, 2 and 3. Signal 1 comprises

the antigen-specific TCR activation whereas signal 2 involves the interaction between the APC

and the T cell itself, resulting in the promotion of survival and expansion of the T cell. In signal 3

differentiations of the effector T cells in response to cytokine stimulation is seen. One of the co-

stimulatory molecules involved in signal 2 is B7 molecules, which act on the CD28 receptor on

the T cell.

Different morphology of naïve vs. activated lymphocytes

Inactivated or naïve lymphocytes are small, has few organelles and most of the chromatin in the

nucleus is inactive. When activated the cell undergo and extreme transformation, they swell

(both the nucleus and the surroundings), the chromatin becomes less dense, nucleoli becomes

visible and a massive mRNA and protein synthesis occurs – the lymphocytes goes from being an

inactive naïve lymphocyte to becoming an activated lymphoblast.

1.5.2.1 T cell subtypes As mentioned previously activation of T cells result in proliferation and differentiation into

different kinds of effector T lymphocytes such as Cytotoxic T cells, Helper T cells or Regulatory T

cells (fig. 8). From the start (after their development in the thymus) T lymphocytes can be

divided into two groups according to the cell surface proteins displayed. One group bears the

surface protein CD4 the other carries CD8, both surface proteins being important for T cell

function. There are two main classes of MHC molecules – MHC class I and MHC class II and

whereas CD8+ T cells recognize MHC class I molecules, CD4

+ T cells recognize antigens shown on

the surface by MHC class II molecules. MHC class I molecules display peptides from the cytosol


Page 28 of 117

while MHC class II molecules display peptides from intracellular vesicles, a difference reflecting

the different tasks of the two T cell subtypes.

CD8+ T cells

Cytotoxic T cells carry the CD8 surface protein (or co-receptor) and kills virus infected cells. They

recognize antigen peptides generated in the cytoplasm of a cell and presented by MHC class I

molecules, which is found on almost all nucleated cells. When activated CD8+ T cells are always

bound to differentiate into cytotoxic T cells.

CD4+ T cells

Whereas CD8+ T cells always become cytotoxic T cells, CD4

+ T cells can differentiate further into

the subsets TH1, TH2, TH17 and regulatory T cells (fig 8). TH1 and TH2 subsets are also known as

helper T cells since they help activate B cells, TH17 are involved in activating a neutrophile

response, whereas the role of regulatory T cells is to suppress the immune system. CD4+ T cells

recognize MHC class II molecules and their general role is to activate other cells such as the B

cell. MHC class II molecules are found on B cells, dendritic cells and macrophages which are all

cells of the immune system and which is in contrast to the MHC class I molecules found on

numerous cell types.

Figure 8: overview of the development of T and B cells and their subsets upon activation Both T and B cells originate from the common lymphoid progenitor. A B cell will stay a B cell but will after activation differentiate into antibody secreting plasma cells. T cells on the other hand can be divided into two main groups; those carrying the CD8 surface protein and those carrying CD4. CD8+ T cells are bound to become cytotoxic T cells upon activation while CD4+ cells can further differentiate into the subsets TH1, TH2, TH17 and regulatory T cells. A small fraction activated T and B cells will become memory cells (not shown on this figure). Own figure.


Page 29 of 117

1.5.3 When the immune system attacks the body Sometimes the immune system turns on us in a way that is not beneficial for us. Factors in the

surrounding environment can be taken for antigens and elicit an unwanted immune response as

seen in e.g. allergic reactions, which as its best means a lot of discomfort for the individual, but

as its worst can be life-threatening. Another type of unwanted immune reaction is not seen in

response to the surroundings but to the body’s own cells and tissue – a response known as

autoimmunity. One of the most important features of the immune system is its ability to

regulate and to generate self-tolerance. During lymphocyte development lymphocytes with

affinity to the individual will be generated since the process is strictly random, but they will be

removed before entering the bloodstream. In autoimmune diseases something in the process of

generating self-tolerance is not working properly with potential catastrophic consequences as a

result. Of T cell mediated autoimmune diseases can be mentioned e.g. type 1 diabetes mellitus

and multiple sclerosis.

1.5.3 Ion channels in T cell activation

When encountering an antigen presented by MHC class proteins on dendritic cells T cells get

activated. This activation leads to clonal expansion of the antigen specific T cells and the

(hopeful) destruction of the invading pathogen. Clonal expansion involves strong proliferation

thereby amplifying the number of T cells bearing the TCR’s relevant for a given infection. The

activation of T cells relies on numerous factors and ion channel function is essential in this

process.

Three ion channels have been well described in T cell activation namely the voltage-activated

Kv1.3 channel, the Ca2+

-activated KCa3.1 channel and the Ca2+

release-activated Ca2+

channel

(CRAC). For more on the subject see e.g. (16; 112; 146). All of these channels and mechanisms

will be further described in the following sections.

1.5.3.1 Ca2+ signaling For a sustained activation of T cells an elevated intracellular Ca

2+ concentration ([Ca

2+]i) is crucial

(59; 152) and is generated by Ca2+

release from intracellular stores together with Ca2+

influx from

the extracellular matrix. MHC class II protein presentation of an antigen and the subsequent TCR

stimulation results in depletion of Ca2+

from intracellular stores to the cytoplasm through two

different signaling pathways - an 1,4,5-inositol triphosphate (IP3) (58) and a cyclic ADP-ribose

(cADPr) (45) mediated signaling pathway. The elevated [Ca2+

]i in turn activates calcineurin – a


Page 30 of 117

Ca2+

-calmodulin-dependent phosphatase which dephosphorylates the transcription factor NFAT

facilitating interleukin-2 (IL-2) transcription and expression, which is important in keeping the T

cell activated in an antigen independent manner. A 1-2 hour rise in [Ca2+

]i is needed for sufficient

NFAT stimulation and IL-2 expression, thus a sustained high intracellular Ca2+

level is therefore

essential for T cell activation (24; 40; 65; 102). For a review on Ca2+

signaling in T cells see (86).

The IP3 mediated pathway

The binding of MHC complexes (bearing antigen peptides) to the T cell receptor results in

recruitment and activation of protein tyrosine kinases (PTK’s) from the families Src, Csk, Tec and

Syk (see (127)). The PTK’s then phosphorylates and activates phospholipase C-γ (PLCγ) leading to

the cleavage of phosphatidylinositol 4,5-bisphosphate (PIP2) into diacylglycerol (DAG) and IP3. IP3

binds to the IP3-receptor in the membrane of the endoplasmatic reticulum (ER) causing release

of Ca2+

from ER stores into the cytosol. When Ca2+

is depleted from ER-stores Ca2+

is released

from of the EF-hand domain of stromal interacting molecule 1 (STIM1) located within the lumen

of ER, thus STIM1 acts as a store depletion sensors (90; 133). The Ca2+

from the ER stores causes

the STIM protein to oligomerize and translocate to activate Orai1 bound to the plasma

membrane. This results in the forming of a Ca2+

selective pore – the CRAC channel which allows

Ca2+

to enter the cell down its concentration gradient (124; 149; 155) (see fig. 9).

The cADPr mediated pathway

Another pathway resulting in Ca2+

release from intracellular thapsigargin insensitive stores is via

cADPr – a pathway much lesser studied and described than the IP3 mediated pathway described

above. The intracellular stores releasing Ca2+

through this pathway are located close to the

plasma membrane (13) and the Ca2+

release is as for the IP3 pathway initiated by tyrosine

kinases in response to receptor coupling see (112).

Signaling events by both pathways result in the uptake of Ca2+

through CRAC channels and

results by Guse et al. (46) suggests that high [Ca2+

]i via the IP3 pathway is important for the

initiation and cADPr signaling for the sustainment of the signal.

1.5.3.2 K+ channels in Ca2+ signaling The sustained high [Ca

2+]i depends on membrane hyperpolarization facilitated by K

+ efflux

through specific K+ channels, thus besides the release from ER stores [Ca

2+]i is also influenced by

voltage sensitive Ca2+

influx from the extracellular matrix through CRAC channels (fig. 9). Ca2+

influx is governed by capacitative Ca2+

entry by which the depletion of intracellular stores

facilitates Ca2+

uptake (126; 144) through CRAC channels situated in the plasma membrane (114)

and once activated the electrochemical gradient determines the Ca2+

influx (159). This also


Page 31 of 117

means that the influx will increase in response to cell

membrane hyperpolarization which can be obtained

by the efflux of K+

(fig. 9).

One of the most well described K+ channels in T cell

activation physiology is the voltage-gated Kv1.3

channel (also known as KCNA3) which was first

described in 1984 (15; 27; 36; 95). The channel

activates at approximately -60mV (94; 147) not far

from the resting potential (148) and further

depolarization causes increased conductance, thus T

cell activation and the following Ca2+

influx results in

a depolarization of the membrane, Kv1.3 activation

and K+ efflux. This in turn results in hyperpolarization

which creates the electrochemical gradient favoring

sustained Ca2+

entry (16; 112).

The Kv1.3 channel is not alone in generating the

membrane potential needed for Ca2+

entry thus the

Ca2+

activated KCa3.1 (also known as hIKCa1, IKCa1

or KCNN4) also play an important role. Activation of KCa3.1 is not dependent on membrane

potential but is activated – as the name implies – with rising [Ca2+

]i which is detected by

calmodulin bound to the C-terminus (33; 67). KCa3.1 activation level is between 200 and 300nM

[Ca2+

]i which is below the concentration found in non-activated T cells (30-100nM Ca2+

(147)).

The channel is activated by the rise in [Ca2+

]i mediated by store depletion and Ca2+

influx via

CRAC channels (fig. 9).

1.5.3.3 Other ion channels described in T cells

There are five main channel types described in T cells; Kv1.3, KCa3.1, ICl,vol, CRAC and TRPM7

channels but other channel types has also been proposed to play a role in T cell physiology.

Amongst them are e.g. channels such as voltage-gated Na+ channel (15) and the potassium

channels TASK-1 and TASK-3 has likewise been reported in T cell function (98) though their roles

still need further investigations and might be by some regarded as questionable (16; 146).

Besides TASK-1 and TASK-3 one of those, in T cell activation relatively recent described K+

channels, is KCNK5 (also known as TASK-2 or K2p5.1), thus in 2010 Bittner et al. published a

paper giving KCNK5 a role in the autoimmune disease multiple sclerosis (10). Though the

presence and functional importance is questioned by some (REF) preliminary data supported

that KCNK5 was expressed in T cells and we thus investigated the expression pattern of KCNK5

Figure 9: Ion channels and T cell activation When activated Ca2+ is released from intracellular stores which in turn causes activation CRACK channels in the plasma membrane, which in turn causes a rise in [Ca2+]i, depolarization, KCa3.1 and Kv1.3 channel activation, K+ efflux and hyperpolarization. The K+ efflux and following hyperpolarization favors further Ca2+ influx via the CRAC channels. All of these mechanisms act to gain a sustained high intracellular Ca2+ needed for the T cell to stay activated. Model adapted from (112).


Page 32 of 117

both on mRNA and protein levels and found a strong up-regulation of the channel upon T cell

activation (see Paper III, fig. 1+2).

Bittner et al. furthermore found a significant up-regulation of KCNK5 in the T cell subtypes CD4+

and CD8+ in multiple sclerosis patients during acute relapses and furthermore they found KCNK5

levels to be increased in CD8+ T cells. We have also looked into the KCNK5 expression pattern in

various T cell subtypes, confirming an up-regulation upon activation though we find no

significant difference in KCNK5 protein expression between the studied subtypes (see fig. 10,

own unpublished results).

1.5.4 T cell proliferation

T cell proliferation is essential for the battle against pathogen intruders, since the activation and

increased number of the right T cells will result in efficient fight. Clonal expansion is one of the

most important dogmas in immunology and describes how T cells will proliferate upon

activation. Being able to control or ultimately stop proliferation has thus gained high interest

since it might be a mean to prevent autoimmune diseases.

1.5.4.1 K+ channels in proliferation K

+ channels are shown to be involved in proliferation in various cell types and though the exact

mechanisms behind the involvement of ion channels in proliferation is still somewhat uncertain

the importance of ion channels are not. It is believed that ion channels help regulate

Figure 10: T cell subtypes KCNK5 protein expression and proliferation upon activation A: SDS-PAGE, western blotting and antibodies against KCNK5 and β-actin was used to measure KCNK5 protein expression pattern in T cell subtypes upon 72 hours stimulation and activation with CD3/CD28 beads (n=5-7). B: T cell proliferation upon 72 hours CD3/CD28 activation was measured using a NucleoCounter NC-100 (n=5).


Page 33 of 117

proliferation through their potential impact on

mechanisms such as membrane potential,

calcium homeostasis and or cell volume

regulation (see below and section 1.4.2.2).

As mentioned previously ion channels and

volume regulation is thought to influence cell

proliferation since a key feature of the dividing

cell is the gain in volume ensuring that the

daughter cells do not get smaller and smaller with

every division (see section 1.4.2.2).

Different K+ channels have been implicated in cell

cycle-control in various cell types where they are

thought to play a role in cell cycle progression by control of the membrane potential thus it is

seen that the membrane potential in early G1 phase (see fig. 11) is depolarized and followed by

a hyperpolarization through G1 to the S phase (see fig. 11) – a hyperpolarization believed to be

governed by K+ channels (143). In MCF-7 breast cancer cells this hyperpolarization depend on

the activation of ATP-sensitive K+ channels (153) and in brown fat cells (113) melanoma cells

(109) and prostate cancer cells (141) voltage-gated K+ channels was shown to be involved. The

same mechanisms was also true for the two pore-domain K+ channel TASK-3 in a human lung

carcinoma cell line (121). For more on the subject see (116). KCNK5 has been implicated in the

regulation of proliferation in human breast cancer cell lines T47D and MCF-7 where it is believed

that a increase in KCNK5 is necessary for the progression of the cells from G1 to the S phase (1).

Even though we cannot draw direct conclusions from our data two things are certain, T cells

proliferate upon activations and KCNK5 is at the same time heavily up-regulated (see fig. 10 A+B

and Paper III).

Figure 11: Progression through the cell cycle See the text for details. Figure from Clinical topics, Inc.


Page 34 of 117

2 Summary and conclusions

During my time as a PhD-student I have worked on the KCNK5 channel in three different set-ups.

I have worked on the signaling pathway from acute cell swelling to activation of KCNK5 and RVD

in Ehrlich cells, I have studied the long-term effects on channel physiology and expression

pattern also in Erhlich cells and last but not least I have studied the expression pattern and

physiology in activated human T cell physiology. I have learned a great deal about the channel

and in the following I will try and summarize and when possible conclude on my findings.

Paper I

- KCNK5 is tyrosine phosphorylated upon swelling-induced activation in a time-

dependent manner

- As tyrosine phosphorylation seem to be involved in activation of the channel tyrosine

phosphatases seem to be involved in the de-activation

- This tyrosine phosphorylation does not seem to be caused by neither Src nor FAK

- The JAK/STAT pathway could be up-streams of the phosphorylation and activation of

KCNK5

Paper II

- Long-term hypotonicity (48h) reduces the maximum current through KCNK5

- Long-term hypotonicity likewise reduces RVD performance upon cell swelling

- This physiological impairment is probably due to an decreased KCNK5 protein

expression

Paper III

- CD3/CD28 activation of human T cells result in proliferation and cell swelling

- KCNK5 is heavily up-regulated both on mRNA and protein levels in activated T cells

- Despite KCNK5 up-regulation activated T cells has impaired RVD

- RVD impairment in activated T cells is probably due to a functional down-regulation of

the volume activated anion channel VRAC in these cells


Page 35 of 117

3 Experimental procedures

Most of the experimental procedures are described in the three articles, but one procedure

deserves a few comments (section 3.1) and some experiments are not published but are

included in this thesis, thus the experimental procedures for these experiments will be explained

in the following section (section 3.2).

3.1 cDNA quantification instead of reference genes In the search of suitable reference genes for normalization of Real-Time qPCR data numerous

candidates were tested but none showed satisfactory results. Instead I looked at alternative

ways of normalizing my data and such an alternative was cDNA quantification using Quant-It

Oligreen (Invitrogen) which was described by Lundby et al. in 2005 (91). One could argue that

since the same amount of mRNA is used for each cDNA sample generated there should be no

particular need for an extra normalization procedure but besides being standard method of

operation when conducting Real-Time qPCR previously results verify the need for an additional

normalization step. The discrepancies between mRNA starting concentration and cDNA end

result could be due partly to pipetting uncertainties or slight differences in PCR reaction in each

tube. Thus cDNA was quantified in each sample as described (91) and the data normalized to

that value.

3.2 T cell subtypes During my studies of KCNK5 in activated T cell physiology I also looked at the KCNK5 expression

pattern in T cell subtypes besides the studies on whole T cell populations. The results are found

in section 1.5.3.3 and in fig. 10.

3.2.1 Purification

T cell subtypes were purified from human peripheral blood mononuclear cells (PBMC’s) using

EasySep enrichment kits from StemCell Tech. PBMC’s were previously purified from human

buffy coats obtained from the blood bank at Rigshospitalet as described in paper III. The

following subtypes were purified in accordance to manufactures descriptions: Human CD8+ T

cells, Human CD4+ T cells, Human Naïve CD8

+ T cells and Human Naïve CD4

+ T. T cell subtypes

were kept in the same media and under the same conditions as the heterogeneous T cell

population (see paper III) and were kept either quiescent or activated using Dynabeads Human

T-activator CD3/CD28 (Invitrogen, Life Sciences) in the ratio 1:1.

3.2.2 Proliferation

Proliferation was simply measured by cell count using the NucleoCounter® NC-100™ machine

(ChemoMetech, Allerød, Denmark).


Page 36 of 117

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4 Paper I

Activation of the TASK-2 channel after cell swelling is dependent on

tyrosine phosphorylation

Am J Physiol Cell Physiol 299: C844–C853, 2010.

First published July 14, 2010; doi:10.1152/ajpcell.00024.2010


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Activation of the TASK-2 channel after cell swelling is dependent on tyrosinephosphorylation

Signe Skyum Kirkegaard,2 Ian Henry Lambert,1 Steen Gammeltoft,2 and Else Kay Hoffmann1

1Section for Cell and Developmental Biology, Department of Biology, University of Copenhagen, Copenhagen;and 2Department of Clinical Biochemistry, Glostrup Hospital, Glostrup, Denmark

Submitted 22 January 2010; accepted in final form 6 July 2010

Kirkegaard SS, Lambert IH, Gammeltoft S, Hoffmann EK. Ac-tivation of the TASK-2 channel after cell swelling is dependent ontyrosine phosphorylation. Am J Physiol Cell Physiol 299: C844–C853,2010. First published July 14, 2010; doi:10.1152/ajpcell.00024.2010.—The swelling-activated K� currents (IK,vol) in Ehrlich ascites tumorcells (EATC) has been reported to be through the two-pore domain(K2p), TWIK-related acid-sensitive K� channel 2 (TASK-2). Theregulatory volume decrease (RVD), following hypotonic exposure inEATC, is rate limited by IK,vol indicating that inhibition of RVDreflects inhibition of TASK-2. We find that in EATC the tyrosine kinaseinhibitor genistein inhibits RVD by 90%, and that the tyrosine phos-phatase inhibitor monoperoxo(picolinato)-oxo-vanadate(V) [mpV(pic)]shifted the volume set point for inactivation of the channel to a lower cellvolume. Swelling-activated K� efflux was impaired by genistein andthe Src kinase family inhibitor 4-amino-5-(4-chloro-phenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) and enhanced by the tyrosine phos-phatase inhibitor mpV(pic). With the use of the TASK-2 inhibitorclofilium, it is demonstrated that mpV(pic) increased the volume-sensitive part of the K� efflux 1.3 times. To exclude K� efflux via aKCl cotransporter, cellular Cl� was substituted with NO3

�. Also underthese conditions K� efflux was completely blocked by genistein. Thustyrosine kinases seem to be involved in the activation of the volume-sensitive K� channel, whereas tyrosine phosphatases appears to beinvolved in inactivation of the channel. Overexpressing TASK-2 inhuman embryonic kidney (HEK)-293 cells increased the RVD rateand reduced the volume set point. TASK-2 has tyrosine sites, andprecipitation of TASK-2 together with Western blotting and antibod-ies against phosphotyrosines revealed a cell swelling-induced, time-dependent tyrosine phosphorylation of the channel. Even though wefound an inhibiting effect of PP2 on RVD, neither Src nor the focaladhesion kinase (FAK) seem to be involved. Inhibitors of the epider-mal growth factor receptor tyrosine kinases had no effect on RVD,whereas the Janus kinase (JAK) inhibitor cucurbitacin inhibited theRVD by 40%. It is suggested that the cytokine receptor-coupledJAK/STAT pathway is upstream of the swelling-induced phosphory-lation and activation of TASK-2 in EATC.

cell volume regulation; Janus kinase; volume-sensitive channels

THE ABILITY of a cell to volume regulate after volume pertur-bations is fundamental for its homeostasis. When exposed to anincrease in the internal or a decrease in the external osmolyteconcentration, most mammalian cells swell and subsequentlycompensate for this swelling by a release of osmolytes throughdifferent membrane transporters followed by a concomitantefflux of water, a process termed regulatory volume decrease(RVD) (19). Especially swelling-activated K� and Cl� chan-nels as well as volume-sensitive transporters of organic os-

molytes (e.g., taurine) are important in the RVD response (16),and it has been estimated that �70% of the loss of osmolytesin Ehrlich ascites tumor cells (EATC) during RVD is due to theloss of KCl (14). Various K� channels have been shown to beimplicated in RVD depending on cell type; i.e., 1) Ca2�-activated small conductance K� (SK) channels (24, 42), inter-mediate conductance K� (IK) channels (3, 7, 24, 25, 46), andBig conductance K� (BK) channels (10, 23); 2) stretch-acti-vated K� channels; 3) voltage-dependent channels; 4) theMinK channel; and 5) the two-pore domain K� channel (4,31–35, 37). The volume-sensitive ion channels in EATC arethe volume-regulated anion channel (VRAC) (38) and theTWIK-related acid-sensitive K� channel-2 (TASK-2) (22, 33,41), which belongs to the family of two-pore domain channels(K2p). TASK-2 is sensitive to clofilium (35), independent ofintracellular Ca2� (35, 41), and highly sensitive to external pH(21). Besides being the swelling-activated K� channel inEATC, TASK-2 has also been found to be involved in volumeregulation in kidney cells (4) and in murine spermatozoa (1).Moreover, TASK-2 has been associated with apoptotic volumedecrease in EATC cells following cisplatin exposure (40). Forfurther details about volume-sensitive K� channels see Refs.19 and 44.

Tyrosine kinases are important under several physiologicalconditions such as cell proliferation, migration, differentiation,metabolism, and immune system function, but also a highnumber of oncogenes are tyrosine kinases (for a review seeRef. 6). There are several evidences for the role of tyrosinekinases in RVD (8). The nonreceptor tyrosine kinases Src andfocal adhesion kinase (FAK) has previously been reported tobe activated during hypotonic stimulation in various cell types;i.e., phosphorylation of an activation site in FAK have previ-ously been reported in, for example, chicken retina cells (9),human umbilical vein endothelial cells (15), and in cerebellargranule neurons (30), whereas hypotonic activation of Src hasbeen reported in cerebellar granule neurons (30), rat hepato-cytes (45), and in rat hepatoma cells (2). There is however noclear evidence linking the activation of tyrosine kinases toactivation of swelling-activated K� currents (IKvol). In EATCpreliminary results showed that the relatively unspecific ty-rosine kinase inhibitor genistein (371 �M) strongly inhibitedRVD, and the tyrosine phosphatase inhibitor monoperoxo(pi-colinato)-oxo-vanadate(V) [mpV(pic)] (10 �M) significantlydecreased the volume set point after RVD (17). Thus tyrosinephosphorylation appears to play a role in RVD in EATC,possibly as a part of the volume-sensing mechanism. Theobjective of this study was thus to examine the involvement oftyrosine kinases/phosphatases in the swelling-induced activa-tion/deactivation of TASK-2 in EATC. We specifically testwhether overexpression of TASK-2 improves the RVD re-

Address for reprint requests and other correspondence: E. K. Hoffmann,Section of Cell and Developmental Biology, Dept. of Biology, The AugustKrogh Bldg, Univ. of Copenhagen, 13, Universitetsparken, DK-2100, Copen-hagen, Denmark (e-mail: [email protected]).

Am J Physiol Cell Physiol 299: C844–C853, 2010.First published July 14, 2010; doi:10.1152/ajpcell.00024.2010.

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sponse and whether TASK-2 is directly tyrosine phosphory-lated following hyposmotic exposure.

EXPERIMENTAL PROCEDURES

Cell Cultures: EATC, EATCC, and Human Embryonic KidneyCells-293

In the present investigation we have used EATC maintained undertwo separate conditions: 1) EATC maintained in first generationhybrids of female NMRI mice by weekly intraperitoneal transplanta-tion of 1.5 � 107 cells per mouse. Six to seven days after transplan-tation the cells were harvested and transferred to isotonic NaCl-Ringercontaining 2.5 U/ml heparin and resuspended at cytocrit 4%. 2) EATCcultures (EATCC) were obtained by transferring EATC to RPMI 1640medium supplemented with 10% serum and 100 U/ml penicillin-streptomycin and grown at 37°C, 5% CO2. EATCC were maintainedby transfer of 8.5 � 105 cells to 10 ml fresh RPMI 1640 mediumevery 3–4 days, and only passages 6–28 were used for experiments.The EATCC were introduced because we wanted to avoid the use ofmice for tumor cell growth. This was supported by The Danish Societyfor the Protection of Laboratory Animals. Propagation of ascites cells wasapproved by “Dyreforsøgstilsynet” 2007/561-1313. 3) Human embryonickidney cells (HEK-293) were grown in Dulbecco’s modified Eagle’smedium (DMEM with GlutaMAX-l, D-glucose, and sodium pyruvate,Invitrogen) with 10% fetal bovine serum and 100 U/ml penicillin-streptomycin. Cells were subcultured every 3–4 days.

Solutions and Materials

For EATC the standard isotonic NaCl-Ringer solution (300 mosM)contained (in mM) 143 NaCl, 5 KCl, 1 MgSO4, 1 Na2HPO4, 1 CaCl2,3.3 MOPS, 3.3 N-tris-(hydroxymethyl) methyl-3-aminoethane sul-fonic acid (TES), and 5 HEPES, pH 7.4. Hypotonic Ringer (150mosM) was obtained from the isotonic solution by dilution withbuffered water (water containing 3.3 mM MOPS, 3.3 mM TES, and 5mM HEPES, pH 7.4). The NO3

� Ringer is a Ringer where all Cl� issubstituted with NO3

�. For HEK-293 cells the isotonic NaCl-Ringer(300 mosM) contained (in mM) 140 NaCl, 4 KCl, 2 CaCl2, 1 MgCl2,and 10 HEPES; pH was adjusted to 7.4. Hypotonic Ringer (150mosM) was obtained as described above. Unless otherwise stated,reagents were analytical grade and obtained from Sigma (St. Louis,MO). [3H]Inulin and 86Rb were from Amersham Bioscience.

Cell Volume Measurements

Cell volume was determined using two different methods. Absolutecell volume of EATC/EATCC was estimated by electronic cell sizingby Coulter counter measurements (tube orifice 100 �m, CoulterMultisizer 3, Coulter, Luton, UK). Relative cell volume of the mono-layer HEK-293 cells was estimated by large angle light scatteringmeasurements (RatioMaster model C44–2000, Photon TechnologyInternational).

Coulter counter measurements. Cell density was 90,000 cells/mlequivalent to a cytocrit at 0.008%. Cell volume was determined as themedian of the cell volume distribution curves after calibration withlatex beads (14.89 �M). The Ringer solutions for the coulter counterexperiments were filtered (Millipore, 0.45 �M) before use. Volumerecovery was estimated as (Vmax � V3– 4min)/(Vmax � Viso), whereVmax, V3– 4min, and Viso are the maximal cell volume, cell volume attime 3 or 4 min and cell volume under isotonic conditions, respectively.

Light scatter measurements. Wild-type or TASK-2 transfectedHEK-293 cells were grown on coated (poly-L-lysine) coverslips until�80% confluence. Light scattering results were obtained using themonochromator settings: excitation 589 nm and emission 595 nm. Forfurther description of the light scatter method see Ref. 39.

86Rb Efflux-Estimation of K� Efflux

86Rb is regarded as a tracer for K�, and 86Rb efflux experimentswere performed as previously described (20). Briefly, EATC wereequilibrated for 30 min with 86Rb� in standard NaCl-Ringer (2 � 104

Bq/ml), washed, and resuspended at a cytocrit of 2% in standardisotonic (300 mosM) or hypotonic Ringer (150 mosM), both contain-ing 0.01% bovine serum albumin (BSA) and 30 �M bumetanide.Bumetanide, added to avoid 86Rb� reuptake via NKCC1, was presentfor the last 5 min of the preincubation and during the efflux experi-ment. [3H]Inulin was added for estimation of extracellular space.86Rb� efflux from the cells was measured as gain in the extracellular86Rb� activity (cpm/ml medium) within the initial 10 min aftertransfer to hypotonic and isotonic Ringer by serially isolating cell-freeefflux medium by centrifugation (15,000 g, 30 s) through a silicone oilphase [300 �l 5:1 (wt/wt) AR200,200 and DC 200/20]. Separatedouble samples were taken at time 1 min for estimation of cellularcontent (�mol/sample) and cell dry weight (g/sample) after correctionfor 86Rb and K� trapped in the extracellular space using the [3H]inu-lin as a marker (20, 27). 3H and 86Rb activities were measured in aliquid scintillation spectrometer (Packard 2000CA Tri-Carp LiquidScintillation Analyzer) in channels ranging from 2.0 to 18.6 kV and19 to 1,500 kV, respectively, using ULTIMA GOLD (Packard) asscintillation fluid. K� content was determined by atomic absorptionflame photometry (model 2380, Perkin Elmer atomic absorptionsspectrophotometer) using samples and K� standards diluted 100-foldwith 1 mM CsCl. 86Rb� activity (cpm/g cell dry wt), estimated fromthe extracellular 86Rb activity (cpm/ml medium) and the cell dryweight (g/ml medium), was plotted versus time, and the rate constantfor 86Rb� efflux (min�1) was determined by fitting the efflux curvebetween 1 and 10 min to an exponential function (18). The 86Rb�

efflux (cpm/ml cell water � min�1) was calculated as the product ofthe rate constant and the cellular concentration (cpm/ml cell water).The K� efflux (�mol/ml cell water � min�1) was calculated from the86Rb� efflux by division with the specific activity (cpm/ml cellwater·�mol K��1·ml cell water�1) and converted to �mol/g dry wt �min�1 by multiplication with the water content (ml cell water/g celldry wt).

Gel Electrophoresis and Western Blotting

EATCC were lysed in 95°C lysis buffer (10 mM Tris·HCl, pH 7.4,1% SDS, and 20 mM EDTA) with protease inhibitors (Roche AppliedScience) and phosphatase inhibitors added. The lysates were subse-quently homogenized, and the supernatant was collected by centrifu-gation. HEK-293 cells transfected with TASK-2 were lysed in 95°Clysis buffer (5 mM Tris·HCl, pH 7.4, 0.5% SDS, and 10 mM EDTA).Protein concentrations were determined using Bio-Rad DC proteinassay. SDS-polyacrylamide gel electrophoresis were performedusing a 10% gel (Invitrogen) and transferred to nitrocellulosemembranes. The membranes were dyed with Ponceau Red (Sigma)and washed in TBST (10 mM Tris·HCl, pH 7.5, 120 mM NaCl, and0.1% Tween 20) before being blocked in a 5% nonfat dry milk andTBST solution. Membranes were incubated with primary antibodyat following concentrations: TASK-2 (Alomone) 1:500, nonphos-phorylated (np) FAK Y397 (Biosource) 1:200, phosphorylated (p)FAK Y397 (Biosource) 1:200, np-Src Y416 (Cell Signaling) 1:250,p-Src Y416 (Cell Signaling) 1:250, np-Src Y527 (Cell Signaling)1:250, p-Src Y527 (Cell Signaling) 1:250. Membranes werewashed in TBST before introduction to secondary antibody(Sigma) 1:5,000. The protein bands were visualized using 5-bro-mo-4-chloro-3-indolyl phosphate-nitro blue tetrazolium (BCIP/NBT Kirkegaard and Perry Lab) and scanned. Protein band densitywas estimated using UN-SCAN-IT software.

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Transient Transfection

A TASK-2 containing vector (pcDNA3.1) was kindly given to usby Dr. Sepúlveda (Chile). The TASK-2 channel was transferred totwo other vectors. The first, pMT2-HA vector using PCR at thefollowing conditions: denaturation at 98°C for 30 s followed by 25cycles with the initial temperature at 98°C for 10 s, annealing at 65°Cfor 30 s extension at 72°C for 40 s, and a end extension at 72°C for7 min. The restrictions sites Xho1 and kpn1 were used together withthe following primers: 5=-ATGGTGGACCGGGGT-3= (sense) and5=-TCACGTGCCCCTGGG-3= (antisense). PCR product was purifiedand resolved by agarose gel electrophoresis. DNA bands were cut outof the gel, purified, and subsequently cloned into the pMT2-HAvector. For all purification steps products from Qiagen was used.Sequencing was performed by Eurofins MWG Operon (Germany).Second, pcDNA3.1/myc-His vector (Invitrogen) using PCR and therestrictions sites BamH1 and Xho1. The following primers weredesigned: 5=-AGTGTGGACCGGGGTCCTTTAC-3= (sense) and 5=-CGTGCCCCTGGGGTTATCTGC-3= (antisense). PCR conditionswere the following: denaturation at 98°C for 5 min followed by 25cycles with the initial temperature at 98°C for 1 min, annealing at62°C for 30 s and extension at 72°C for 40 s, and at the end a 7 minextension at 72°C. The rest of the procedure was identical to the onedescribed above.

Light scatter. HEK-293 cells for transfection were grown on coated(poly-L-lysine) coverslips to �50–80% confluence and transfectedwith a TASK-2 containing vector (pMT2-HA). For transfection weused 1 �g DNA/3 �l FuGENE 6 Transfection Reagent (RocheApplied Science) according to the manufacturer’s instructions. Exper-iments were performed �24 h after transfection.

Precipitation of TASK-2. HEK-293 cells for transfection weregrown in coated (poly-L-lysine) wells (9.6 cm2/well) to �50–80%confluence and transfected with a TASK-2-containing vector(pcDNA3.1/myc-His). For transfection we used 1 �g DNA/3 �lFuGENE 6 Transfection Reagent (Roche Applied Science) accordingto the manufacturer’s instructions. Experiments were performed �36h after transfection. It should be noted that we tried to use the HA tagto immunoprecipitate the TASK-2 without success.

Western blot analysis. HEK-293 cells for transfection were grownto 50–80% confluence in six-well trays and transfected as describedabove.

TASK-2 Phosphorylation

HEK-293 cells were grown in coated (poly-D-lysine) six-well traysto 50–80% confluence and transfected with a pcDNA3.1/myc-Hisvector (Invitrogen) containing TASK-2 as described above. The cellswere either hypotonically (150 mosM) or isotonically (300 mosM)(controls) stimulated for 1, 5, or 10 min at 37°C and subsequentlylysed using a Radio Immuno Precipitation Assay (RIPA) buffer (1%NP-40, 1% sodium deoxycholate, 0,1% SDS, 150 mM NaCl, 5 mMTris·HCl, 2 mM EDTA, and 10 mM imidazole). The TASK-2 channelwas precipitated using Ni-NTA agarose (Qiagen), and the specifictyrosine phosphorylation was visualized through SDS-PAGE separa-tion and Western blotting (see above) using the phosphospecifictyrosine antibody pY-100 (1:500, Cell Signaling) and a TASK-2-specific antibody (1:500) (Alomone). Visualization of TASK-2 bandswas performed using BCIP/NBT (Kirkegaard and Perry), whereas thepY100 band was visualized using Lumigen solutions A and B (GEHealthcare) and chemiluminiscens. Protein band density was esti-mated using UN-SCAN-IT software.

Statistically Analysis

The values are given as means � SE. Statistically significance wasanalyzed by the use of Student’s t-test or ANOVA and shown as oneasterisk when significant at a 5% significance level and two asteriskswhen significant at a 1% significance level.

RESULTS

Effect of Tyrosine Kinase Inhibitors on RVD and onSwelling-Activated K� Efflux

In the present investigation we used EATC grown in cellculture (EATCC) or in the abdominal cavity of mice (EATC)to investigate the effect of the tyrosine kinase inhibitorgenistein on the RVD and the swelling-induced K� loss. FromFig. 1, A and B, it is seen that genistein is an effective inhibitorof RVD in EATC grown both in culture and in the abdominalcavity. Genistein was dissolved in DMSO, which can bepotentially toxic to the cells, but experiments showed thatthe concentrations used (0.34%) did not affect the ability of thecells to perform RVD (data not shown). With the use of therecovery level after 3 min (EATC) or 4 min (EATCC), it isestimated that genistein reduced the RVD from 73% to 22% inEATC (Fig. 1C) and from 61% to 10% in EATCC (Fig. 1D).It is noted that the swelling response to hyposmotic media ismuch faster (in seconds) in the EATCs than that in theEATCCs, which probably reflects a difference in the numberof aquaporins. This has not been tested. To investigatewhether genistein directly affects the swelling-activated K�

efflux, we measured the K� efflux in EATC in hypotonicmedium in the presence and absence of genistein. 86Rb wasused as a tracer for K�. Figure 2, A and C, shows thatgenistein reduces the measured 86Rb efflux and the calcu-lated K� efflux down to about 30% upon hypotonic expo-sure; i.e., the control cells had a mean K� loss of 100�mol·g protein�1·min�1, and the treated cells had a meanloss of 33.3 �mol·g protein�1·min�1. To exclude contribu-tion to the K� efflux by the KCl cotransporter, we substi-tuted all internal and external Cl� with NO3

� by preincu-bating the cells for 60 min in NO3

� Ringer. From Fig. 2, Band D, it is seen that the swelling-activated K� efflux, nowrepresenting only efflux via K� channels, is completely inhib-ited by genistein. Substitution of NO3

� for Cl� reduced the K�

efflux from 222 � 13 to 171 � 10 �mol·mg dry wt�1·min�1

(n � 11 for each series), indicating a K� efflux via the KClcotransporter equal to 51 �mol·mg dry wt�1·min�1; i.e., 23%.This is quantitatively similar to the genistein-insensitive K�

flux seen in NaCl medium (Fig. 2C).

Effect of Tyrosine Phosphatase Inhibitor on RVDand Loss Of K�

As inhibition of tyrosine kinases hinders RVD (Fig. 1) andinhibits the activation of volume-sensitive K� channels (Fig.2), we investigated whether inhibition of tyrosine phosphatasescould potentiate the RVD in EATC. Cell volume measure-ments were performed in the presence of the tyrosine phos-phatase inhibitor mpV(pic) (10 �M). From Fig. 3A it is seenthat in control cells a new steady-state cell volume was ob-tained after 3 min exposure to hypotonic medium (150 mosM),which was about 20% larger than the original cell volume. Inthe presence of the tyrosine phosphatase inhibitor mpV(pic),the volume recovery at time 4 min improved and amounted to90% compared with the 70% in control cells (Fig. 3B). Thesefindings were further substantiated through K� efflux measure-ments. When treated with mpV(pic), the mean swelling-acti-vated K� efflux in EATC was increased to 150% comparedwith nontreated cells (Fig. 3C). Adding clofilium, a known

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TASK-2 blocker (35), the K� efflux was inhibited to 27% ofthe K� efflux value in nontreated control cells. In the presenceof clofilium plus mpV(pic), the K� efflux was reduced to theefflux value seen in the presence of clofilum alone. Thus ampV(pic)-sensitive phosphatase seems to be involved in theinactivation of the volume-sensitive K� channel (TASK-2) inEATC.

To test whether inhibition of tyrosine phosphatase couldprevent the effect of inhibition of tyrosine kinase, wemeasured the RVD response in cells treated with genisteinalone and genistein in combination with the phosphataseinhibitor mpV(pic). From Fig. 3D it is seen that inhibition ofthe tyrosine phosphatase indeed prevented the effect ofkinase inhibition.

Fig. 1. The general tyrosine kinase inhibitorgenistein inhibits volume recovery in Ehrlichascites tumor cells (EATC) and EATC cultures(EATCC). A and B: EATC and EATCC wereplaced in either isotonic Ringer (300 mosM) orhypotonic Ringer (150 mosM), and the absolutevolume was observed as a function of time.EATC and EATCC were preincubated with thetyrosine kinase inhibitor genistein (371 �M) for45 min before they were transferred at time 0 tohypotonic medium also containing genistein(371 �M). C and D: mean recovery after 3 minof EATC and EATCC in hypotonic mediumwas estimated from 3 independent sets of ex-periments and shown as means � SE. **Signif-icantly reduced from control cells ability toregulate their volume.

Fig. 2. Genistein inhibits swelling-induced, Cl�-indepen-dent K� efflux. EATC were incubated in isotonic NaClRinger (A and C) or isotonic NaNO3 Ringer (B and D).86Rb, genistein (100 �M) and [3H]inulin were added 60,45, and 5 min before initiation of efflux estimation,respectively. Cells were subsequently washed once inisotonic Ringer (300 mosM, NaCl/NaNO3), and at time 0resuspended in hypotonic Ringer (150 mosM, NaCl/NaNO3) containing no genistein (control) or 100 �Mgenistein. Twenty samples were drawn within the first 10min and centrifuged through a silicone oil phase as indi-cated in Experimental Procedures. Double samples wereused to estimate the cellular K� and 86Rb content within1 min following hypotonic exposure. 86Rb� activity(cpm/g cell dry wt) was plotted versus time (A: NaClRinger; B: NaNO3 Ringer), and the rate constant for86Rb� efflux (min�1) was determined by fitting the effluxcurve to an exponential function. The K� efflux (�mol·gdry wt�1·min�1), calculated by multiplying the 86Rb-rateconstant (min�1) by the K� content (�mol/g dry wt), wasestimated from 5 and 8 sets of experiments in NaCl Ringer(C) and NaNO3 Ringer (D), respectively and shown asmean values � SE. **Significantly reduced from controlcells ability to regulate their volume (P � 0.01).



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Functional Effect of Overexpressing TASK-2 on RVD

To test the effect of TASK-2 overexpression on RVD, weused HEK-293 cells, which according to Niemeyer and co-workers (33) show no swelling-activated K� current. FromFig. 4A it is seen that overexpression of TASK-2 cloned fromEATC in HEK-293 was obtained after 24, 36, and 48 h. Usingthe light scattering technique, we found that TASK-2 overex-pressing HEK-293 cells (Fig. 4C) had a faster volume recoverythan the wild-type cells (Fig. 4B). It was estimated thatTASK-2 overexpression resulted in a twofold faster volumerecovery (Fig. 4D). It is noted that the TASK-2 overexpressingcells made an overshot in the RVD (Fig. 4C), which could betaken to indicate that the cellular signaling system involved inclosing of the TASK-2 channel was not effective in theHEK-293 cells. This was not investigated further. It is alsonoted that the transfected cells showed a strong post-RVDregulatory volume increase (RVI) response when reexposed toisotonic Ringer (Fig. 4C) in contrast to wild-type HEK-293cells (Fig. 4B), indicating a far greater ion loss during the RVDperiod compared with the wild-type cells. Thus expression ofTASK-2 contributed significantly to the net loss of K� fol-lowed by Cl� and water.

Swelling-Induced Tyrosine-Phosphorylation of TASK-2

To investigate whether TASK-2 is tyrosine-phosphorylatedfollowing hypotonic exposure, we transfected HEK-293 cellswith a TASK-2 containing pcDNA3.1/myc-His vector, stimu-lated the cells hypotonically for 1, 5, or 10 min, and subse-quently precipitated the TASK-2 channel using Ni-NTA aga-rose (Qiagen). Tyrosine phosphorylation was detected throughSDS-PAGE and Western blotting using a phospho-specifictyrosine antibody (pY100). From Fig. 5 it is seen that TASK-2expression was identical in isotonic- and hypotonic-treatedcells (Fig. 5A), whereas the phosphorylation of TASK-2(pY100) increased within 1 min following hypotonic exposureand appeared to be maximal after 5 min (Fig. 5B). It is notedthat ANOVA test on the total experimental data set indicatedno difference of the pY100 band among 1, 5, and 10 minisotonic medium. The relative specific activity for tyrosine-phosphorylated TASK-2 under hypotonic conditions (the ratiobetween pY100 band intensity and the TASK-2 band intensitygiven relative to the ratio in isotonic medium) was increasedfour times within the first minute (P � 0.14), four times within5 min (P � 0.01), and three times after 10 min (P � 0.03)hypotonic exposure. Hence, the TASK-2 channel was still

Fig. 3. Possible involvement of mpV(pic)-sensitive tyrosine phosphatases in regulatory volume decrease (RVD) and swelling-activated K� efflux in EATC.EATC were preincubated with the tyrosine phosphatase inhibitor mpV(pic) (10 �M) for 30 min before they were transferred to hypotonic medium also containingmpV(pic) (10 �M). A: EATC were placed in either isotonic Ringer (300 mosM, closed circles) or hypotonic Ringer (150 mosM) without mpV(pic) (open circles)or with mpV(pic) (closed triangles), and the absolute cell volume was measured as a function of time. B: mean values for volume recovery of EATC after 4 minhypotonic exposure was calculated from 3 to 8 independent experiments and shown as means � SE. C: K� efflux was estimated as indicated in Fig. 2. Cellswere preincubated with 100 �M clofilium and/or 10 �M mpV(pic) for 30 min, and both inhibitors were present under the efflux experiment. Flux are given asmean values � SE and represent 12 control [mpV(pic)], 6 (clofilium), and 6 [clofilium � mpV(pic)] independent experiments. In the case of clofilium the K�

efflux was calculated from 86Rb efflux within the initial 5 to 6 min after which the efflux suddenly increased by a yet undescribed pathway (data not shown).**Value is significantly different compared with control (P � 0.01). #Significantly reduced compared with mpV (pic) alone (P � 0.01). D: EATCC were exposedto the tyrosine kinases inhibitor genistein (371 �M) in the absence or presence of the tyrosine phosphatase inhibitor mpV(pic) (10 �M). The rate of RVD wasestimated as the reduction in cell volume within the period 1 to 3 min following hypoosmotic exposure (150 mosM) using the Coulter counter. Genistein, whenpresent (black and grey bars), was added to the cells 45 min before hypotonic exposure. mpV(pic) was added at the same time as genistein (light shaded bar)or 30 min (preincubation) before addition of genistein (dark shaded bar). All inhibitors were present during estimation of RVD. The number of experimentsrepresents 6 (control), 4 (genistein), and 3 [genistein � mpV(pic)]. *Value is significantly different compared with control. #Significantly increased comparedwith genistein alone.



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tyrosine phosphorylated 10 min after hypotonic exposure, i.e.,at that time the K� efflux was still high (Fig. 2, A and B).

Putative Tyrosine Kinases Involved in Swelling-InducedActivation of TASK-2

Src and FAK. Various tyrosine kinase inhibitors were usedin an attempt to narrow down the potential tyrosine kinase(s)involved in channel activation. As seen in Fig. 6A the volume-activated K� efflux was reduced with 25% in the presence ofthe Src kinase inhibitor PP2 compared with controls with noinhibitor present. Hypotonic medium with and without PP2present indicates a small but significant inhibition of theswelling-activated K� efflux in EATC. The RVD, estimated asthe initial reduction in cell volume (10�15 l/min), in EATCCwas significantly reduced to 35% (Fig. 6B), indicating aneventual role of Src in the RVD response. To pursue a role ofSrc in activation of the swelling-induced K� channels andRVD we used Western blot analysis with phospho-specificantibodies against 1) Src Y416, which is in the kinase domainand is phosphorylated during activation, and 2) Src Y527,which has to be dephosphorylated to activate the kinase. FromFig. 6, C (left) and D, it is seen that 1 and 5 min of hypotonicstimulation elicited a significant decrease in the phosphoryla-tion of Src Y416, reflecting a decreased Src activity afterosmotic cell swelling. Looking at the inactivating site Y527, nochange in the phosphorylation levels was observed over a10-min time period (Fig. 6, C, right, and E). The eventualeffect of cell swelling on FAK was likewise investigatedusing Western blot analysis and a phospho-specific FAKY397 antibody. From Fig. 7, A (Western blot) and B(quantification), it is seen that initially (1 min after stimu-lation), there was no change in the phosphorylation of FAK,which would be expected if the kinase was activated duringcell swelling. On the contrary there was a significant de-crease in the phosphorylation levels at 5 and 10 min after

hypotonic stimulation (Fig. 7B). Thus an increase in the Srcand FAK kinase activities does not seem to be involved inactivation of volume-sensitive channels K�, in EATCC, andhence RVD.

JAK2 and receptor-coupled tyrosine kinases. To substantiatethe functional role of tyrosine kinases, we investigated theeffect of inhibition of JAK2 [cucurbitacin I (5)], the insulin-like growth factor I receptor [picropodophyllotoxin (13)], andthe epidermal growth factor receptor [GW583340, dihydro-chloride (11)] on the volume recovery after cell swelling. FromFig. 8 it is seen that cucurbitacin I reduced the recovery levelafter 4 min by 40%, whereas the other inhibitors had nosignificant effect. This indicates that the JAK/STAT3 pathwaycould be involved in the tyrosine phosphorylation of TASK-2and hence in the RVD response.

DISCUSSION

Tyrosine Kinases are Involved in the Initiationof RVD

In volume measurements on EATC and EATCC using theCoulter counter, it was seen that the control cells did not reachthe perfect osmometer value, which is due to the fact that thecells activate their volume-sensitive ion channels already afterno more than a 5% swelling. A RVD response will thus hinderthe cells in reaching the perfect osmometer value. On thecontrary EATCC treated with genistein did in fact reach theperfect osmometer value, indicating that their ability to volumeregulate was completely inhibited. Even after 4 min there wasno sign of a RVD response. The inhibition of RVD was evidentand similar in both cell lines. These results may be explainedby the inhibition of either one or both of the volume-activatedion channels, which in EATC and EATCC are VRAC (ICl,vol)and TASK-2 (IK,vol) (see INTRODUCTION) upon hypotonic swell-ing. The K� permeability increases much less when compared

Fig. 4. TWIK-related acid-sensitive K� channel-2(TASK-2) overexpression improves volume recovery.A: HEK-293 cells transfected with TASK-2 contain-ing plasmid were harvested 24, 36, and 48 h aftertransfection. The success of the transfection was visu-alized through SDS-PAGE and Western blot analysisusing antibody against TASK-2. B and C: wild typeHEK-293 cells (control, B) and cells transfected withTASK-2 for 24 h (C) were grown on coated coverslipsuntil �80% confluence, and cell volume was mea-sured using the light scatter technique. When stablemeasurements in isotonic Ringer were seen, the me-dium was shifted to a hypotonic Ringer for 10–15min. Volume measurements were carried out every1–2 s. After this period of time the Ringer was shiftedback to the isotonic Ringer and measurements contin-ued for another 10–15 min. D: volume recovery fol-lowing 4 min hypotonic exposure was estimated in 5(wild type) to 6 (TASK-2) independent experimentsand given as mean values � SE. *Significantly in-creased compared with control cells (P � 0.05).



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with the Cl� permeability during cell swelling, which makesthe K� permeability the limiting factor for the RVD rate (27).Inhibition of RVD will thus often reflect an inhibition of theswelling-activated K� channel. However, it should be notedthat an almost complete inhibition of VRAC would lead to thesame results. Results from efflux measurements comply withthe volume measurements, and it was seen that the swelling-activation of K� efflux in EATC was strongly inhibited whencells were treated with the tyrosine kinase inhibitor genistein.This was true also when all intracellular Cl� was substitutedwith NO3

� to assure that all measured K� efflux was via a K�

channel and not involving the KCl cotransporter. It is thus clearthat one or more tyrosine kinases are responsible for swelling-activation of the K� channels, which in EATC and EATCC areknown to be TASK-2 channels. It has previously been shownthat TASK-1 background current is inhibited by genestein andthat an inhibitor of protein tyrosine phosphatase reduces thiseffect (12). In the present investigation it is demonstrated thatinhibition of tyrosine phosphatases prevents the inhibitoryeffect of the tyrosine kinase inhibitor genistein on RVD. Asimilar phenomenon has been demonstrated on the volume-sensitive release of organic osmolytes (26). We assume that itis a balance between tyrosine kinase and tyrosine phospha-tase activities that determines the degree of phosphorylationof TASK-2 and hence its activation following osmotic cellswelling.

Tyrosine Phosphatases are Involved in the Inactivationof RVD

When using the tyrosine phosphatase inhibitor mpV(pic),the set point for RVD in EATC (defined as the volume wherethe RVD response ends) was significantly lowered comparedwith control cells. The treated cells seemed to have lost moreions than the control cells, which could indicate that the K�

channel (being the inhibiting factor in RVD) was preventedfrom closing due to the impairment of tyrosine phosphatases.These findings were substantiated by K� efflux measurements,in which it was found that inhibition of tyrosine phosphataseswith mpV(pic) resulted in a significantly increased swelling-activated K� efflux. Taken together these results stronglyindicate that tyrosine phosphatases are involved in the closingof the volume-activated K� channel TASK-2 in EATC. In acouple of experiments it was not possible to see an effect inEATCC treated with mpV(pic). We have no explanation onthis apparent difference between EATC and EATCC.

Overexpression of TASK-2 Accelerates the RVD

As mentioned earlier the K� conductance is rate limitingduring RVD, and the insertion of more channels would intheory increase the ability of the cells to volume regulate. Inaccordance with this theory, the TASK-2-transfected cellsshowed a significantly increased RVD response, both in regardto end point and velocity. This reflects a greater ion loss duringRVD in cells transfected with TASK-2 channels than in wild-type HEK-293 cells. Therefore when reintroduced to isotonicRinger, the transfected cells responded with a strong RVI likeresponse, a so-called post-RVD RVI response. When returningthe cells to the isotonic Ringer, this now appeared hypertonicdue to the excessive loss of ions during RVD, causing the cellsto shrink beneath their original set point. The increased post-RVD RVI response in the cells overexpressing TASK-2 wastaken to indicate a greater loss of ions during RVD in trans-fected cells than in the wild type. We thus have a model systemin which the RVD rate can be used to evaluate effects on theTASK-2 channel expressed in this system. The clear effect ofgenistein on this RVD response is thus a clear evidence fortyrosin kinases being involved in activation of TASK-2 aftercell swelling. Further experiments using mass spectrometryanalysis will hopefully give us a more detailed picture of theactivation pattern of the channel.

Swelling-Induced Tyrosine Phosphorylation of TASK-2

Several tyrosine kinases have been demonstrated to beactivated following cell swelling, and tyrosine kinase inhibitorshave been shown to block VRAC as well as the volume-sensitive efflux pathway for organic osmolytes (19, 28, 29, 36).With respect to tyrosine kinases and swelling-activated K�

channels, it has been shown that Src modulates the activity of,for example, the swelling-activated Kv channels (1.3 and 1.5)and the BK channels (19). In the present investigation we findthat swelling of HEK-293 cells overexpressing TASK-2 chan-nels resulted in a time-dependent phosphorylation of the chan-nel, which was evident within the period 1 to 10 min afterhypotonic treatment. Patch-clamp experiments performed oncell-attached patches with EATC have indicated that the vol-ume-sensitive K� channel is activated within 65 s after hypo-

Fig. 5. Tyrosine phosphorylation of the TASK-2 channel following hypotonicexposure. TASK-2 expression and tyrosine phosphorylation of TASK-2 inHEK-293 cells transfected with a TASK-2 containing pcDNA3.1/myc-Hisvector was measured under either isotonic or hypotonic conditions (1, 5, and10 min). The TASK-2 channel was precipitated using Ni-NTA agarose(Qiagen) and TASK-2 expression together with TASK-2 tyrosine phosphory-lation (pY100) visualized through SDS-PAGE and Western blotting (A).ANOVA test indicated no difference between isotonic pY100 values at 1, 5,and 10 min. The relative specific activity for tyrosine phosphorylated TASK-2under hypotonic conditions was estimated as the ratio between pY100 bandintensity and the TASK-2 band intensity given relative to the ratio in isotonicmedium (B). Values are given as means from 8, 9, and 6 sets of experimentstaken at time 1, 5, and 10 min, respectively, �SE. *Significantly increasedcompared with isotonic conditions (t-test, P � 0.05). #Significantly increasedcompared with isotonic conditions (ANOVA, P � 0.05).



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tonic exposure (7). Whole cell patch-clamp experiments inEATC cells revealed that the volume-sensitive K� channelswere detectable within 200 s after shift in solution (35). Wholecell patch-clamp experiments cannot be used to evaluate howlong the channels remain open after hypotonic challenge be-

cause the cells do not volume regulate. The rate constant,calculated from our efflux data shown in Fig. 2B, was high andunaltered in the time period 1 to 10 min after hypotonicexposure, indicating that the K� channel was activated withinthe whole time frame. This is in accordance with the tyrosinephosphorylation pattern for TASK-2. Although it might not bepossible to compare time scales for TASK-2 activation in

Fig. 7. Possible phosphorylation of the tyrosine kinase focal adhesion kinase(FAK) after cell swelling. SDS-page and Western blot analysis were performedon EATCC lysates made of cells exposed to isotonic or hypotonic Ringer for1, 5, or 10 min. A: FAK phosphorylation (pFAK) was visualized using specificantibodies against the site Y397. B: bar graphs represent the mean values of 6independent experiments � SE. All calculations of phosphorylation are showncompared with their respective isotonic controls.

Fig. 6. Possible involvement of the tyrosineSrc kinase in RVD and swelling-activated K�

efflux in EATC. EATC in suspension werepreincubated with the Src kinase inhibitor4-amino-5-(4-chloro-phenyl)-7-(t-butyl)pyra-zolo[3,4-d]pyrimidine (PP2, 5 �M) for 30min before they were transferred to hypotonicRinger also containing PP2. A: K� efflux wasestimated as described in Fig. 2. B: volumerecovery in EATCC was estimated as de-scribed in Fig. 1. Experiments were also per-formed with the use of 10 �M PP2 but withno significantly difference compared with theuse of 5 �M (data not shown). Volume re-covery is shown as the water loss (flow/min)obtained by linear regression on cell volumesmeasured within the first 3 min after maximalcell swelling in hypotonic Ringer. Values arecalculated from 3 independent experiments andshown as means � SE. C: SDS-page and West-ern blot analysis performed on EATCC lysatesfrom cells exposed to isotonic or hypotonicRinger for 1, 5, or 10 min. The Src phosphor-ylation was visualized using specific antibodiesagainst the activating site: SrcY416 (C, left)and the inactivating site: SrcY527 (C, right).D and E: hypotonic values for the phosphor-ylation of SrcY416 (D) and SrcY527 (E) aregiven relative to the isotonic values and thebars represent the mean values of 3 indepen-dent experiments � SE.

Fig. 8. The Janus kinase inhibitor cucurbitacin I (CC1) inhibits volumerecovery in EATCC. EATCC were placed in either isotonic Ringer (300mosM) or hypotonic Ringer (150 mosM), and the absolute volume wasobserved as a function of time. Cells were preincubated for 4 h with the JAK2inhibitor CC1 (10 �M), the insulin-like growth factor I receptor inhibitorpicropodophyllotoxin (PPPT, 1 �M), and the epidermal growth factor receptorinhibitor dihydrochloride (DC, 1 �M), and the inhibitors were present duringvolume estimations. Mean recovery after 4 min in hypotonic medium wasestimated from 3 independent sets of experiments and shown as means � SE.*Significantly reduced from control cells ability to regulate their volume.



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EATC cells with time scales for tyrosine phosphorylation ofTASK-2 in HEK-293 cells, we suggest that direct tyrosinephosphorylation is involved in swelling-induced activation ofTASK-2. We cannot exclude that tyrosine phosphorylation isalso involved upstream in the signaling sequence from cellswelling to activation of the K� channel.

Tyrosine Kinases Involved in RVD

As genistein is a known broad-spectrum tyrosine kinaseinhibitor, the results give no information to which tyrosinekinase(s) could be involved. Experiments using the morespecific Src kinase inhibitor PP2 showed a significant inhibi-tion of the volume-sensitive K� efflux in EATC (Fig. 6A) aswell as of the concomitant RVD response (Fig. 6B) whenintroducing the cells to a hypotonic solution together with theapplication of PP2. As these experiments are performed in thepresence of intracellular Cl�, it might however be possible thatthis small inhibition of the swelling-activated K� efflux couldrepresent an inhibition of the swelling-activated KCl cotrans-porter. We had bumetanide present in these experiments, butbumetanide, which is an excellent inhibitor of NKCC1, is notvery efficient toward KCl cotransport. Even though PP2 (a Srckinase family inhibitor) showed a small, but significant, inhi-bition of the RVD and the K� efflux, neither the Src kinase norFAK seemed to be activated during 10 min of hypotonicstimulation in EATCC when analyzed by Western blotting. Asdescribed in the INTRODUCTION, swelling-induced activation ofthese kinases has been reported in different cell types, but theydo not seem to play an important role in RVD in EATCC. Withrespect to tyrosine kinase activity coupled to membrane-boundreceptors, we found a significant inhibition of the RVD fol-lowing addition of cucurbitacin I, an inhibitor of the JAK/STAT pathway that is associated with cytokine signaling.Direct roles of cytokine receptors in volume sensing has beenreported (43), although the knowledge is yet rather limited butinteresting to pursue. Several reports points to a role for growthfactor receptors in cell volume sensing (19). However, we seeno effect of the growth factor receptor tyrosine kinase inhibi-tors picropodophyllotoxin and dihydrochloride (GW583340)on the cell volume recovery in the present investigation. Fromthe present data a likely working hypothesis is that cell swell-ing activates the cytokine receptor coupled JAK/STAT path-way, which is upstream of the swelling-induced phosphoryla-tion and activation of the TASK-2 in EATC.

ACKNOWLEDGMENTS

The technical expertise by Birgit B. Jørgensen, Birthe J. Hansen, BirteKofoed, and Dorthe Nielsen is acknowledged.

GRANTS

The present work was supported by The Danish Council for IndependentResearch/Natural Sciences (Grants 272-07-0530, 272-08-0170, and 21-04-0535). The Danish Council for Independent Research/Medical Sciences (Grant271-08-0520 to I. H. Lambert). Lundbeck Foundation (J Nr R32-A3102 to E.K Hoffmann). The Danish Society for the Protection of Laboratory Animals (toI. H Lambert and E. K Hoffmann).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

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Page 63 of 117

5 Paper II

KCNK5 is functionally down-regulated upon long-term hypotonicity in Ehrlich ascites tumor cells

The paper has been submitted to “Cellular Physiology and Biochemistry”

Status 09-30-2013: The paper has been found in “principle acceptable for publication”.


Page 64 of 117

1

KCNK5 is functionally down-regulated upon long-term hypotonicity in Ehrlich ascites tumor cells

Signe Skyum Kirkegaard1, Tune Wulff1+2, Steen Gammeltoft3 and Else Kay Hoffmann1

1Section of Cell and Developmental Biology, Institute of Biology, University of Copenhagen,

Denmark, 2National Food Institute, Technical University of Denmark, Kgs. Lyngby, Denmark,

3Molecular Biology, Sleep Research Unit, Diagnostic Department, Glostrup Hospital, Denmark

Keywords: KCNK5, TASK-2, long-term volume regulation, RVD

Running title: long-term hypotonicity and volume regulated channels

Correspondence to:

Prof. Else K Hoffmann


Department of Biology, The August Krogh Building


13, Universitetsparken, DK-2100, Copenhagen Ø

email: [email protected]

2

Abstract Background/aims: Regulatory volume decrease (RVD) in response to acute cell swelling is well

described and KCNK5 (also known as TASK-2 or K2P5.1) has been shown to be the volume

sensitive K+ channel in Ehrlich cells. Very little is on the other hand known about the effects of

long-term hypotonicity on expression and function of KCNK5, thus we have investigated the

effect of long-term hypotonicity (24h – 48h) on KCNK5 in Ehrlich cells on the mRNA, protein and

physiological levels.

Methods: Physiological effects of long-term hypotonicity were measured using patch-clamp and

Coulter counter techniques. Expression patterns of KCNK5 on mRNA and protein levels were

established using Real-Time qPCR and western blotting respectively.

Results: The maximum swelling-activated current through KCNK5 was significantly decreased

upon 48h of hypotonicity and likewise the RVD response was significantly impaired after both 24

and 48h of hypotonic stimulation. No significant differences in the KCNK5 mRNA expression

patterns between control and stimulated cells were observed, but a significant decrease in the

KCNK5 protein level 48h after stimulation was found.

Conclusion: The data suggest that the strong physiological impairment of KCNK5 in Ehrlich cells

after long-term hypotonic stimulation is predominantly due to down-regulation of the KCNK5

protein synthesis.

3

Introduction Mammalian cells, with some exceptions, are susceptible to changes in intra- or extracellular

amount of osmolytes due to the combination of aquaporins present in the membrane and an

osmotic pressure across the membrane. An acute decrease in extracellular or increase in

intracellular osmolarity respectively will in most cells result in water uptake and thus in cell

swelling. The cell homeostasis is very vulnerable to volume changes and to counteract swelling

the cell possess different regulatory mechanisms involving efflux of osmolytes through ion

channels and membrane transporters – a response known as regulatory volume decrease (RVD).

It is estimated that 70% of the osmolyte efflux in Ehrlich Ascites Tumor (EAT) cells during RVD is

KCl efflux [3] and the remaining 30% is due to the efflux of organic osmolytes [4]. For a detailed

description of the mechanisms of volume regulation see [1, 2]. In EAT cells and Ehrlich Lettré

Ascites (ELA) cells the volume sensitive channels are the two-pore domain K+ channel KCNK5

(also known as TASK-2 - TWIK-related Acid-Sensitive K+ channel 2 or K2p5.1) [5-7], and the

Volume Regulated Anion Channel (VRAC, ICl,vol) [8]. KCNK5 has besides in Ehrlich cells [5-7] also

been shown to be involved in RVD in other cell types including mouse proximal tubules [9], T

lymphocytes [10, 11], murine spermatozoa [12] and retinal glial cells [13]. It has previously been

shown that the rate limiting factor in RVD in EAT cells is the volume sensitive K+ efflux and thus

the efflux through the KCNK5 channel [14], making an altered RVD response very likely to be due

to changes related to this channel. For a recent review on KCNK5 see [15].

Response to hypotonicity can be divided into an acute and a long-term phase. The acute phase

which includes RVD happens within minutes of cell swelling. During long-time exposure to

osmolarity changes, the cell uses other mechanisms to ensure a steady intracellular

environment, such as an altered gene expression and an altered protein synthesis [16]. The role

of KCNK5 in acute volume regulation is well described in different cell types (see above). It has

previously been shown how the KCNK5 channel is tyrosine phosphorylated after acute cell

swelling in an time dependent manner [17] and recently it has been shown how KCNK5 is

inhibited by Gβγ subunits of heteromeric G protein and thereby how a G protein coupled

mechanism also can regulate the KCNK5 channel [18].

Effects of long-term hypertonicity is well studied and is known to elicit an increase in gene

transcription of a number of osmoregulatory genes, which will affect the uptake and synthesis of

organic osmolytes (see [2, 19]). On the other hand only very limited work has been done on the

effects of long-term hypotonicity on cells, thus a potential role for KCNK5 in long-term

regulation has never been studied. Preliminary studies (referred in [2]) using micro array

screening suggested that the expression of KCNK5 is 2.7 times down-regulated after 48 hours in

a hypotonic medium correlating with preliminary measurements of the maximum swelling-

4

activated KCNK5 current. The purpose of this study was thus to examine the effect of long-term

hypotonicity on mRNA-, protein- and functional levels of the KCNK5 channel.

Experimental procedures

Solutions and materials

Hypotonic media (180mOsm) for long-term hypotonic stimulation was obtained by diluting

growth media with buffered water, containing 5 mM HEPES, 10% fetal bovine serum (FBS) and

1% penicillin/streptomycin (p/s), pH 7.4. The solution was sterile filtered before use. Patch-

clamp experiments: hypotonic NaCl Ringer’s solution (180mOsm) contained in mM: 71 Na+, 5 K

+,

1 Mg2+

, 1 Ca2+

, 38 Cl-, 33 gluconate, 10 HEPES, pH 7.4. Isotonic Ringer’s solution (300mOsm) was

generated by addition of 110 mM mannitol and contained only 5 mM HEPES. The pipette

solution contained in mM: 2 Na+, 116 K

+, 1.2 Mg

2+, 42.4 Cl

-, 62 gluconate, 10 HEPES, 30 mannitol,

10 EGTA, 2.5 ATP and 0.1 GTP. Coulter counter experiments: hypotonic Ringer’s solution

(180mOsm) contained in mM: 72 Na+, 74.5 Cl

-, 2.5 K

+, 0.5 Mg

2+, 0.5 SO4

2-, 0.5 HPO4

-, 0.5 Ca

2+, 3.3

MOPS, 3.3 TES and 5 HEPES, pH 7.4. Isotonic Ringer’s solution (300mOsm) was obtained by the

addition of sucrose.

Cell cultures

ELA cells and EAT cells were cultured in RPMI1640 media supplemented with 10% FBS and 1% p/s

at 37 oC and 5% CO2. EAT and ELA cells were maintained by transferring 1 ml cell suspension to

10 ml fresh RPMI1640 medium every 3-4 days. ELA cells were loosened from the surface by

trypsination, 1.5 ml 0.25% trypsine/EDTA solution per T-75 culture flask. Only passages 6-30

were used for experiments.

Patch-clamp measurements

ELA cells were kept in isotonic (300mOsm) or hypotonic medium (180mOsm) for 24 or 48 hours

and transferred to 25 mm cover-slips after which they were placed in a chamber mounted on an

inverted microscope (Zeiss Axiovert 10, Carl Zeiss, Germany). Solution shifts were generated

using gravity-fed and pump-suction mechanisms. The current was measured using standard

whole-cell patch-clamp using a suitable amplifier (Axopatch 200B, Axon instruments, California,

USA). A Digidata 1200 Interface board and pClamp7 software (Axon Instruments) were used to

generate voltage-clamp command voltages, and to digitize data. In the cell attached

configuration, prior to establishing the whole-cell configuration the fast capacity transients were

eliminated. Following whole-cell formation, the series resistance and the whole-cell capacitance

were compensated and annulled with mean values that did not change significantly during

experiments [20]. Pipettes were made from Vitrex glass capillary tubing with an outside

5

diameter of 1.7 mm (Modulohm, Herlev, Denmark) using a Narishige PP-830 puller (Tokyo,

Japan) and had resistances of ≈4 MΩ. All experiments were carried out at 37 °C.

IK was measured as previously described in [5]. Briefly, the membrane potential was clamped in

pulses of 500 ms at the equilibrium potential for Cl– which was 5 mv during isotonic conditions

and 0 mv under hypotonic conditions and these reversal potentials have previously proven to be

good estimates [5, 6, 21]. The voltage correction was due to a dilution of the intracellular ion

compositions as a result of hypotonicity and the following cell swelling. The current increase was

measured after 300 sec of hypotonic stimulation and taken relative to the cell membrane

surface (pF).

mRNA measurements

ELA and EAT cells were stimulated with hypotonic media (180 mOsm) or isotonic media (300

mOsm) for 24 or 48 hours and lysed in RNA lysis buffer (Machery-Nagel, Düren, Germany). RNA

was purified from lysates using NucleoSpin® RNA II (Macherey-Nagel, Düren, Germany)

according to manufacturer’s instructions. Reverse transcriptase PCR with SuperScript II and

oligo(dT)12-18 primer (Invitrogen, Life Technologies, Naerum, Denmark) was used to generate

cDNA from the purified mRNA and performed on an Eppendorf Mastercycler. In a total of 10 µl

ddH2O, dNTP mix (500 µM) and oligo(dT)12-18 primer (500 ng) was mixed on ice, 1 µg mRNA was

added and incubated for 5 min at 65 °C. The solution was put on ice and briefly centrifuged. 4 µl

5 x first strand buffer, DTT (10 µM) and 1 µl ddH2O was subsequently added and the solution

incubated for 2 min at 42 °C. 1 µl SuperScript II was added and incubated at 42 °C for 50 min

followed by incubation at 70 °C for 15 min and ending at 4 °C. Real-time qPCR was performed in

triplicates using the Stratagene MX4000 PCR system, Brilliant® II SYBR® Green QPCR Master Mix

(Stratagene, Agilent Technologies) and the following primers: mKCNK5 forward: 5’-GTC AAG GCC

ACT TGG TGA GG-3’ and mKCNK5 reverse: 5’-TGC TGG TGA AGG TGG ACT CA-3’ (KCNK5 GenBank

accession number NM_021542) and ARP: mARP forward: 5’-CGA CCT GGA AGT CCA ACT AC-3’

mARP reverse: 5’-ACT TGC TGC ATC TGC TTG-3’ (GenBank accession number NM_007475),

which was used as reference gene. 10 µl Master Mix, 0.4 µl forward and reverse primer (200

nM final concentration), 0.4 µl 500 x diluted ROX II reference dye (40 nM final concentration),

7.8 µl ddH2O and 1 µl cDNA was mixed and quantification was done using the following cycles:

95 ˚C 10 min and 95 ˚C 30 sec, 58 ˚C 1 min, 72 ˚C 30 sec x 40. Standard curves were done in

order to measure primer efficiency which was adjusted for in calculations. Primers were selected

using Primer3 software and purchased from MWG Eurofins (Germany). Quantification was

carried out using the Pfaffl method [22].

6

SDS-PAGE and western blotting

ELA and EAT cells were stimulated with isotonic or hypotonic media for 24 or 48 h and

subsequently lysed in 95°C lysis buffer (10 mM Tris-HCl pH 7.4, 1% SDS and 20 mM EDTA) with

protease inhibitors (Roche Applied Science) and phosphatase inhibitors added. SDS-PAGE and

western blotting were performed as previously described [17]. Briefly, we used a SDS-PAGE-

western blotting system form Invitrogen (Lfe technologies) and with the following antibodies;

KCNK5 (Alomone Lab., Israel) 1:250 and β-actin (Sigma-Aldrich) 1:1000. Since we use two

different antibodies on every blot, membranes were cut around 50kDa letting us visualize both

KCNK5 and β-actin (65 and 42 kDa respectively) using BCIP/NBT. Protein bands were quantified

using UN-SCAN-IT software.

Cell membrane protein labeling and purification

EAT and ELA cells were grown in 40% hypotonic medium (180mOsm, standard medium diluted

with buffered water, see above) or isotonic medium (300mOsm, standard medium) for 48h

before cell membrane purification. Cell membrane proteins were biotinylated and purified using

Pierce Cell Surface Protein Isolation Kit (Thermo Fisher Scientific) according to manufactures

instructions, with two exceptions; after lysis and before purification a sample was drawn from

the lysate and protein concentration measured. These samples were used as a control of total

KCNK5 protein content under the two circumstances. SDS-PAGE and western blotting was used

to analyze the amount of KCNK5 in whole cell lysate as well as purified samples (see above).

Cell volume measurements

Absolute cell volume was measured by electronic cell sizing using the Coulter Multisizer ll

(Coulter, Luton, UK) with a tube orifice of 100 μm. For each experiment 2.5x106 cells were used

and cell volume was determined as the median of the cell volume curves after calibration with

latex beads (15 µm). Cells were either kept in isotonic (300mOsm) medium or in a 40%

hypotonic medium for 24 or 48h. Prior to cell volume measurements cells were centrifuged and

resuspended in standard medium (isotonic) were they were kept for 30 min in order to

acclimatize before experiencing a hypotonic chock. The duration in standard medium allowed

the cells to adapt to the changed environment without influencing protein synthesis and gene

transcription. It should be mentioned that the cells shrink when transferred from the hypotonic

into the isotonic medium and thus perform a RVI process. As KCNK5 channels are inhibited in

shrunken cells [7, 18] we have included the 30 min isotonic acclimatization period and have

controlled that there is no significant difference between the cell volumes in the three

experimental groups. The mean isotonic, 24h hypotonic pre-treatment and 48h hypotonic pre-

treatment cell volumes where 803.7±61.5 µm3, 829.7±58 µm

3 and 783.7±46.9 µm

3 respectively,

with no significant difference between the control and pre-treated cells.

7

All Ringer’s solutions were micro-filtered (Millipore, 0.45 µM) before use. Volume recovery was

estimated as (Vmax-V4min)/(Vmax-Viso), where Vmax, V4min and Viso are the maximal cell volume, cell

volume at time 4 min and cell volume under isotonic conditions, respectively.

Statistics

Student’s T-test and one-way ANOVA was used to test for statistical significance. 95% and 99%

levels of significance were shown by one star or two stars respectively.

Results

Patch-clamp studies revealed a decreased maximum swelling-activated current in ELA cells

upon 48h of hypotonicity

Fig. 1 shows the maximum swelling-activated K+ current through the volume sensitive K

+ channel

and it is seen that after 24h there is a decreased (not significant) K+ current. This is supported by

experiments measuring K+ efflux after 24h hypotonic incubation using

86Rb

+ as a tracer. We

found that the rate constant for the swelling-activated K+ efflux in stimulated cells was lower

than in control cells. At 12 minutes after cell swelling the rate constant was 0.0134 in stimulated

cells compared to 0.0154 in the control cells (data not shown), thus supporting the results

obtained by patch-clamp. After 48h of hypotonicity the maximum swelling-activated current

through KCNK5 was significantly decreased from 28.7 ± 7.2 pA/pF to 7.0 ± 2.1 pA/pF

corresponding to a decrease of 75.6%.

Long-term exposure to hypotonicity (24 and 48 h) decreased RVD in EAT cells

In addition we studied the effect of 24 and 48 hours hypotonicity (180mOsm) on RVD in EAT

cells (Fig. 2) and found that after both 24h and 48h RVD was significantly decreased both when

looking at % recovery after 4 min (fig. 2A+B) and at the initial rate of RVD (fig. 2A+C). 24h of

hypotonic stimulation resulted in a decreased RVD when re-exposing the cells to a hypotonic

shock, with a recovery after 4 minutes of 19.3 ± 1.33% compared with 41.5 ± 2.51% seen in the

control cells (fig. 2B), corresponding to a 53% decrease. When looking at the initial rate of RVD,

24h of hypotonicity resulted in a relative decrease in the rate of RVD to 46.5±3.79% compared

to untreated control cells (fig. 2D). After 48h the initial rate of RVD was further decreased to

30.8±3.69% compared to untreated control cells equal to a 69% RVD inhibition (fig. 2D). The %

recovery after 4 min was decreased to 12±1.51% compared to 41.5±2.51% of the control cells

corresponding to a 71% decrease after 48h of hypotonicity.

There was no significantly change in mRNA levels in EAT and ELA cells upon long-term

hypotonicity

To further elucidate the nature of the decreased physiological function of the KCNK5 channel we

investigated the expression of KCNK5 on mRNA and protein levels.

8

Real-Time qPCR was performed on treated (48h of hypotonicity) and untreated (control) EAT

and ELA cells and fig. 3 shows the mRNA expression data for ELA (Fig. 3A) and EAT (Fig. 3B) cells

upon long-term hypotonicity. It is seen that even though there is a 10.6% decrease in mRNA

levels in 48h hypotonically treated ELA cells compared to the untreated control cells the

decrease is not significant. In the EAT cells an apparent increase in KCNK5 mRNA was detected,

though the difference was also not significant.

KCNK5 protein levels were significantly decreased in EAT and ELA cells upon 48h of

hypotonically exposure

Western blot analysis on EAT and ELA cells revealed a significant decrease in KCNK5 protein

expression levels after 48h, but not after 24h of hypotonically stimulation. Fig. 4A shows how

the mean KCNK5 protein level in EAT cells is approximately the same in both control cells and

after 24h of hypotonicity, but is significantly decreased to 70±4.4% after 48h of treatment. The

same analysis in ELA cells likewise revealed a significant difference between protein amount in

control and treated cells (fig. 4B). After 48h of hypotonicity a ≈30% decrease to 70.9±12.2% was

seen, while an apparent increase (not significant) after 24h was observed. The results were

confirmed in a single experiment by measuring cell surface KCNK5 protein amount using

membrane protein biotinylation and showing how 48h of hypotonic treatment decreases the

amount of KCNK5 protein inserted into the membrane by approximately 50%, thus confirming

and not very different from the decrease seen in total KCNK5 protein (see fig 4C).

Discussion Since the KCNK5 channel is a very important player in the RVD response in Ehrlich cells, we

investigated a possible effect of long-term hypotonicity on RVD and on expression of KCNK5. The

RVD measurements were performed on the EAT cell line using a Coulter Counter. EAT cells were

chosen for these volume measurements since the most accurate volume measurements are

obtained on cells in suspension. A significant impairment of RVD was found both after 24 and 48

hours of long-term exposure to the hypotonic medium. The inhibited RVD performance in EAT

cells could reflect a decreased function of either KCNK5 (K+) or of VRAC (Cl

-). However since the

KCNK5 channel is the rate limiting factor for RVD in these cells [14] and since patch-clamp

measurements showed a decreased maximum swelling-activated current through KCNK5 upon

long-term hypotonic stimulation, the results were taken to indicate a down-regulation of the

KCNK5 channels or reduced ability of the KCNK5 channel during RVD.

The physiological impairment of RVD and of the maximum swelling-activated K+ current through

KCNK5 channels could be due to regulation on a number of levels such as I) a down-regulation of

KCNK5 protein synthesis II) KCNK5 mRNA transcription III) post-translational modifications

9

resulting in an inhibited channel function IV) an unknown regulatory mechanism “turning off”

the channel V) an internalization of membrane embedded channels or a combination of the

above. We found that there was no significant difference between the mRNA amount of KCNK5

in control cells compared to hypotonic stimulated cells which were true in both EAT and ELA

cells. Since there was no significant difference in the mRNA expression pattern between control

and stimulated cells we conclude that the physiological changes most likely are not due to

regulation on the transcriptional level. It might be argued that cells with down-regulated KCNK5

expression could have died before 48h and this is why no decrease in mRNA level is seen.

However, the fact that we do see a clear down-regulation at the protein level argues against the

possibility.

Although there was no effect of long-term stimulation on gene transcription levels, we found

significant lower levels of KCNK5 protein after 48h of hypotonicity in both cell lines, thus

indicating a higher degree of KCNK5 protein degradation or decreased synthesis of the KCNK5

protein. Our results were further substantiated in one experiment, where it was shown how 48h

of long-term hypotonicity resulted in a decrease in KCNK5 protein present in the plasma

membrane, thus supporting the theory that long-term hypotonicity decreases KCNK5 protein

expression and may in addition cause an altered KCNK5 sorting to the membrane. As the

decrease in membrane bound KCNK5 was not very different from the total decrease in KCNK5

protein, we have not looked further into this.

At least three factors could influence this change in KCNK5 protein expression level: the lowered

ion strength, the decrease in K+ concentration and the decreased Cl

- concentration all generated

by the response to the hypotonic medium. We speculate if e.g. the decreased K+ concentration

could prompt the cells to down-regulate the K+ leak-channel KCNK5 in order to govern the

smaller amount of K+ more strictly. The lowered Cl

- concentration could result in the same

mechanisms, but since we are dealing with a significant decrease in KCNK5 protein expression,

we suggest that the physiological changes are predominantly due to alterations in potassium

rather that in chloride level.

To our knowledge not much work has been published on long-term effects of hypotonicity on

protein synthesis but it should be noted that it has been shown that protein synthesis is up-

regulated in hepatocytes of air-breathing walking catfish upon hypotonicity (≈ 2 hours) [23].

Since we are dealing with a down-regulation of the KCNK5 protein this is thus not likely to be

caused by a general effect on the protein synthesis but rather by a specific effect on the KCNK5

protein synthesis or breakdown.

10

There is a time discrepancy between the physiological data and the protein expression data

obtained in EAT cells, since there is a significant impairment of RVD in EAT cells already after 24h

of hypotonicity but the down-regulation of KCNK5 protein amount is first detectable after 48h of

hypotonicity. It is likely that this is caused by a regulatory inhibition of the channel followed by a

later decrease in protein synthesis. This has not been further investigated.

Taken together our results show that the clear physiological changes of the KCNK5 channel in

Erhlich cells upon long-term hypotonic stimulation is predominantly due to a decreased protein

synthesis. We cannot rule out other regulatory mechanisms as contributors to the down-

regulation of the maximum swelling-activated KCNK5 current seen after long-tem hypotonicity.

We have previously shown how protein tyrosine kinases are vital in KCNK5 channel opening and

that tyrosine phosphatases are important for the closing of the channel upon a swelling-

mediated activation of the channel [17], and it has furthermore been suggested that KCNK5

activation can be modulated by G protein Gβγ subunits [18]. Thus an altered tyrosine

kinase/phosphatase profile or changes in the G protein coupled mechanism could potentially be

involved in the functional inhibition of KCNK5.

The technical skills of Dorthe Nielsen and Birthe Juul Hansen (University of Copenhagen) is

gratefully acknowledged and appreciated. This work was supported by The Danish Council for

Independent Research/Natural Sciences (grants 09–064182 and 10–085373) and The Lundbeck

Foundation, Denmark (J Nr R32-A3102).

Conflict of interest

The authors declare no conflict of interest.

11

References 1 Lang F, Busch GL, Ritter M, Völkl H, Waldegger S, Gulbins E, Häussinger D: Functional Significance of

Cell Volume Regulatory Mechanisms. Physiol Rev 1998;78:247-306. 2 Hoffmann EK, Lambert IH, Pedersen SF: Physiology of Cell Volume Regulation in Vertebrates. Physiol

Rev 2009;89:193-277. 3 Hendil KB, Hoffmann EK: Cell volume regulation in Ehrlich ascites tumor cells. J Cell Physiology

1974;84:115-125. 4 Hoffmann Else K, Hendil Klavs B: The role of amino acids and taurine in isoosmotic intracellular

regulation in Ehrlich ascites mouse tumour cells. J Comp Physiol 1976;108:279-286. 5 Riquelme G, Sepúlveda FV, Jørgensen F, Pedersen S, Hoffmann EK: Swelling-activated potassium

currents of Ehrlich ascites tumour cells. Biochim Biophys Acta 1998;1371:101-106. 6 Hougaard C, Niemeyer MI, Hoffmann EK, Sepúlveda FV: K

+ currents activated by leukotriene D4 or

osmotic swelling in Ehrlich ascites tumour cells. Pflügers Archiv European Journal of Physiology 2000;440:283-294.

7 Niemeyer MI, Cid LP, Barros LF, Sepúlveda FV: Modulation of the Two-pore Domain Acid-sensitive K

+

Channel TASK-2 (KCNK5) by Changes in Cell Volume. J Biol Chem 2001;276:43166-43174. 8 Pedersen SF, Prenen J, Droogmans G, Hoffmann EK, Nilius B: Separate Swelling- and Ca

2+-activated

Anion Currents in Ehrlich Ascites Tumor Cells. J Membr Biol 1998;163:97-110. 9 Barriere H, Belfodil R, Rubera I, Tauc M, Lesage F, Poujeol C, Guy N, Barhanin J, Poujeol P: Role of

TASK2 Potassium Channels Regarding Volume Regulation in Primary Cultures of Mouse Proximal Tubules. J Gen Physiol 2003;122:177-190.

10 Bobak N, Bittner S, Andronic J, Hartmann S, ühlpfordt F, Schneider-Hohendorf T, Wolf K, Schmelter C,

Göbel K, Meuth P, Zimmermann H, Döring F, Wischmeyer E, Budde T, Wiendl H, Meuth SG, Sukhorukov VL: Volume regulation of murine T lymphocytes relies on voltage-dependent and two-pore domain potassium channels. Biochim Biophys Acta 2011;1808:2036-2044.

11 Andronic J, Bobak N, Bittner S, Ehling P, Kleinschnitz C, Herrmann AM, Zimmermann H, Sauer M,

Wiendl H, Budde T, Meuth SG, Sukhorukov VL: Identification of two-pore domain potassium channels as potent modulators of osmotic volume regulation in human T lymphocytes. Biochimica et Biophysica Acta (BBA) - Biomembranes 2013;1828:699-707.

12 Barfield JP, Yeung CH, Cooper TG: The Effects of Putative K

+ Channel Blockers on Volume Regulation of

Murine Spermatozoa. Biology of Reproduction 2005;72:1275-1281. 13 Skatchkov SN, Eaton MJ, Shuba YM, Kucheryavykh YV, Derst C, Veh RW, Wurm A, Iandiev I, Pannicke T,

Bringmann A, Reichenbach A: Tandem-pore domain potassium channels are functionally expressed in retinal (Müller) glial cells. Glia 2006;53:266-276.

14 Lambert IH, Hoffmann EK, Jørgensen F: Membrane potential, anion and cation conductances in Ehrlich

ascites tumor cells. J Membr Biol 1989;111:113-131.

12

15 Cid LP, Roa-Rojas HA, Niemeyer MI, González W, Araki M, Araki K, Sepúlveda FV: TASK-2: a K2P K+

channel with complex regulation and diverse physiological functions. Frontiers in Physiology 2013;4:1-9.

16 Cohen DM: SRC family kinases in cell volume regulation. Am J Physiol Cell Physiol 2005;288:C483-

C493. 17 Kirkegaard SS, Lambert IH, Gammeltoft S, Hoffmann EK: Activation of the TASK-2 channel after cell

swelling is dependent on tyrosine phosphorylation. American Journal of Physiology - Cell Physiology 2010;299:C844-C853.

18 Añazco C, Peña-Münzenmayer G, Araya C, Cid LP, Sepúlveda F, Niemeyer M: G protein modulation of

K2P potassium channel TASK-2. Pflugers Arch - Eur J Physiol 2013;1-12. 19 Burg MB, Kwon ED, Kültz D: Regulation of gene expression by hypertonicity. Annual Review of

Physiology 1997;59:437-455. 20 Hougaard C, Jørgensen F, Hoffmann EK: Modulation of the volume-sensitive K

+ current in Ehrlich

ascites tumour cells by pH. Pflügers Archiv European Journal of Physiology 2001;442:622-633. 21 Niemeyer MI, Hougaard C, Hoffmann EK, Jørgensen F, Stutzin A, Sepúlveda FV: Characterisation of cell

swelling-activated K+ selective cunductance of ehrlich ascites tumour cells. J Physiol 2000;524:757-

767. 22 Michael W.Pfaffl: A new mathematical model for relative quantification in real-time RT–PCR. Nucleic

acids Research 2001;29:2002-2007. 23 Biswas K, Jyrwa L, Häussinger D, Saha N: Influence of cell volume changes on protein synthesis in

isolated hepatocytes of air-breathing walking catfish (Clarias-ábatrachus). Fish Physiol Biochem 2010;36:17-27.

13

Fig. 1 Activity of KCNK5 current in ELA cells upon long-term hypotonic stimulation

Cells were kept in isotonic (300mOsm) or hypotonic medium (180mOsm) for 24 or 48 hours. The

maximum swelling-activated K+ current was measured using standard whole-cell patch-clamp.

The current increase was measured after 300 sec of hypotonic stimulation (n=3) and taken

relative to the cell membrane surface (pF). Student’s T-test was used to test for statistical

significance and one star (*) indicate a 95% significance level.

Fig. 2 RVD in EAT cells

A Coulter counter was used to measure the volume of isotonic control (300m) and long-term (24

or 48h) hypotonically (180 mOsm) treated EAT cells. A: representative figure showing relative

RVD over time (sec) for untreated control cells and cells kept in hypotonic medium for 24 or 48

h. B: Mean volume recovery of n=5 experiments after 4 min was calculated as (Vmax-V4min)/(Vmax-

Viso), where Vmax, V4min and Viso are the maximal cell volume, cell volume at time 4 min and cell

volume under isotonic conditions, respectively. C: initial rate of RVD or the slope was calculated

(n=5) using linear regression on the linear part of the RVD curve, from maximum volume to the

end of linearity. One-way ANOVA was used to test for statistical significance and two stars (**)

indicate a 99% significance level.

Fig. 3 mRNA levels of KCNK5 in ELA and EAT cells

ELA (A) (n=4) and EAT (B) (n=5) cells were stimulated with hypotonic (180 mOsm) or isotonic

media (300 mOsm) for 24 or 48 hours and Real-Time qPCR was performed. Student’s T test or

one-way ANOVA was used to test for statistical significance.

Fig. 4 KCNK5 protein levels in Ehrlich ascites tumor and Ehrlich Lettré ascites cells.

Cells were isotonic (300mOsm) or hypotonically (180mOSm) treated for 24 or 48 hours and SDS-

PAGE and western blotting was performed on the lysates using antibodies against KCNK5 and β-

actin. A: representative western blot and mean protein levels (n=3) in EAT cells. B:

representative western blot and mean protein levels (n=9) in ELA cells. C: membrane purification

(n=1) showing KCNK5 protein amount inserted into the membrane under control and 48h

hypotonic conditions. Student’s T-test was used to determine statistical significance and one

star (*) represents a statistical significance level of 95%.


Page 82 of 117


Page 83 of 117

6 Paper III

The potential role of KCNK5 in activated T cell physiology with specific focus on cell volume control

Paper in preparation.


Page 84 of 117

1

The potential role of KCNK5 in activated T cell physiology with specific focus on cell volume control

Signe S. Kirkegaard1, Pernille Dyhl Strøm1, Anker Jon Hansen2, Steen Gammeltoft3 and

Else K. Hoffmann1

1Section for Cell and Developmental Biology, University of Copenhagen, Denmark.

2Novo

Nordisk, Måløv, Denmark. 3Molecular Biology Sleep Research Unit, Diagnostic Department,

Glostrup Hospital, Denmark

Running title: KCNK5 and T cell activation

Correspondence to:

Else K Hoffmann, PhD


Department of Biology, The August Krogh Building


13, Universitetsparken, DK-2100, Copenhagen Ø

email: [email protected]

2

Summary

The potential role of the two-pore domain potassium channel KCNK5 (also known as TASK-2 and

K2P5.1) in activated T cell physiology has only recently been described and for years the main

focus of potassium channels in T cell physiology have been on the voltage-gated K+ channel

Kv1.3 and the Ca2+

-activated KCa3.1. So far KCNK5 has been described to be up-regulated in

multiple sclerosis patients and to be implicated in the volume regulatory mechanism regulatory

volume decrease (RVD) in T cells. KCNK5 has furthermore been shown to be involved in RVD in

Ehrlich cells, proximal tubules and spermatozoa.

Here we further elucidate on the potential role of KCNK5 in activated T cell physiology, thus we

investigated the time-dependent expression pattern of KCNK5 in activated T cells together with

its role in RVD.

We find that KCNK5 is indeed up-regulated in CD3/CD28 activated T cells both on mRNA and

protein levels, but despite this strong up-regulation we find the RVD response to be inhibited in

activated T cells compared to non-activated control cells. In addition we find that T cells in

response to activation increase their volume and swell more than non-activated control cells

when subjected to hypotonicity. We furthermore find the swelling-activated Cl- permeability in

activated T cells to be strongly decreased and thus find the RVD inhibition predominantly to be

due to the decreased Cl- permeability. Our results suggest that the up-regulation of KCNK5 in

activated T cells does not play a volume regulatory role and we speculate that it might play a

role in hyperpolarizing the cell membrane and thus increasing the Ca2+

influx.

3

Introduction

The importance of ion channels in T cell activation and activated T cell function is established.

Especially the role of the voltage-gated Kv1.3, the Ca2+

-activated Kca3.1 and Ca2+

release-

activated Ca2+

channel (CRAC) is well known. For reviews on ion channels and T cells see (8; 30;

34). Activation of the T cell when encountering an antigen requires a sustained rise in

intracellular Ca2+

concentration ([Ca2+

]i) which is generated by IP3 mediated Ca2+

release from

stores in ER. This in turn mediates the activation of CRAC channels allowing Ca2+

entry from the

cytosol. This Ca2+

influx and subsequent rise in [Ca2+

]i activates KCa1.3 and also causes a

depolarization activating the voltage sensitive Kv1.3 K+ channel. Activation of Kv1.3 and KCa3.1

causes K+ efflux and hyperpolarization of the plasma membrane allowing further Ca

2+ entry via

the CRAC channels in the membrane. The sustained increase in [Ca2+

]i is essential for the

transcription and expression of interleukin 2 (IL-2) which in turn is essential for keeping the T

cells activated without the requirement of further antigen stimulation (see (23)). Other ion

channels, however, have been implicated in T cell activation. The leak conductance channel

(TREK-2) was described in WEHI-231 cells which is a murine immature B cell line (27; 37). Further

studies have also revealed the presence of two-pore domain K+ channels in lymphocytes e.g.

TASK-1 and TASK-3 (25) and of special interest to this study Bittner and co-workers suggested

that the two-pore domain potassium channel KCNK5 (also known as TASK-2 or K2P5.1) has a role

in T cell activation and in multiple sclerosis (5).

KCNK5 has previously been shown to be volume sensitive and to be implicated in the volume

regulatory process of regulatory volume decrease (RVD) in response to cell swelling in various

cell types and tissues including Ehrlich Ascites Tumor (EAT) cells (19; 29), mouse proximal

tubules (4), human and murine spermatozoa (2; 3) and murine T lymphocytes (6). Recently it

was shown that two-pore domain potassium channels are also involved in volume regulation in

human T cells (1). For a recent review on KCNK5 see (12).

T cells like most other mammalian cells are subject to changing intracellular environment

causing potential volume changes and subsequent regulation. T cells are in constant movement

around the body and thereby subjected to extracellular osmotic changes e.g. in the kidney and

as such, T cells are as most other mammalian cells capable of performing RVD in response to cell

swelling (7; 10; 11; 14).

As seen in all volume regulating cells the volume regulated anion channel (VRAC) is responsible

for the swelling-activated Cl- current (8) whereas different K

+ channels have been suggested to

play a part in T cell volume regulation including the voltage-gated potassium channel Kv1.3 (9;

22), the Ca2+

-activated KCa3.1 (33), and two-pore domain potassium channels (1; 6).

4

KCNK5 is up-regulated during T cell activation and is also involved in volume regulation (1; 5) but

there is no description on the time dependency of the expression pattern of KCNK5 during T cell

activation and its physiological consequences. This is the subject of the present paper and in

addition we demonstrate a strong decrease in the swelling-activated Cl- permeability during the

activation which inhibits the RVD process in the large activated T cells.

Experimental procedures

Solution and Materials

Volume measurements: Hypotonic Ringer’s solution (180mOsm) contained (in mM): 71.5 NaCl,

2.5 KCl, 0.5 MgSO4, 0.5 Na2HPO4, 0.5 CaCl2, 3.3 MOPS, 3.3 TES and 5 HEPES, pH 7.4. Isotonic

Ringer’s solution (300mOsm) was obtained by addition of sucrose. Cl- permeability: the

hypotonic (150mOsm) low Na+ Ringer’s solution contained (in mM): 70 NMDGCl

-, 0.8 NaCl, 5 KCl,

1 K2HPO4, 1 MgSO4, 1 CaCl2, 3.3 MOPS, 3.3 TES and 5 HEPES, pH 7.4.

Gramicidin and Clofilium was purchased from Sigma and used in the concentrations 1µM and

100µM respectively

Purification and maintenance of cells

Human T cells were purified from buffy coats from healthy donors. Human buffy coats were

obtained from the blood bank at Rigshospitalet, Copenhagen, Denmark. All procedures were

performed at room temperature. The T cell population was purified from buffy coats using

RosetteSep™ (Human T Cell Enrichment Cocktail, StemCell Tech.) accordingly to manufactures

description. After purification any residual red blood cells was lysed using RBC lysis buffer

(eBioscience). Purified T cells were kept in RPMI1640 GlutaMax medium supplemented with 30

U/ml IL-2, 10% FBS and 1% P/S at 37 oC and 5% CO2.

RNA purification and Real-Time qPCR

T cells were stimulated for 2, 4, 8, 12, 24, 48, 72 and 144 hours CD3/CD28 beads (Invitrogen) and

RNA was purified from lysates using NucleoSpin® RNA II (Macherey-Nagel) according to

manufacturer’s instructions. Reverse transcriptase PCR with SuperScript II (Invitrogen) and

oligo(dT)12-18 primer (Invitrogen) was used to generate cDNA from the purified mRNA and

performed on a Eppendorf Mastercycler as previously described (20). Real-Time qPCR was

performed using a Stratagene MX4000 real-time PCR system, Brilliant® II SYBR® Green QPCR

Master Mix (Stratagene, Agilent Technologies) and the following primers: hKCNK5 forward: 5’-

ACCACCCACTCATCTTCCAG-3’, hKCNK5 reverse: 5’-AGTGCTGGTGAAGGTGGACT-3’, hKv1.3

forward: 5’-CACTTCAGGTTTCAGCAGCA-3’, hKv1.3 reverse: 5’-TGTCTCCCGGTGGTAGAAGT-3’,

hKCa3.1 forward: 5’-ACTGGGCACCTTTCAGACAC-3’ and KCa3.1 reverse: 5’-

5

ACGTGCTTCTCTGCCTTGTT-3’. A total volume of 20 μl containing 1 μl of the cDNA, 200 nM of

primers, and 10 μl 2× MasterMix was used.

Despite testing numerous potential reference genes none of them lived up to standards and

instead determination of total cDNA concentration was used to correct the data obtained. To

determine cDNA content upon PCR reaction and to normalize samples Quant-iT™ OliGreen®

ssDNA Assay Kit was used as described in (24). Standard curves were also done to measure

primer efficiency which was corrected for in calculations. Primers were selected using Primer3

software and purchased from MWG Eurofins (Germany). Quantification was carried out using

the Pfaffl method: (26)

Western blotting

T cells were stimulated for 2h, 4h, 8h, 12h, 24h, 48h, 72h or 144h days with CD3/CD28 beads

and lysed in 95°C lysis buffer (10 mM Tris-HCl pH 7.4, 1% SDS, 20 mM EDTA) with protease

inhibitors (Roche Applied Science) and phosphatase inhibitors added. SDS-PAGE and western

blotting was performed as previously described (19). We used the following antibodies and

concentrations: KCNK5 (TASK-2) (Alomone Lab., Israel) 1:100, β-actin (Sigma-Aldrich) 1:1000.

Volume measurements by Coulter Counter

Volume measurements: T cells were stimulated with CD3/CD28 beads for 2h, 4h, 8h, 12h, 24h,

48h, 72h or 144 before the absolute cell volume was measured by electronic cell sizing using

the Coulter Multisizer ll (Coulter, Luton, UK) with a tube orifice of 80 μm. Before measurements

calibration was performed using 15 µm latex beads. Approximately 2.5x106 cells were used per

experiment and cell volume was determined in either isotonic or hypotonic Ringer’s solutions

(all micro-filtered before use). Volume recovery was estimated in two different ways, by %

recovery after 3 min as previously described (19) and by calculating the initial rate of RVD

measured as the slope of the recovery part of the RVD curve. Cl- permeability: to determine if

the Cl- permeability was decreased in activated T cells we used a method previously described in

(17) with some minor alterations, thus we used clofilium as a blocker of potassium channel

KCNK5 and a NMDG Na+ substitution Ringer’s solution (see above). Briefly, the principle of the

experiments is as follows: the addition of clofilium blocks the KCNK5 channel allowing the cells

to swell when subjected to hypotonicity but inhibiting K+ efflux and thus RVD. When adding

gramicidin a high cation permeability is introduced making Cl- the limiting factor. This way makes

it possible to indirect measure the Cl- permeability since a cell shrinkage will reflect the cells Cl

-

permeability which then can be measured in relative values.

6

Statistical methods

Statistical significance was determined by Student’s T-test or one-way analysis of variance

(ANOVA) where one star represent statistical significance at a 95% level, two stars at a 99% level

and three stars at a >99% significance level.

Results

KCNK5 and KCa3.1 is highly up-regulated on mRNA level in CD3/CD28 activated T cells

When activating the T cells for 2, 4, 8, 12, 24, 48, 72 and 144 hours with CD3/CD28 beads both

KCa3.1 and KCNK5 were up-regulated while no significant change was seen in Kv1.3 (fig. 1A)

mRNA expression level compared to un-stimulated controls using Real-Time qPCR. An initial

down-regulation of KCa3.1 (fig. 1B) mRNA level was observed upon 2 and 4 h of stimulation

followed by an increasing up-regulation after 8, 12 and 24 h reaching a stable level of a 13 fold

up-regulation after 48 h and with statistic significant values after 72 and 144 h (p<0.05). KCNK5

(fig. 1C) showed the largest up-regulation of the three with a 115 fold peak-increase in mRNA

level following 24 h of T cell activation when comparing with the non-activated T cells. No initial

down-regulation was observed regarding KCNK5 thus a 4 fold increase was already seen after 2

h. Statistical significance was only seen at 24h (p<0.05) when using ANOVA test though the up-

regulation seem clear. This could be due to the rather large standard deviations seen in the data.

When using Student’s T test on the data-set comparing each test value to its respective control

statistical significance is found at times 2, 4, 8 and 144h (p<0.05).

KCNK5 is also highly up-regulated on protein level

Figure 2 shows a biphasic KCNK5 protein expression pattern with an initial significant decrease

(p<0.001) during the initial stimulation (2 and 4h) of the cells with CD3/CD28 beads followed by

a still increasing protein expression after 8, 12, 24 and 48 h. The expression peaks upon 72 h of

stimulation with a 277% increase compared to the quiescent control cells with a subsequent

slight decrease to 209% at 144 h of activation both time-points showing significant more KCNK5

protein in activated T cells than in the controls (p<0.001 and p<0.05 respectively). The activation

of the T cells resulted in fast proliferation of more than 300% measured 72 h after CD3/CD28

stimulation compared to non-stimulated control cells (seen in two experiments, data not

shown).

Activation causes isotonic cell swelling, decreased RVD ability and increased hypotonic

swelling

Since the KCNK5 channel is a known volume regulator we tested the activated T cells ability to

perform regulatory volume decrease (RVD) upon hypotonicity. From the volume measurements

7

depicted in fig. 3 it is seen that the activated T cells when measured in isotonic Ringer’s solution

are larger (fig. 3A+B) – as described many times before, but it is also seen that in hypotonic

Ringer’s solution the activated cells swell more than the non-activated control cells (fig. 3C).

After 8 h of CD3/CD28 stimulation T cells in isotonic Ringer’s solution begin increasing their

volume and after 24 h the mean volume is significantly (p<0.001) larger than the control cells

with a mean volume of 171.6 µm3 compared to 118.8 µm

3of the control cells. A plateau is seen

at times 72 and 144 h where the activated cells have significantly (p<0.001) increased their

volume by 213% (fig. 3B).

Figure 3A+C shows how both activated and non-activated cells swell in hypotonic Ringer’s

solution but to a different degree. CD3/CD28 stimulated T cells have increased maximum

swelling starting at 8 h of stimulation, with highest measured values after 72 h and a small

decrease seen after 144 h. At 72 h their maximum swelling reaches a value of 309.3 µm3

compared to a gain of volume of 71.9 µm3 seen in control cells equivalent to a 4 times increase.

Maximum swelling values 24, 72 and 144 h after stimulation are significantly larger than control

values (p<0.001).

Besides swelling more when hypotonically challenged, activated T cells have poorer RVD

performance compared to control cells. As seen from fig. 3A both stimulated and non-

stimulated T cells swell and perform RVD but when measuring % recovery after 3 min (19) and

the initial rate of RVD (see experimental procedures) stimulated T cells show much less RVD

capability than unstimulated control cells. As a control the RVD response in non-stimulated

control cells was measured as a function of time, and it is seen that the recovery percentages of

non-activated T cells are not significantly different from each other at the measured time-points

(average recovery of 36% after 3 min). From fig. 3D it is seen how the relative recovery of

activated T cells decreases beginning after 8 h of stimulation and with significant decrease at 12,

24, 72 and 144 h. After 72 h of CD3/CD28 stimulation the cells have the lowest ability to perform

RVD with a recovery of 13.6% compared to 35.7% (data not shown) of the un-stimulated control

cells and equivalent to a 60% decrease (fig. 3D). After 144 h the cells seem to regain a small

fraction of RVD performance now with an absolute mean recovery of 15.5% or 53% relative to

the control. When measuring RVD by the initial rate of shrinkage the results are similar –

activated T cells have a lesser RVD performance than control cells (fig. 3E). It is seen that the

initial rate of RVD is relatively faster in all controls when comparing them to the activated T cells

though the values are only significantly lower for 8, 12, 24, 72 and 144 h. 8 h of CD3CD28

stimulation results in a 26% decreased RVD rate of (p<0.05) followed by a further decrease after

12 and 24 h to 56% and 53% respectively (p<0.001). Stimulation for 72 and 144 h results in a

slightly faster RVD with rates of 67% (p<0.01) and 63% (p<0.001) compared to that of the control

cells.

8

The Cl- permeability of activated T cells are inhibited

Even though KCNK5 is a well-known player in RVD, the considerable up-regulation of the

channel in activated T cells does not seem to increase the cells ability to perform RVD on the

contrary RVD is inhibited in CD3/CD28 activated cells. Since RVD is driven by the extrusion of KCl

we speculated if the Cl- permeability of the activated cells could be decreased. To determine if a

decreased Cl- permeability could be responsible for the inhibited RVD we used the method

described in materials and methods and in (17). As described in (17) we use the rate of RVD

after addition of gramicidin in a low Na+ media as a measure of the swelling-activated Cl

-

permeability. As seen in fig. 4 the addition of gramicidin to hypotonically swollen non-stimulated

control cells result in a significant faster RVD (higher Cl- permeability) with a 34.3±1.02%

recovery compared to -0.102±1.24% (statistically not different from 0) seen in the CD3/CD28

activated T cells. Thus there is a significant decrease (p<0.05) in the Cl- permeability in non-

stimulated T cells when comparing them to their non-activated counterparts.

Discussion

In the current study we find that the activated T cells enlarge upon activation which is a well

described feature in the activated and proliferating T cells (31; 36). We find that this

enlargement occurs in a time-dependent manner which coincides with the massive up-

regulation of KCNK5 protein. The strong up-regulation is in agreement with earlier findings (1;

5). Thus we here confirm these findings while adding a time profile. The time profile described

here shows an initial (after 2 and 4 hours of CD3/CD28 stimulation) and significant decrease in

KCNK5 protein expression in activated T cells which correlates with a minimal though not

significant early shrinkage and decreased maximum swelling when comparing the activated T

cells to non-activated control cells.

CD3/CD28 T cell activation causes an initial loss of cell number which in two experiments was

found to be of 56% and 41% after 2 and 4 hours of stimulation respectively. This could reflect

that only the activated cells survive whereas the stimulated but non-activated cells undergo

programmed cell death. The initial decrease in cell number is only seen in activated cells and is

therefore not due to purification procedures or culturing conditions. The loss of cell number

after 2 and 4 hours correlates with the decrease in KCNK5 protein and we speculate if this

decrease could occur in order to protect the T cells from apoptosis. It should be noted that the

initial cell loss does not influence the results obtained by western blot, since protein expression

measurements are performed on the same amount of total protein at each time-point. It has

been shown that the same channels involved in RVD are also active in apoptosis thus apoptotic

volume decrease (AVD) is a vital signal in the cells commitment to apoptosis (21). We therefore

speculate if the down-regulation of KCNK5 protein expression seen in the heterogenic T cell

9

population including both apoptotic and surviving/proliferating cells reflect an initial protection

against apoptosis whereas the later up-regulation of KCNK5 protein is involved in the increased

proliferation in the activated T cell

CD3/CD28 activated T cells not only get larger when measured in isotonic Ringer’s solution they

also swell significantly more when subjected to a hypotonic Ringer’ solution as if they have an

inhibited KCl efflux. Despite the up-regulation of the potent volume regulator KCNK5 we also

find the activated T cells to have an inhibited RVD performance upon cell swelling compared to

the control cells. Since an up-regulation of KCNK5 protein expression with a decreased RVD

might seem contradictory we speculate if the up-regulation occurs in order to facilitate

proliferation. Proliferating cells are enlarged to keep daughter cells the same size as the parental

cell a feature vital for successful proliferation, thus a strong RVD mechanism would be

problematic for the activated T cell whose main objective is to increase in size and number to

sufficient fight off a pathogenic intrusion. The KCNK5 up-regulation will help insure a

hyperpolarization of the cell membrane and thus a sustained Ca2+

influx vital for the long-term

activation of the T cells keeping them activated in an antigen-independent manner (13; 16; 18;

28; 35) this would facilitate optimal conditions for the T cell expansion. Andronic et al. find the

RVD performance in stimulated and non-stimulated T cells to be the same, an observation

contradicting our findings (1), but Andronic et al only measure the 20 min volume recovery and

from their figure 2 we calculate the initial rate of volume recovery to be slower in stimulated

cells compred to non-stimulated cell as found in the present study. With respect to maximum

swelling we and Deutch and Lee (15) find the stimulated cells to swell more than non-stimulated

control cells an observation contradicting what was found by Andronic et al (1). We do not have

an explanation for these apparent differences.

Since the RVD in activated T cells was found to be inhibited despite the strong up-regulation of

KCNK5 protein we speculated whether the swelling-mediated Cl- permeability in activated T cells

could be inhibited thereby setting a limit for the cells RVD capacity. We tested this and found

that the Cl- permeability through the volume sensitive anion channel VRAC indeed was inhibited

in the activated T cells compared to control cells. This means that regardless of the increase in

KCNK5 protein quantity the cells will not perform RVD as long as there is a functional down-

regulation of VRAC since the KCl efflux during RVD is an electroneutrale process. It should be

noted that our results are in conflict with those published by Deutsch and Lee in 1988 (15)

where it is shown how phytohemagglutinin activated human peripheral blood lymphocytes (PBL)

respond to gramicidin in a set-up similar to ours by decreasing cell volume, and it is thus

speculated that K+ is the rate limiting factor of RVD in activated T cells. A major difference seems

to be that Deutsch and Lee are not adding gramicidin until 30+ minutes after cell swelling

whereas we add it immediately after cell swelling when the channels are maximally stimulated.

10

We have previously shown in Ehrlich cells that the volume activated channels close before 15

min after swelling activation (17) and Sarkadi and co-workers show that it is also true in human

lymphocytes (32). Another difference between our experiments and those presented by

Deutsch and Lee is the fact that they used PBL’s which contained T cells, B cells and monocytes

whereas we use purified T cells.

In summary we suggest that the KCNK5 up-regulation detected in CD3/CD28 activated human T

cells helps ensure a hyperpolarization of the membrane thus favoring a sustained Ca2+

influx and

T cell activation. We further suggest that to keep the cells from loosing volume thereby

supporting a strong proliferation the Cl- channel VRAC is functionally down-regulated.

Acknowledgements

The technical skills of Dorthe Nielsen (University of Copenhagen) and Pia Birn (Novo Nordisk) is

gratefully acknowledged and appreciated. This work was supported by The Danish Council for

Independent Research/Natural Sciences (grants 09–064182 and 10–085373) and The Lundbeck

Foundation, Denmark (J Nr R32-A3102).

Conflicts of interest

The authors declare that they have no conflict of interest.

11

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14

Fig. 1: mRNA levels of KCNK5, KCa3.1 and Kv1.3 determined by Real-Time qPCR

mRNA levels were determined using Real-Time qPCR and primers against KCa3.1 (A), Kv1.3 (B)

and KCNK5 (C). ANOVA test was used to test for statistical significance on 5-8 independent

experiments (n=2 for 48h). One star (*) indicates a 95% significance level.

Fig. 2: KCNK5 protein expression

T cells were purified from human buffy coats and stimulated with CD3/CD28 activation beads for

2h, 4h, 8h, 12h, 24h, 48h, 72h or 144h. SDS-PAGE and western blotting was performed using

antibodies against KCNK5 and β-actin. Protein bands were visualized and pixel density

calculated. ANOVA was used to determine statistical significance on 3-10 independent

experiments and one star (*) represents a statistical significance level of 95% whereas (***)

indicate a significance level >99%. No significance was seen after 2 and 4 h using the statistical

test ANOVA but when using Student’s T-test both times showed significance here represented

by (###) indicating a level of significance of >99%.

Fig. 3: RVD in CD3/CD28 stimulated T cells and non-activated controls

T cells were purified from human buffy coats and stimulated with CD3/CD28 activation beads for

2, 4, 8, 12, 24, 72 or 144 hours before absolute volume was measured using a coulter counter.

Stimulated T cells and non-stimulated controls were measured in either isotonic (300mOsm) or

hypotonic (160mOsm) Ringer’s solution. A: representative figure showing RVD over time (min)

for untreated control cells and CD3/CD28 stimulated T cells. B: Initial mean volume measured in

isotonic Ringer’s solution C: Mean maximal swelling seen in hypotonic Ringer’s solution. D:

Mean volume recovery after 3 min was calculated as (Vmax-V3min)/(Vmax-Viso), where Vmax, V3min

and Viso are the maximal cell volume, cell volume at time 3 min and cell volume under isotonic

conditions, respectively. E: initial rate of RVD (the slope) was calculated using linear regression

on the linear part of the RVD curve, from maximum volume to the end of linearity. ANOVA test

was used to test for statistical significance on 5-7 independent experiments and one star (*)

represent a 95% significance level whereas two (**) and three stars (***) indicate significance

levels of 99% and >99% respectively.

Fig. 4: Cl- permeability in RVD in CD3/CD28 activated and non-activated T cells

T cells were purified from human buffy coats and stimulated with CD3/CD28 beads for 72 hours.

Activated T cells and non-activated control cells were treated with 100µM clofilium for 30 min to

block the potassium channels. The cells were swollen in hypotonic low Na+ NMDG Ringer’s

solution and their volume was measured using a coulter counter. After one min. 1 µM

gramicidin was added to facilitate cation flux thereby letting the Cl- flux being the limiting factor.

Student’s T-test was used to test for statistical significance on 3 independent experiments and

two stars (**) indicate a significance level of 99%.

18

***


Page 103 of 117

7 Final thesis discussion, future work and perspectives

Throughout my time as a PhD student I have worked with the two-pore domain potassium

channel KCNK5 in various cell systems and with different focus points in mind. My contribution

to the field has mainly been on the channels role in cell volume control, and I have thus worked

with its short time activation, effect of long-term anisotonicity and the potential role of KCNK5

in activated T cell physiology. In this section I will focus on the perspectives of my work

presented in the three papers included in the thesis. Each of the papers holds their own

discussion section and I will thus refer to those, but at the same time try to look a bit further

into the perspectives and future work to be done.

Protein tyrosine phosphorylation of KCNK5 upon acute cell swelling

In Paper I we show how over-expression of KCNK5 channel protein enhances RVD in human

embryonic kidney (HEK)-293 cells which confirms earlier findings (104), indicating that the K+

efflux upon swelling is rate-limiting for the RVD response in this set up (Paper I, fig. 4). We

furthermore show how protein tyrosine kinases (PTK’s) play an important role in the opening of

the channel, since inhibiting PTK’s resulted in an inhibited RVD response in response to

hypotonic cell swelling (Paper I, fig. 1) and how protein tyrosine phosphatases are important in

the closing of the channel (Paper I, fig. 3). We test whether the Src kinase (Paper I, fig. 6) or the

focal adhesion kinase (FAK) (Paper I, fig. 7) could be the PTK’s involved, but our results suggest

that neither of them are likely candidates. On the contrary we find the Janus kinase (JAK) could

be a potential candidate (Paper I, fig. 8). PTK phosphorylation could be involved at different

steps in pathway, from cell swelling to KCNK5 channel (called TASK-2 in the paper, see section

1.3) activation and K+ efflux (53), and we found that cell swelling resulted in a direct tyrosine

phosphorylation of KCNK5 itself with a time course following the time course of the RVD

response (Paper I, fig. 5). We have not tested the potential role of PTK’s in other steps of the

pathway and thus cannot rule out the possibility that tyrosine kinases and tyrosine

phosphatases could also be involved somewhere else in the pathway from cell swelling to

KCNK5 activation – a question that could be interesting to look further into.

Our findings opens up for a lot of new questions, as to which tyrosine phosphorylation site (or

sites) are phosphorylated upon swelling-activation of the channel and which kinase (or kinases)

are involved?

When submitting the KCNK5 sequence (GenBank accession number NM_021542) to the

NetPhos 2.0 server predicting tyrosine phosphorylation sites, 5 possible sites are identified (fig.

12).


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Future experiments using e.g. mass spectrometry could help answer the questions of which

site/sites are phosphorylated upon swelling-induced activation of the channel and which kinases

are involved. Answering those questions would be really beneficial for the further and full story

of PTK’s in the swelling-activation of KCNK5.

NetPhos 2.0 Server - prediction results

Technical University of Denmark

502 Sequence

MVDRGPLLTSAIIFYLAIGAAIFEVLEEPHWKEAKKNYYTQKLHLLKEFPCLSQEGLDKILQVVSDAADQGVAITGNQTF 80

NNWNWPNAMIFAATVITTIGYGNVAPKTPAGRLFCVFYGLFGVPLCLTWISALGKFFGGRAKRLGQFLTRRGVSLRKAQI 160

TCTAIFIVWGVLVHLVIPPFVFMVTEEWNYIEGLYYSFITISTIGFGDFVAGVNPSANYHALYRYFVELWIYLGLAWLSL 240

FVNWKVSMFVEVHKAIKKRRRRRKESFESSPHSRKALQMAGSTASKDVNIFSFLSKKEETYNDLIKQIGKKAMKTSGGGE 320

RVPGPGHGLGPQGDRLPTIPASLAPLVVYSKNRVPSLEEVSQTLKNKGHVSRPLGEEAGAQAPKDSYQTSEVFINQLDRI 400

SEEGEPWEALDYHPLIFQNANITFENEETGLSDEETSKSSVEDNLTSKEQPEQGPMAEAPLSSTGEFPSSDESTFTSTES 480

ELSVPYEQLMNEYNKADNPRGT 560

......................................Y......................................... 80

....................Y........................................................... 160

.............................Y.................................................. 240

............................................................Y................... 320

..................................................................Y............. 400

................................................................................ 480

...................... 560

Phosphorylation sites predicted: Tyr: 5

Tyrosine predictions

Name Pos Context Score Pred

_________________________v_________________

Sequence 15 AIIFYLAIG 0.019 .

Sequence 38 AKKNYYTQK 0.061 .

Sequence 39 KKNYYTQKL 0.789 *Y*

Sequence 101 TTIGYGNVA 0.740 *Y*

Sequence 118 FCVFYGLFG 0.076 .

Sequence 190 EEWNYIEGL 0.917 *Y*

Sequence 195 IEGLYYSFI 0.248 .

Sequence 196 EGLYYSFIT 0.418 .

Sequence 219 PSANYHALY 0.124 .

Sequence 223 YHALYRYFV 0.014 .

Sequence 225 ALYRYFVEL 0.007 .

Sequence 232 ELWIYLGLA 0.075 .

Sequence 301 KEETYNDLI 0.965 *Y*

Sequence 349 PLVVYSKNR 0.042 .

Sequence 387 PKDSYQTSE 0.958 *Y*

Sequence 412 EALDYHPLI 0.210 .

Sequence 486 LSVPYEQLM 0.104 .

Sequence 493 LMNEYNKAD 0.194 .

_________________________^_________________

Figure 12: NetPhos tyrosine phosphorylation prediction NetPhos server 2.0 was used to predict tyrosine phosphorylation sites on KCNK5 (mus musculus GenBank accession number NM_021542). 5 possible tyrosine phosphorylation sites were predicted by the software. http://www.cbs.dtu.dk/services/NetPhos/


Page 105 of 117

As described in section 1.3.3 there are various other gating and activation mechanisms involved

in the swelling-induced activation of KCNK5 and we cannot exclude the possibilities of other

gating mechanisms being involved.

Long-term hypotonic stimuli and its consequences for KCNK5

Paper II deals with the long-term effects of hypotonicity on KCNK5 channel expression and

physiology. In patch-clamp (Paper II, fig. 1) and Coulter counter experiments (Paper II, fig. 2) we

find that there is a decreased maximum current through the channel and at the same time, we

see an inhibited RVD response after cell swelling, when subjecting the cells to long-term

hypotonicity. The RVD results could also reflect an inhibition of VRAC or perhaps a combination

of decreased KCNK5 and VRAC activities. Since the molecular identity of VRAC remains

unknown, it would not be possible to measure mRNA or protein expression, but the volume

activated Cl- current through VRAC is measurable and it would be interesting to see what

potential effect long-term hypotonicity would have on VRAC. It should though be noted that K+

efflux have been shown to be rate-limiting of RVD in Ehrlich cells, thus it is very likely that the

physiological changes seen is due to K+ channel regulation.

Since we speculated that the physiological impairment was likely to be caused by changes

related to K+ efflux, we studied the KCNK5 expression pattern both on mRNA and protein levels.

We found a down-regulation of the KCNK5 channel protein (Paper II, fig. 4) suggesting that the

functional inhibition of the K+ current and the RVD response is indeed likely to be due to

lowered amount of KCNK5 protein.

Not much work has to my knowledge been done on the long-term effects of hypotonicity, thus

we haven’t got much to relate our findings to, nor to help explain the exact mechanisms behind,

which again leaves some questions un-answered. Future experiments could, and should, aim at

explaining the mechanisms behind the functionally down-regulation of the KCNK5 channel. We

suggest that the physiological impairment is the effect of a decreased KCNK5 synthesis or

increased KCNK5 protein degradation, but we cannot exclude the possibility of a contribution

from one or more still un-known regulatory mechanisms. Such mechanisms could e.g. involve

direct or indirect regulation of the channel by one or more of the mechanisms described in

section 1.3.3., a decreased protein insertion into the membrane by an altered protein sorting on

its way to the membrane, internalization of KCNK5 proteins already embedded in the membrane

or post-translational modifications. Since our results show a lowered KCNK5 protein expression

and since we (in one experiment) show an approximately equal decrease in KCNK5 inserted into

the membrane we do favor the idea that an altered KCNK5 protein expression is the main

reason for the changes observed rather than a decreased recruitment to the membrane.


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We find that there is some discrepancy between the physiological data and the protein

expression in which a physiological effect is already seen after 24 hours of stimulation whereas

the inhibited protein expression is first detectable upon 48 h of hypotonicity, thus suggesting

that something else causes initial impairment of the channel. We therefore speculate that the

cell uses other regulatory mechanisms (see section 1.3.3 and above) initially in the response to

the acute environmental change until an altered KCNK5 protein expression can be effective.

As to the question of why the cell down-regulates the KCNK5 protein and thus introduce an

impaired physiological function in response to long-term hypotonicity different factors could be

involved. As the long-term hypotonic stimulated cell experiences a loss of cellular ionic strength

and a decrease in cellular KCl concentration it is very likely that the cell down-regulates

potassium channels to protect the cell potassium homeostasis. As described in section 1.2 K+

channels and thus potassium ions are involved in numerous physiological mechanisms e.g.

setting the membrane potential, proliferation secretion of hormone and transmitters, thus the

cellular potassium concentration is important for many cellular functions. Furthermore a

decrease in intracellular K+

is seen in apoptosis (see section 1.4.2.1) thus a functional down-

regulation of the channel in a low K+ media could help protect the cell.

KCNK5 in activated T cell physiology

We got the opportunity to work together with Novo Nordic on the project of KCNK5 in T cell

activation and thus the previously cell systems studied were changed from a murine cell line to

primary human T cells and the focus point changed to T cell activation. I have not regretted

taking a directional shift since the work on T cell physiology proved to extremely exciting and the

chance to work together with an industrial firm to be very educational. I can only regret that

limited time means that there is still work to be done before this story is complete.

While changing cell type and host species we still worked on KCNK5 and its activation,

involvement in volume regulation and expression pattern. A potential role for KCNK5 and other

two-pore domain K+ channels in lymphocyte biology is by some regarded as questionable (112;

146), but recent publications from different groups have made the presence and physiological

function of two-pore domain K+ channels including KCNK5 in lymphocytes more than likely (3;

10; 11; 35; 98; 100). Paper III ads to the increasing evidence for a role for KCNK5 in activated T

cell physiology, thus we find the channel to be massive up-regulated both on mRNA and protein

levels upon T cells activation (Paper III, fig 1C and fig. 2). The roles of the two potassium

channels Kv1.3 and KCa3.1 in T cell activation has long been known and is undoubtedly of great

importance in T cell activation and physiology, thus we also looked at the mRNA expression of

Kv1.3 and KCa3.1. We found Kv1.3 mRNA to be expressed in T cells but with no change in

expression level upon T cell activation. KCa3.1 mRNA is on the other hand, like KCNK5 mRNA,


Page 107 of 117

was also up-regulated in activated T cells. Kv1.3 is believed to be of most importance in

regulating membrane potential in quiescent T cells, whereas KCa3.1 is up-regulated upon

activation, though some variation in expression profile is seen in different T cell sub-populations

(see (16)).

Since we find that KCNK5 is so heavily up-regulated with no resulting increase in RVD (Paper III,

fig. 3) we suggest that KCNK5 holds another function in activated T cells namely as a important

player in hyperpolarizing the plasma membrane upon activation and thus facilitating a sustained

Ca2+

influx (see section 1.5.3.) in co-operation with Kv1.3 and KCa3.1 and possible other K+

channels. Since the RVD response in activated T cells is inhibited even though KCNK5 is up-

regulated we also investigated the volume sensitive Cl- permeability after T cell activation in

order to see if a down-regulation of the volume sensitive Cl- current could account for the

decreased RVD performance seen in activated T cells, and we did find this to be the case (Paper

III, fig. 4).

In future experiments it would be interesting to perform patch-clamp measurements of the Cl-

current upon T cell activation, to confirm our indirect findings of a decreased permeability.

Furthermore membrane potential and proliferation measurements on KCNK5 knock-down T

cells would help elucidate the potential importance of KCNK5 in activated T cell function.

All in all new knowledge has come from the work presented here though it has also raised

several new questions as research often does and only time can tell if and how these questions

will be answered.


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8 Appendix

Co-authorship statement for papers I, II and III can be found on the following pages.

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