INSTITUTE FOR EXPERIMENTAL MEDICAL RESEARCH,

OSLO UNIVERSITY HOSPITAL,

FACULTY OF MEDICINE, UNIVERSITY OF OSLO, NORWAY

TANDEKILE LUBELWANA HAFVER

The Sodium-Calcium Exchanger 1:

Novel Regulatory Mechanisms and Interacting Partners

THESIS SUBMITTED FOR THE DEGREE OF PHILOSOPHIAE DOCTOR

February 2017

© Tandekile Lubelwana Hafver, 2017 Series of dissertations submitted to the Faculty of Medicine, University of Oslo ISBN 978-82-8377-025-4 All rights reserved. No part of this publication may be reproduced or transmitted, in any form or by any means, without permission. Cover: Hanne Baadsgaard Utigard. Print production: Reprosentralen, University of Oslo.

iii

Acknowledgements

The work for my PhD thesis was carried out from 2012 to 2016 at the Institute for Experimental

Medical Research (IEMR), Oslo University Hospital, Ullevål and the University of Oslo, under the

supervision of Dr. Cathrine Rein Carlson and Professor Ole Mathias Sejersted. Primary financial

support was provided by Helse Sør Øst.

I would like to express my deepest gratitude to my supervisors, Cathrine Carlson and Ole

Sejersted, for mentoring me throughout my PhD studies. Your knowledge, guidance and support have

been invaluable to me. Throughout my time at IEMR I participated in national and international

conferences and appreciate these valuable experiences.

I also wish to express my deepest gratitude to all the co-authors of the papers contained in this

thesis. I am grateful for your contributions which have led to the completion of this work. I wish to

thank all my colleagues at IEMR for their friendship, my office mates for a nice office environment,

Lisbeth Winer and Jo-Ann Fabe Larsen for excellent administration, and the technical team for their

support.

Lastly, I wish to thank my family and friends: To the Hafvers, thank you for all your support

and encouragement throughout these years. To the Lubelwanas and my extended family in South

Africa, thank you for your support, encouragement, enthusiasm and love. To my mother (Nontsikelelo

Elizabeth Lubelwana née Sokopo Hermanus), your sacrifices over the years have not gone unnoticed.

Thank you for your support and love. To my husband Andreas, I would not choose anyone else to be

with me on this journey. Thank you for your encouragement, the scientific discussions and joy you

bring to my life. I am truly blessed to have you as my life partner.

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I dedicate this thesis to the memory of my grandparents, Siphokazi Mary Hermanus née

Nkowane (23.10.1922 to 19.08.2005) and Rev. Dr. Jonathan Tim Hermanus (04.04.1919 to

12.05.2006), who have had an enormous influence in my life. My grandfather was an astute

community leader (under the former Ciskei Government) who always put the interests of others before

his. He was detained as a political prisoner in the infamous Robben Island prison (02.07.1969 to

01.07.1976) for his role in the liberation of his people in Apartheid South Africa. Despite his

incarceration, this never discouraged him, and during this time he obtained his Bachelor of Theology

(Th.B.) in 1971, Master of Theology (Th.M.) in 1973 and Doctor of Theology (Th.D.) in 1976, with

the thesis entitled: “The arrival of missionaries in Africa, with special emphasis on the southern tip of

Africa in the 19th century”. He also went on to obtain a Doctor of Philosophy in Theological (PhD) in

1997. Jola, JoliNkomo, Qengeba, you serve as a source of inspiration, achieving against all odds.

‘uYehova ngumalusi wam’

Oslo, Norway, 6th February 2016

Tandekile Lubelwana Hafver

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Table of Contents

Acknowledgements ................................................................................................................................ iii

Abbreviations ........................................................................................................................................ vii

List of papers .......................................................................................................................................... ix

1 Introduction ................................................................................................................................. 1

1.1 Brief historical perspective...................................................................................................... 1

1.2 The sodium–calcium exchanger 1 (NCX1)............................................................................. 2

1.2.1 Molecular biology of NCX1 and splice variants of NCX ............................................... 2

1.2.2 Topology and structure of NCX1.................................................................................... 3

1.2.3 Function........................................................................................................................... 5

1.2.3.1 NCX1 role in excitation-contraction coupling ............................................................ 5

1.2.3.2 NCX1 in pathophysiology........................................................................................... 6

1.2.4 Regulation ....................................................................................................................... 7

1.2.4.1 Regulation by ions....................................................................................................... 7

1.2.4.2 Regulation by calpain .................................................................................................. 8

1.2.4.3 Regulation by protein phosphatase 1 (PP1)............................................................... 10

1.2.4.4 Regulation by phospholemman (PLM) ..................................................................... 12

1.2.4.5 Regulation by other cytosolic factors ........................................................................ 14

2 Aims of the thesis ...................................................................................................................... 15

3 Methodological considerations.................................................................................................. 17

3.1 Human myocardial biopsies .................................................................................................. 17

3.2 Animal models ...................................................................................................................... 18

3.3 In vitro cell models................................................................................................................ 19

3.4 Biochemical and molecular biology techniques.................................................................... 20

3.4.1 Fractionation.................................................................................................................. 20

3.4.2 Co-immunoprecipitation (Co-IP) and pull-down assays ............................................... 20

3.4.3 Peptides ......................................................................................................................... 22

3.4.4 Proximity ligation assay (PLA) ..................................................................................... 23

3.4.5 Tobacco etch virus (TEV) protease............................................................................... 23

3.4.6 Phosphatase activity assay............................................................................................. 24

3.4.7 Affinity-purification coupled to mass spectrometry...................................................... 24

3.5 Computational methods......................................................................................................... 25

3.6 Biophysical methods ............................................................................................................. 26

3.6.1 Surface plasmon resonance (SPR)................................................................................. 26

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3.6.2 Patch clamp ................................................................................................................... 27

3.6.3 Measurement of intracellular Ca2+................................................................................. 28

4 Summary of results.................................................................................................................... 29

4.1 Paper 1................................................................................................................................... 29

4.2 Paper 2................................................................................................................................... 29

4.3 Paper 3................................................................................................................................... 30

4.4 Paper 4................................................................................................................................... 31

5 Discussion ................................................................................................................................. 32

5.1.1 Insights into calpain regulation of NCX1...................................................................... 32

5.1.2 PP1c mediates indirect regulation of NCX1.................................................................. 34

5.1.3 Insight into the NCX1-PLM disruptor peptide (Opt-pep) ............................................. 37

5.1.4 Insights into the NCX1 interactome .............................................................................. 38

5.1.5 Therapeutic targeting of NCX1..................................................................................... 40

6 Conclusion................................................................................................................................. 43

Reference list......................................................................................................................................... 44

Appendix: Papers 1-4 ............................................................................................................................ 55

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Abbreviations

AB Aortic bandingACE Angiotensin-converting enzymeAM Acetoxymethyl AS Aortic stenosisAsn AsparagineAsp Aspartic acidATP Adenosine triphosphateBIAcore Biomolecular interaction analysisbpm Beats per minuteCABG Coronary artery bypassCaCA Ca2+/cationCAD Coronary artery diseaseCaMKII Ca2+/calmodulin dependent kinase IICaMPDB Calpain for modulatory proteolysis databaseCBD Ca2+-binding domaincDNA Complementary deoxyribonucleic acidCICR Ca2+ induced Ca2+ releaseCLD Catenin-like domainCo-IP Co-immunoprecipitationCys CysteineDARPP-32 dopamine- and cyclic-AMP-regulated phosphoprotein of molecular weight

32,000DAVID Database for annotation, visualization and integrated discoveryDNA Deoxyribonucleic acidFRET Fluorescence resonance energy transferGlu Glutamic acid GO Gene ontology GST Glutathione S-transferaseHEK293 Human embryonic kidney 293HEPES N-2-Hydroxyethylpiperazine-N'-2-Ethanesulfonic acidHF Heart failureHis HistidineHis-TF-NCX1cyt

His-trigger factor-NCX1 cytosolic loop

HPRD Human protein reference databaseI-1 Inhibitor-1I-2 Inhibitor-2IP ImmunoprecipitationITC Isothermal titration calorimetrykDa KilodaltonKEGG Kyoto encyclopedia of genes and genomesLTCC L-type Ca2+ channelLV Left ventricleLys LysineMet MethioninemM MillimolarmRNA Messenger RNA

viii

MS Mass spectrometry NCX Sodium calcium exchangerNKA Sodium/potassium-transporting ATPasenM NanomolarNMR Nuclear magnetic resonanceOpt-pep NCX1-PLM disruptor peptide (developed in paper 3)PDB ID Protein data bank identificationPhe PhenylalaninePI PhosphatidylinositolPIP2 Phosphatidylinositol-4,5-bisphosphatePKA Protein kinase APKC Protein kinase CPLA Proximity ligation assayPLM PhospholemmanPLN PhospholambanPMA Phorbol 12- myristate 13-acetatePMCA Plasma membrane Ca2+ ATPasePNUTS PP1 nuclear targeting subunitPP1 Protein phosphatase 1PP1c Protein phosphatase 1 catalytic subunitPP2A Protein phosphatase 2APro ProlinepSer-68-PLM Phospho serine-68-phospholemmanPVDF Polyvinylidene difluoride RNA Ribonucleic acidRYR2 Ryanodine receptors 2SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresisSer SerineSERCA2 Sarcoendoplasmic reticulum Ca2+ ATPase 2SLC Solute carrierSPR Surface plasmon resonanceSR Sarcoplasmic reticulumSTRING Search tool for the retrieval of interacting genes TEV Tobacco etch virusThr Threonine TM Transmembrane Tyr TyrosineμM MicromolarWT Wild typeXIP Exchanger inhibitory peptideYFP Yellow fluorescent protein

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List of papers

1. Molecular Basis of Calpain Cleavage and Inactivation of the Sodium-Calcium

Exchanger 1 in Heart Failure

Pimthanya Wanichawan, Tandekile Lubelwana Hafver, Kjetil Hodne, Jan Magnus Aronsen,

Ida Gjervold Lunde, Bjørn Dalhus, Marianne Lunde, Heidi Kvaløy, William Edward Louch,

Theis Tønnessen, Ivar Sjaastad, Ole Mathias Sejersted and Cathrine Rein Carlson. J Biol

Chem. 2014 Dec 5; 289(49):33984-98.

2. Protein Phosphatase 1c Associated with the Cardiac Sodium Calcium Exchanger 1

Regulates Its Activity by Dephosphorylating Serine 68-phosphorylated Phospholemman

Tandekile Lubelwana Hafver, Kjetil Hodne, Pimthanya Wanichawan, Jan Magnus Aronsen,

Bjørn Dalhus, Per Kristian Lunde, Marianne Lunde, Marita Martinsen, Ulla Helene Enger,

William Fuller, Ivar Sjaastad, William Edward Louch, Ole Mathias Sejersted and Cathrine

Rein Carlson. J Biol Chem. 2016 Feb 26; 291(9):4561-79.

3. Development of a high-affinity peptide that prevents phospholemman (PLM) inhibition

of the sodium/calcium exchanger 1 (NCX1)

Pimthanya Wanichawan, Kjetil Hodne, Tandekile Lubelwana Hafver, Marianne Lunde, Marita

Martinsen, William Edward Louch, Ole Mathias Sejersted and Cathrine Rein Carlson.

Biochem J. 2016 Aug 1; 473(15):2413-23.

4. Mapping the in vitro interactome of cardiac Na+-Ca2+ exchanger 1 (NCX1)

Tandekile Lubelwana Hafver, Gustavo Antonio de Souza, Pimthanya Wanichawan, Marianne

Lunde, Marita Martinsen, Ole Mathias Sejersted and Cathrine Rein Carlson.

Manuscript in revision. PROTEOMICS-pmic.201600417

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1

1 Introduction

1.1 Brief historical perspectiveThe function of the heart is a topic that has interested scholars since antiquity, but William Harvey

(1578-1657) is commonly cited as the pioneer of modern cardiovascular research. Harvey challenged

previous beliefs that blood is continuously produced from digested food and consumed by the organs

in the body, and postulated instead that the blood flows in a cycle, and that the heart’s function is to

pump the blood through the circulatory system (1). Harvey’s theories soon gained acceptance, but

another question remained a puzzle: What drives the heartbeat?

Over the next centuries, two competing theories emerged: On the one hand, the myogenic

theory held that excitation originates in the heart itself. On the other hand, the neurogenic theory

proposed that excitation originates from nerve connections or ganglions. This debate was eventually

settled in favor of the myogenic theory: Walter Gaskell (1847–1914) observed that an isolated strip of

tortoise ventricular muscle, devoid of ganglions or nervous connections, continued to pulsate at a rate

similar to the intact heart, and concluded that “The rhythmic capacity of every part of the heart

depends not upon the presence of ganglion cells but rather upon the persistence of a primitive

condition of heart muscle” (2). Consequently, through a sequence of discoveries by, among others, Jan

Evangelista Purkinje (1787–1869), Wilhelm His Jr (1863–1934) and Sunao Tawara (1873–1952) (3), a

network of electrically conducting pathways in the heart was revealed. In 1907, Arthur Keith and

Martin Flack identified the sinus node, which contains special pacemaker cells that produce and

transmit the electrical impulses that drive the heartbeat, the so called action potential (4).

The next big question was how the action potential translates into contraction of the heart

muscle. The first hints had been provided by Sydney Ringer, in a series of four papers in the 1880s (5-

8). Ringer demonstrated that the force of contraction of frog hearts was influenced by the

concentrations of various ions in the perfusion solution he used in his experiments. Crucially, he

discovered that the isolated frog hearts could not be made to contract in the absence of Ca2+. Thus,

Ca2+ was identified as a crucial mediator of contraction.

In 1948, Willibrandt and Koller (9) reported that cardiac contraction increased in the presence

of low concentrations of Na+. The importance of Na+ for contractility was further recognized when, in

1953, Schatzmann discovered that cardiac glycosides (such as digitalis, or foxglove, which had been

used for centuries as a remedy for treating heart conditions (10)) work by inhibiting K+ and Na+

transport (11). In 1958, Lüttgau and Niedergerke reported that the force of contraction in fact

depended on the ratio of extracellular Ca2+ and Na+ (12,13). A decade later, Reuter and Seitz (1968)

and Baker and Blaustein et al. (1969), through experiments on guinea pig atria (14) and squid giant

2

axons (15) respectively, were the first to show a coupling between influx and efflux of Ca2+ and Na+

across the plasma membrane and documented the existence of a Na+-Ca2+ exchange counter-transport

system. This coupling between Na+ and Ca2+ transport explained how the blocking of Na+ transport by

cardiac glycosides could regulate contractility by affecting the Ca2+ loading of cardiac cells (16).

Over the past half century, the role of Ca2+ and Na+ transport in cardiac excitation-contraction

coupling has been further characterized and clarified, but it still remains an active focus of research in

elucidating disease mechanisms.

1.2 The sodium–calcium exchanger 1 (NCX1)The sodium–calcium exchanger (NCX) is a ubiquitously expressed ion-transporting plasma membrane

protein that plays an important role in maintaining cytosolic Ca2+ homeostasis. Splice variant NCX 1.1

(referred to as NCX1 in this thesis) is cardiac specific, and plays an important role in excitation-

contraction coupling in cardiomyocytes as it is the dominant cellular Ca2+ efflux mechanism

mediating diastolic relaxation and preparing the cell for the next contraction cycle (17).

The physiological importance of NCX1 in early development is illustrated by the observation

that ubiquitous deletion of NCX1 brings about early death of embryos because of a lack of heartbeat

(18). Studies have also reported increased NCX1 messenger RNA (mRNA) and protein levels in end-

stage human heart failure (HF) (19,20), but, interestingly, this is not always correlated with increased

NCX1 activity (21). Such observations have elicited interest in NCX1’s role in pathophysiological

conditions and as a potential therapeutic target (21). Two features which makes NCX1 particularly

interesting in this regard are the facts that it can exchange Ca2+ for Na+ in both directions, and that it

operates at a stoichiometry of one Ca2+ exchanged for three Na+’s, making its transport electrogenic.

This means that NCX1 can play a role both in Ca2+ import and removal, and by carrying charge, it can

also influence the configuration of the action potential and contributing to arrhythmias (22). However,

if NCX1 is to be a viable therapeutic target, a better understanding of its regulation is warranted,

motivating the work contained in this thesis.

1.2.1 Molecular biology of NCX1 and splice variants of NCX

An important step in the study of NCX1 was the successful purification of NCX1 in 1988 (23). The

isolation of the exchanger paved the way for the production of polyclonal antibodies which were used

in the cloning of NCX1 from canine hearts in 1990 (24). This has allowed subsequent studies of the

exchanger at the molecular level.

The initial molecular cloning of NCX1 was followed by the identification of several related

proteins through sequence database mining and low-stringency library screening. It was established

that NCX1 is a member of a larger gene family, the solute carrier (SLC) 8 family, which, in turn,

forms part of the Ca2+/cation (CaCA) antiporter superfamily (25,26). Three gene members of the SLC8

3

family are expressed in mammals: SLC8A1 (NCX1) (24), SLC8A2 (NCX2) (27), and SLC8A3 (NCX3)

(28). These give rise to at least 17 NCX1 and 5 NCX3 alternative splice variants, but no splice variants

of NCX2 are known. The splice variants can be attributed to six small exons (A, B, C, D, E, and F), of

which exons A and B are mutually exclusive and appear in every splice-variant for NCX1 and NCX3,

resulting in tissue specific expression (29). Although first discovered in cardiac cells, NCX1 is the

most broadly expressed SLC8 family member, present at low levels in most tissues, and expressed at

high levels in the brain and kidneys, in addition to the heart (26). The three mammalian SLC8 family

members share about 70% amino acid identity overall, and more than 80% identity within the

predicted transmembrane segments (30). The latter indicates a common function of ion exchange.

1.2.2 Topology and structure of NCX1

Human NCX1 is 973 amino acids long, with the first 32 amino acids representing a signaling peptide

that is cleaved off during processing. This signal sequence was originally believed to ensure the

correct insertion of the exchanger into the cell membrane, however, the exchanger is correctly targeted

even when this sequence is removed (31). The N-terminus of the protein is glycosylated, which does

not affect exchanger function, but helps to define the exchanger topology (32). On sodium dodecyl

sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis, the NCX1 protein displays three

bands with apparent molecular masses of 70, 120, and 140 kilodalton (kDa). The 120-140 kDa band

corresponds to the full-length mature protein, whereas the ~70 kDa band represents a proteolytic

fragment (23). The 120-140 kDa full-length doublet is attributed to a mobility shift due to the

formation of an intramolecular disulfide bond between Cys-792 and either Cys-14 or Cys-20 (33).

Additionally, intermolecular cross-linking (34) and fluorescence resonance energy transfer (FRET)

(35) suggests that NCX1 forms dimers. It has even been suggested that NCX1 exists as a higher-level

multimer in the membrane (36). The physiological implication of these NCX1 multimers remains

unknown.

Members of the SLC8 family are characterized by a conserved overall membrane topology,

with two clusters of hydrophobic transmembrane (TM) domains, joined by a cytosolic loop whose

length varies between the family members (26). Based on accessibility experiments and epitope

mapping, the current model for mammalian NCX1 has nine TM domains; five in the first hydrophobic

cluster and four in the second (37,38). This has been challenged, however, by recent studies reporting

that the crystal structure of a prokaryotic homologue contains 10 TM domains (39). There is strong

precedent for prokaryotic and eukaryotic homologues of membrane proteins to have a similar topology

(40).

4

The conserved hydrophobic TM domains of NCX1 (residues 1–217 and 727–903) (41) contain

- - -repeats are believed to have

arisen from an ancient gene-duplication event (42) -

repeat regions catalyze ion translocation (43). Crosslinking experiments have shown that the helices

predicted to flank the -repeats are in close proximity (44,45), and accessibility experiments have

indicated that the two -repeats are oriented in opposite directions with respect to the membrane,

possibly with their central regions forming membrane-reentrant loops with limited accessibility from

either side of the membrane (37,38). More than half of the NCX1 protein is made up of a ~ 550 amino

acid hydrophilic cytosolic loop between TM5 and TM6. Deletion of this cytosolic loop does not affect

ion translocation, however, the loop mediates regulation of the exchanger by associating with various

cytosolic factors (46-51). The cytosolic loop of NCX1 can be broken down into different domains: 1)

The N-terminal exchanger inhibitory peptide (XIP) domain (residues 219–238 ), which binds

calmodulin (31). 2) The catenine-like domain (CLD) (residues 218–370 and 651–726) (41), which

contains binding sites for phospholemman (49), a calpain cleavage site (paper 1) and an endogenous

XIP site (52). 3) Regulation sites for two Ca2+-binding domains (CBD), CBD1 (residues 371–500) and

CBD2 (residues 501–650) (41), where Ca2+ binding alters the structure and motional dynamics of

these domains (41,53-55). Recently, CBD1 has been shown by us to have anchoring sites for calpain

(paper 1) (48) and protein phosphatase 1 catalytic domain (PP1c) (paper 2) (50). The amino acid

numbering used above excludes the signaling peptide sequence. The nuclear magnetic resonance

-sandwich, formed by two

-sheets (41) (Fig. 1). The structures of CBD1 and CBD2 have been elucidated, but the

three-dimensional structures of the CLD remain unsolved.

Figure 1. Predicted topology of NCX1. The figure shows that NCX1 is composed of 10 TM domains,

The CBD1 and CBD2 are arranged as anti- -sheets and bind Ca2+ with high affinity. Figure obtained from (56) and modified to show the presence of 10 TM domains. Reprinted with permission from the publisher: Portland Press Ltd.

5

1.2.3 Function

NCX1 plays an important role in maintaining intracellular Ca2+ homeostasis, as it is the primary

contributor to Ca2+ efflux. In the heart, NCX1 mediates diastolic relaxation by transporting Ca2+ across

the plasma membrane in exchange for Na+, at a stoichiometry 1:3. The exchanger can operate in both

Ca2+ efflux (forward mode) and Ca2+ influx (reverse mode), depending on the internal and external

concentration of both Na+ and Ca2+, as well as on the membrane potential (57).

1.2.3.1 NCX1 role in excitation-contraction coupling

The mechanical contraction of the heart is linked to the electrical action potential through Ca2+, as

illustrated in Fig. 2. In brief, the ventricular action potential begins with the depolarization of the cell

membrane, caused by an electrical impulse transmitted from a neighboring cardiomyocyte. This

causes voltage gated Na+ channels to open and Na+ to enter the cell, raising the membrane potential.

As the membrane potential increases, voltage gated L-type Ca2+ channels (LTCCs) are activated,

allowing extracellular Ca2+ to flow down their Ca2+ gradient into the cytosol. Additional Ca2+ may also

enter the cell via NCX1 operating in reverse mode (17,58). Cytosolic Ca2+ ions diffuses across the

dyadic cleft, and binds to clusters of ryanodine receptors (RYR2) localized on the membrane of the

sarcoplasmic reticulum (SR). This triggers the RYR2 to open, and Ca2+ is released from the SR in a

series of sparks, a process called Ca2+ induced Ca2+ release (CICR) (59). This process rapidly increases

cytosolic Ca2+ concentrations and Ca2+ binds to Troponin C of the myofilaments and induces a

conformational change that induces the interaction between actin and myosin which allows cross-

bridges to form, resulting in contractile force generation. In order for the cardiomyocyte to relax and

prepare for the next contraction cycle, Ca2+ must be removed from the cytosol. Most of the Ca2+ is re-

sequestered into the SR, mediated by sarcoendoplasmic reticulum Ca2+ ATPase 2 (SERCA2), while a

small amount of Ca2+ is taken up by the mitochondria and plasma membrane Ca2+ ATPase (PMCA).

The rest of the Ca2+ is extruded from the cell, mainly via NCX1. Similarly, Na+ balance is restored by

the sodium/potassium-transporting ATPase (NKA) (Fig. 2).

Note that NCX1 plays several roles in the excitation-contraction coupling cycle: Firstly, it

serves as the primary Ca2+ extrusion mechanism during diastole. Secondly, the contribution from

reverse mode operation of NCX1 in systole, although small, may prime dyadic RYR2 in its vicinity

for CICR. Furthermore, as its transport is electrogenic (carries net charge), NCX1 also modulate the

action potential.

6

Figure 2. Schematic figure of excitation-contraction coupling in ventricular cardiomyocyte. The figure shows the main participating proteins in excitation-contraction coupling, as described in the text above. The inset shows the progression of the action potential and Ca2+ transient, which drives the contraction. Figure obtained from (17). Reprinted with permission from the publisher: Nature Publishing Group.

1.2.3.2 NCX1 in pathophysiology

The various ion channels in the heart participate in complex interplay, and the correct balance between

different ion transport mechanisms is important to ensure proper functioning of the heart. Improper

handling of Ca2+ is a common cause of both contractile dysfunction and arrhythmias in HF.

Increased NCX1 mRNA and protein levels have been shown in end-stage human HF (19,20),

and elevated activity of NCX1 has been linked to dysfunctional Ca2+ handling in chronic heart disease

(29). Overexpression of NCX1 in HF can lead to increased Ca2+ efflux through forward mode NCX1

activity, which combined with SERCA2 dysfunction can lead to depletion of SR Ca2+ and result in

reduced contractile force (60). Abnormal efflux of Ca2+ during diastole could also lead to delayed

afterdepolarizations and contribute to arrhythmias. In addition, increased reverse mode NCX1 activity

can also contribute to the triggering of spontaneous and unsynchronized Ca2+ release from the SR

(61,62).

Insights regarding NCX1 function and the therapeutic potential of targeting the exchanger can

be learned from transgenic mouse models. Global knock-out of NCX1 is embryonically lethal (18) due

to a lack of heartbeat, indicating a crucial function of NCX1 in early development. Surprisingly,

however, mice with conditional knock-out of NCX1 from the ventricle myocytes survive, with only

20-30% reduction in contractility (63). The survival can be attributed to two adaptations (64): Firstly,

reduced NCX1 Ca2+ efflux is compensated by a ~80% reduction of Ca2+ influx via LTCC, and Ca2+

expulsion is mediated by PMCA, while the SR Ca2+ is not affected (65). Secondly, the action potential

is shortened, which further reduces the inflow of Ca2+ (64). These adaptations serve to maintain both

7

cytosolic Ca2+ homeostasis and contractility. In summary, the knock-out of NCX1 represents a gain in

contractile efficiency (since less Ca2+ influx is required to maintain contraction), but at the cost of

depressed Ca2+ transient amplitude, elevated diastolic Ca2+ concentration and shorter action potentials

(64). Similarly to the conditional knock-out mice, NCX1 overexpressing mice also display almost

normal cardiac function, but with opposite adaptations: Increased Ca2+ extrusion via NCX1 is

compensated by increased Ca2+ influx via LTCC, and the mice exhibit smaller Ca2+ transients and a

prolonged action potential (66).

Whether up-regulation of NCX1 during cardiac hypertrophy and HF is adaptive or

maladaptive is not fully understood, not least because the heart function and the remodeling that

occurs during HF involves a multitude of proteins interacting in complex ways. In HF, increased

NCX1 activity could contribute to arrhythmia, as well as Ca2+ depletion of the SR and reduced

contractility, but, on the other hand, this may also protect against diastolic Ca2+ overload (67).

Inhibition of NCX1 was reported to be protective against cardiac ischemia–reperfusion injury (68),

which may be explained by reduced Ca2+ entry via NCX1.

1.2.4 Regulation

1.2.4.1 Regulation by ions

The most prominent regulatory mechanisms of NCX1 are induced by Na+ and Ca2+, which, in addition

to being the substrates for transport, also have separate regulatory roles. On the one hand, binding of

intracellular Ca2+ to CBD1 and CBD2 induce conformational changes that causes allosteric activation

of the exchanger, as first observed by DiPolo in 1979 (69). Activation occurs in the physiological

range, just above resting Ca2+ level, 150–400 nM (70). Ottolia et al. 2004 showed in FRET

experiments that the Ca2+ activation of the exchanger is fast enough to regulate NCX1 on and off on a

beat to-beat basis (71). Ca2+ gating could be a mechanism to ensure that cytosolic Ca2+ does not

decline too much between beats. On the other hand, Na+ inactivates the outward exchange current

when applied to the intracellular surface of the cell membrane (72,73). This mechanism involves the

XIP region in the cytosolic loop of NCX1. Mutations in the XIP region alters the susceptibility of the

exchanger to inactivation by Na+, where the F223E mutant makes the exchanger hypersensitive, while

the K229Q mutant makes the exchanger resistant to Na+ inactivation (74). Binding of

phosphatidylinositol-4,5-bisphosphate (PIP2) to the XIP region disrupts the Na+-dependent inactivation,

and adenosine triphosphate (ATP), which stimulates synthesis of PIP2 from phosphatidylinositol (PI),

can therefore protect against inactivation of the exchanger (75). A rise in cytosolic Ca2+, on the other

hand, relieves the Na+-dependent inactivation (76). Na+-inactivation is not believed to be important on

a beat-to-beat basis or under normal physiological conditions, as the wild type exchanger only

responded to Na+-dependent inactivation when the cytosol was acidified (73). It is instead believed

that this mechanism might play a protective role in conditions of high pH and high Na+, such as in

8

ischemia, by inhibiting NCX1 mediated Ca2+ influx (77). In addition, NCX1 is also regulated by other

divalent and trivalent cations which have been shown to inhibit Ca2+ transport by the exchanger

(78,79).

1.2.4.2 Regulation by calpain

Proteases are enzymes that catalyze the cleavage of peptide bonds by hydrolysis. Calpains are a family

of non-lysosomal calcium-dependent cysteine proteases, whose enzymatic effect is to cleave its

substrates in response to high Ca2+ concentrations. Calpain is often referred to as a ‘modulator

protease’, because, unlike lysosomal proteases, it does not digest its substrates. Rather, calpain

proteolysis is selective and is limited to specific sites, modulating the function or activity of its

substrates (80).

The nomenclature for calpains has evolved with the discovery of new members. It is common

to classify calpains into classical/conventional/typical and non-classical/unconventional/atypical

calpains, where the former includes calpain-1 and calpain-2 (81). The classical calpains are

ubiquitously expressed heterodimeric proteins with two distinct subunits; 1) a large catalytic subunit

(~80 kDa) and 2) a small regulatory subunit (~28 kDa). The regulatory subunit is common for calpain-

1 and calpain-2, whereas their catalytic subunits are similar, but distinct, with 55-65% sequence

homology (80,82). On SDS-PAGE calpain migrates as a doublet, as autolysis reduces the mass of

calpain-1 to 76 kDa and calpain-2 to 78 kDa (83). The domain structure of the catalytic subunit of

calpain-1 and calpain-2, as well as the regulatory subunit, is shown in Fig. 3.

An important regulator of calpain activity is Ca2+. Calpains undergo conformational changes

in response to Ca2+ binding, causing the alignment of the active site. The classical calpain isoforms

differ in their Ca2+ activation thresholds: Calpain-1 has a half-maximal activity at 3- 2+, and is

often called micro- -calpain). Calpain-2 has a half-maximal activity at 400- 2+, and

is often called milli-calpain (m-calpain) (84). The Ca2+ concentrations required to activate both

calpain-1 and calpain-2 is significantly higher than resting concentrations in cells. This means that

calpain activation only can occur locally near influx points of Ca2+, and the activation is temporarily

limited by diffusion and removal of Ca2+ by ion pumps (85). Calpain activity is also regulated by an

endogenous inhibitor, calpastatin, which inhibits calpain activation, preventing unspecific activation

by normal Ca2+ flux. In the presence of excess Ca2+ levels, calpastatin will dissociate and allow

calpains to autolyse and become an active protease against various substrates (86). The small

regulatory subunit of calpain mediates its regulation by ensuring proper folding of the large catalytic

subunit and is also essential for the proteolytic activity of calpain-1 and calpain-2 (87).

Through proteolytic processing, calpains affect a range of cellular processes by modifying its

substrates. Although the exact functions of calpain remain elusive, it is linked to signal transduction,

cell cycle progression, regulation of gene expression, apoptosis (80) and cleavage of enzymes, and

9

cytoskeletal/membrane proteins (88). Calpain has also been implicated in various pathologies.

Specifically, activation of calpain-1 has been shown to cause destruction of contractile filaments in

fibrillating atrial muscle (89), and increased calpain activity is reported to exacerbate pathologies

relating to lack of Ca2+ control (84). Inhibition of calpain at the onset of reperfusion was shown to

have cardioprotective effects (90). Insights into the function of calpain may be gained from studies in

transgenic mouse models. Calpain-1 knock-out mice are viable and fertile, with some defects in

platelet aggregation (91), while calpain-2 knock-out mice exhibit embryonic lethality. This suggests a

crucial role of calpains, but suggests that calpain-2 may be able to compensate for calpain-1. However,

knockout of the regulatory subunit in both calpain-1 and calpain-2 is embryonically lethal (92),

indicating an important role of the regulatory subunit as an endogenous regulator of calpain activity.

Figure 3. Domain structure of calpains. In the catalytic subunit, the N-terminal anchor helix region is designated as domain I. Domain II contains the Cys, His, and Asn amino acid residues necessary for proteolytic function. Domain III consists of residues that associate with plasma membranes and binding Ca2+. Domain IV contains five EF hand sequences responsible for Ca2+ binding. The small regulatory subunit (CAPNS) is common to both μ-calpain (calpain-1) and m-calpain (calpain-2) isoforms and is composed of Domains V and VI. These domains contain hydrophobic residues that help to achieve membrane interaction and additional Ca2+

binding. The catalytic and regulatory subunits remain associated under inactive conditions. Upon Ca2+ binding, μ-calpain (calpain-1) and m-calpain (calpain-2) self-cleave an N-terminal portion of the large and small subunits to achieve full proteolytic activity. Figure obtained from (82). Reprinted with permission from the publisher:John Wiley and Sons. Of note, a new nomenclature was proposed for the domains and is summarized in (93).

As mentioned above, calpain mediates proteolysis of cytoskeletal/membrane proteins, and

NCX1 has been identified as a substrate for calpain in the brain (94). However, the molecular

mechanisms regulating calpain cleavage of NCX1 in the heart remained unknown. In paper 1 of this

thesis we have shown that treatment of left ventricle (LV) tissue lysates with calpain resulted in a ~75

kDa proteolytic fragment (95). Further, we mapped the calpain binding and cleavage site in NCX1 and

investigated the functional consequence of NCX1 cleavage in human embryonic kidney 293 (HEK293)

cells.

10

1.2.4.3 Regulation by protein phosphatase 1 (PP1)

The balance between the activity of protein kinases and phosphatases regulates diverse

physiological processes, such as cell division, cell differentiation, neuronal activity, muscle

contraction and metabolic functions (96). 3% of all human genes encode protein kinases or

phosphatases, indicating their importance (97).

Protein phosphatase 1 (PP1) is a ubiquitously expressed ~38.5 kDa serine/threonine

phosphatase which counters the effects of serine/threonine kinases (98). Mammalian genomes encode

(99).

The isoforms show 85% sequence identity, but the N- and C-terminal extremities show amino acid

differences, allowing for distinct tissue distribution and subcellular localization (99).

The first crystal structure of the PP1-microcystin complex was shown by Goldberg et al. 1995

and revealed that the PP1 catalytic domain (residues 7- - - - -

active site (100). This active site contains two metal ions which catalyze the dephosphorylation

reaction. Additionally, the active site is surrounded by various substrate-binding grooves, which

increase the number of possible substrates recognized by PP1, as shown by the surface representation

in Fig. 4. The catalytic domain (PP1c) achieves substrate specificity by forming holoenzymes with

more than 200 regulatory proteins (101). These regulatory proteins localize PP1c to specific

subcellular domains and fine-tune its activity, allowing for substrate specific effects (101). Regulatory

proteins can affect the activity of PP1 by serving as inhibitors, pseudosubstrate, substrates or substrate-

specifiers (102). PP1 activity is regulated by toxins such as microcystin (103) and okadaic acid (104),

which inhibit PP1 activity potently, but not specifically. PP1 is also inhibited by protein inhibitors,

such as inhibitor-1 (I-1), dopamine- and cyclic-AMP-regulated phosphoprotein of molecular weight

32,000 (DARPP-32) and inhibitor-2 (I-2). I-1 and DARPP-32 must first be phosphorylated by protein

kinase A (PKA) in order to convert them into potent inhibitors.

90% of PP1 regulatory proteins interact with PP1 via a short degenerate RVxF-docking motif

with consensus sequence [RK]x0-1[VI]{P}[FW], where x denotes any residue and {P} any residue

except proline (105). The RVxF motif serves as an anchor for the initial recruitment of PP1 and

enables the subsequent establishment of secondary PP1 interactions. Additional recently identified

docking motifs have been described, such as the SILK and MyPhoNE motifs found in six of the 200

known PP1 regulator proteins. The former anchors PP1, while the latter is important for substrate

selection (106,107). Choy et al. 2014 defined an extended PP1 binding motif with consensus sequence

RVxF-X5–8- -X8–9-R, where the RVxF motif is followed by a variable, two-residue phi-phi

(108). The conserved arginine in the PP1 nuclear targeting

subunit (PNUTS)-PP1 holoenzyme has been shown to work as a substrate selectivity filter, which

blocks access to the C-terminal binding groove in PP1 (108).

11

Figure 4. The structure of PP1 from the PP1-microcystin complex is shown as a surface representation. A: 2+, but likely Fe2+ and Zn2+ in vivo) (103)

(pink spheres), which catalyzes the dephosphorylation reaction and lies at the Y-shaped intersection of three substrate-binding grooves; the hydrophobic (blue), the acidic (orange), and the C-terminal (red). B: The rotation of (A) shows the binding sites for the PP1-docking motifs RVxF (purple), SILK (cyan) and MyPhoNE (wheat)(PDB ID 1FJM). Figure obtained from (101). Reprinted with permission from the publisher: Elsevier Ltd.

The physiological relevance of PP1 in the heart was shown by the use of okadaic acid, a non-

specific PP1 inhibitor, which enhanced contractility in guinea-pig isolated ventricular muscles (109).

impaired cardiac contractility, ventricular dilatation, increased cardiac fibrosis and re-expression of

fetal genes (110). Overexpression of constitutively active forms of I-1 (111) or I-2 (112) shows the

opposite phenotype as in overexpression of PP1. Active I-1 and I-2 lowered PP1 activity, restored

adrenergic responsiveness, enhanced cardiac contractility and is protective against the development of

cardiac hypertrophy.

PP1 mediates regulation in cardiac electrophysiology and in key contractile proteins by

controlling their dephosphorylation after phosphorylation by protein kinases. PP1 has been shown to

target the K+ channel macromolecular complex composed of the pore-

bunit K+ -adrenergic signaling

(113). RYR2 is phosphorylated on Ser-2030 and Ser-2808 by PKA (114) whereas

calcium/calmodulin-dependent protein kinase II (CaMKII) phosphorylates Ser-2814 (115).

Dephosphorylation of RYR2 is mediated by PP1, which is targeted to the complex by the regulatory

protein spinophilin. The absence of spinophilin decreases PP1-mediated dephosphorylation of RYR2

and leads to RYR2 hyperactivity, contributing to atrial fibrillation (116). PP1 also mediates regulation

of phospholamban (PLN), the accessory protein which regulates the activity of SERCA2 (117,118).

Dephosphorylation of phospho serine-68-phospholemman (pSer-68-PLM) by PP1 was recently shown

to modulate phospholemman (PLM) regulation of NKA (119). pSer-68-PLM also mediates regulation

of NCX1 (see section 1.2.4.4), but it is unknown whether PP1 is involved in NCX1 regulation. In

paper 2 of this thesis we investigated whether a direct and functional NCX1-PP1c interaction is a

prerequisite for dephosphorylation of the pool of pSer-68-PLM which regulates NCX1.

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1.2.4.4 Regulation by phospholemman (PLM)

PLM is a sarcolemmal phospho protein that was purified and cloned in 1991 (120). Early studies on

PLM showed that it is an anion-selective channel, which regulates cell volume in noncardiac tissues

(121). Later studies showed that it is part of the PFXYD family (named after a conserved Pro-Phe-X-

Tyr-Asp motif in the extracellular N-terminus domain) of regulators of ion transport, and this extended

the role of PLM to excitable tissue (122). PLM has been shown to be expressed in the heart, skeletal

muscle, brain, liver and kidneys.

PLM, also known as FXYD1, is synthesized as a 92 amino acid peptide, with the first 20

amino acids being a signal peptide sequence that is cleaved off during processing to form the mature

protein containing 72 amino acid residues. The topology of the mature protein shows that it is

composed of the signature PFXYD motif, which is located in the extracellular N-terminus and is

resistant to protease degradation, a single hydrophobic transmembrane domain and a cytosolic tail

(120). The cloned complementary deoxyribonucleic acid (cDNA) of PLM, from cardiac muscle,

revealed that it has a molecular weight of 8409 Dalton. On SDS-PAGE, however, it migrates with

molecular weight 15-kDa, due to the slow migration of the PLM transmembrane domain (123). In

human and rat, the cytosolic tail of PLM contains three serines (at residues 62, 63, and 68) and one

threonine (at residue 69) (Fig. 5A), but Thr-69 is replaced by serine in mouse (122). NMR showed that

-helices which are rigidly connected, where helix 3 and 4 are

connected by a flexible linker (Fig. 5B) (124). PKA phosphorylates Ser-68, whereas protein kinase C

(PKC) phosphorylates Ser-63 and Ser-68 of PLM (125). In vitro studies suggest that Ser-63 and Thr-

69 may be additional phosphorylation targets for PKA and PKC, respectively (126). Phosphorylation

of Ser-68-PLM has been shown to enhance palmitoylation at Cys-40 and Cys-42, leading to NKA

inhibition (127,128). Cys-42 can also be glutathionylated (129). The basal phosphorylation level of

PLM, by endogenous kinases, has been reported to be 30–40% in adult rat myocytes and HEK293

(47,130).

PLM is proposed as a novel cardiac stress protein which modulates myocardial contractility by

regulating the activity of proteins involved in excitation-contraction coupling. PLM regulates the

activities of LTCC (131), NKA (132) and NCX1 (130) in mammalian hearts. PLM modulates Ca2+

entry through LTCC in adult LV myocytes by reducing peak LTCC current amplitude and enhancing

voltage-dependent inactivation. Only the transmembrane and the extracellular (signature PFXYD

motif) domains of PLM mediates the regulation of LTCC (133). Unphosphorylated PLM exerts

inhibition on NKA, whereas phosphorylation relieves this inhibition (132). It is PLM’s transmembrane

domain (Phe-28) which interacts with TM9 (Glu- -subunit of NKA (134) and

phosphorylation alters the PLM-NKA interaction, but does not mediate disassociation of PLM from

NKA (135). PLM overexpression by adenovirus-mediated gene transfer decreased NKA current in

adult rat myocytes (136), whereas PLM knock-out mice expressing the phosphomimetic PLM Ser-68-

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Glu (S68E) mutant inhibited NCX1 current without any effect on NKA activity (137).

Unphosphorylatable PLM reduces NKA activity and exacerbates Na+ overload, resulting in contractile

dysfunction and adverse remodeling following aortic constriction in mice (138).

Figure 5. PLM domains and structure. A: PLM domain organization and human sequence is shown. PLM is composed of a signal peptide sequence (in blue) that is cleaved off during protein processing; the extracellular domain which has the signature FXYD motif (in bold red) which is located in the extracellular N-terminus; a single hydrophobic transmembrane domain (in grey) and a cytosolic tail with the palmitoylation site at Cys-40and Cys-42, glutathionylated sites at Cys-42 and phosphorylation sites at Ser-63, Ser-68 and Thr-69 (in bold). B: Ribbon diagram of PLM -helices which are rigidly connected where helix 3 and 4 are connected by a flexible linker (PDB ID: 2JO1).

The role of PLM in NCX1 regulation was first described by Song et al. 2002, where they

found that overexpression of PLM altered Ca2+ transients and reduced contractility (136). This led to

speculations that PLM directly regulates NCX1 independently of NKA (136). Ahlers et al. 2005

identified PLM as an endogenous inhibitor of cardiac NCX1 (139), specifically that it is the

phosphorylation of PLM at Ser-68 which mediates this inhibition (125,130). The PLM binding region

on NCX1 was constrained to the PASKT (residues 248–252) and QKHPD (residues 300–304)

containing sequences, and mutating these binding regions resulted in loss of PLM induced NCX1

inhibition in HEK293 (49). The possibility that PLM indirectly regulates NCX1 via Ca2+ was excluded

by the fact that NCX1 lacking both CBD1 and CBD2, expressed in HEK293, were still inhibited by

PLM (140). Upon examination of the now known PLM binding region in NCX1, this finding is

explained by the fact that the PLM anchoring site on NCX1 is outside the CBDs. The physiological

significance of PLM is shown under stress conditions (141): Relieved inhibition of NKA by pSer-68-

PLM can prevent Ca2+ and Na+ overload, which can reduce the risk of arrhytmogenesis. The latter

reduces contractility, but this is counteracted by simultaneous inhibition NCX1. The PLM-NCX1

mechanism can therefore serve to improve or maintain contractility under stress conditions.

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In paper 3 of this thesis we designed a high-affinity peptide for PLM, which is derived from NCX1,

and investigated its role in disrupting the PLM-NCX1 interaction.

1.2.4.5 Regulation by other cytosolic factors

Various cytosolic factors interact with the cytosolic loop of NCX1 and mediate regulation of the

exchanger. We only mention the factors where the interaction site in NCX1 has been mapped down to

amino acid level. Annexin V, has been shown to interact with the Ca2+-activation site (371-525) in

NCX1, where this interaction locally decreases the amount of Ca2+ available for NCX1 (142,143).

Calmodulin-1-3 interacts with the XIP domain (251-270) (31) and inhibits NCX1 activity (31). Further,

calmodulin-1-3 binds to the 1-5-8-14 calmodulin binding segment motif in NCX1 (716–735) (144)

and releases the auto-inhibitory domain which enhances NCX1 transport activity. Caveolin has been

shown to interact with the endogenous XIP site in NCX1 (145) and forms part of a macromolecular

complex with annexin A5. Creatine kinase M-and S-type interact with NCX1 (231-381) and NCX1

(226–380) respectively, and this interaction recovers reverse-mode NCX1 activity under energy-

compromised conditions (146). As mentioned above (section 1.2.4.4), PLM interacts with PASKT

(248-252) and/or QKHPD (300-304) in NCX (49), and phosphorylation of Ser-68-PLM inhibits

NCX1 activity (125,139,141). Calcineurin interacts with NCX1 (407–478) and decreases NCX1

activity (147). NCX1 XIP region also interacts with phospholipid, PIP2, which activates NCX1, as

PIP2 eliminates the Na+-dependent inactivation (148). Phosphorylation of NCX by different protein

kinases, such PKA (149) and PKC (150) has been demonstrated in the heart to increase NCX1 activity,

however other laboratories have failed to see these kinase dependent effects (42,151). Our laboratory

has shown that the PKA phosphorylation site in NCX1 is inaccessible for phosphorylation in vitro (95).

Cys-739 in NCX1 has been shown to be palmitoylated in cardiac muscle, and it is suggested that

palmitoylation is required for the XIP domain to fully inhibit NCX1 exchange activity. This shows

that palmitoylation regulates NCX1 inactivation (152). In paper 4 of this thesis we used a proteomic

approach to screen for novel cytosolic factor which may interact with NCX1 and mediate its

regulation.

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2 Aims of the thesisNCX1 participates in numerous physiological and pathophysiological processes. Altered expression,

regulation and activity of NCX1 contributes to distorted Ca2+ homeostasis in cardiomyocytes affecting

excitation-contraction coupling. There are no selective NCX1 modulators that are in clinical use, and

pharmacological targeting of NCX1 has been desired for a long time. In this thesis we identify and

validate novel NCX1 interacting partners. We elucidate the mechanism of regulation and design a new

pro-drug.

Paper 1

Molecular Basis of Calpain Cleavage and Inactivation of the Sodium-Calcium Exchanger 1 in

Heart Failure

NCX1 has previously been identified as a substrate for Ca2+ activated proteases such as calpains,

however, little was known about the precise molecular mechanisms and biological consequences of

calpain-dependent cleavage of NCX1. In this study we hypothesized that calpain is an important

regulator of NCX1 in response to pressure overload. We aimed to identify the molecular mechanisms

and functional consequences of calpain binding and cleavage of NCX1 in conditions of hypertrophy

and HF relative to normal heart.

Paper 2

Protein Phosphatase 1c Associated with the Cardiac Sodium Calcium Exchanger 1 Regulates Its

Activity by Dephosphorylating Serine 68-phosphorylated Phospholemman

Direct regulation of NCX1 by kinases is controversial; instead it has been suggested that

phosphorylation and dephosphorylation occurs on accessory proteins in the NCX1 macromolecular

complex. PLM, an accessory phosphoprotein, regulates the activity of both NCX1 and NKA when

phosphorylated at Ser-68. PP1 has been shown to modulate pSer-68-PLM regulation of NKA, but it is

unknown whether this mechanism is involved in NCX1 regulation. In this study we hypothesized that

a direct and functional NCX1-PP1c interaction is a prerequisite for dephosphorylating the pool of

pSer-68-PLM which interacts with NCX1. We aimed to identify the molecular mechanisms and

functional consequences of the NCX1-PP1c-PLM interaction in the heart.

16

Paper 3

Development of a high-affinity peptide that prevents phospholemman (PLM) inhibition of the

sodium/calcium exchanger 1 (NCX1)

NCX1 has been shown to bind to pSer-68-PLM, which inhibits NCX1 activity. Other studies, however,

have failed to demonstrate a functional interaction of these two proteins. In this study we hypothesized

that NCX1 and PLM are direct, functional interacting partners. We aimed to design a high affinity

blocking peptide targeted towards the pSer-68-PLM-NCX1 protein interaction, and to analyze the

biological function of this interaction.

Paper 4

Mapping the in vitro interactome of cardiac Na+-Ca2+ exchanger 1 (NCX1)

Dysregulation of NCX1 is observed in human end-stage HF. Currently, no selective NCX1 modulators

are in clinical use. The cytosolic loop of NCX1 mediates its regulation by associating with various

cytosolic factors. In this study we hypothesized that elucidation of NCX1 protein interactions will

broaden our understanding of its regulation and this knowledge may be of use in developing specific

NCX1 modulators. We used a proteomic approach to screen for novel NCX1 interacting partners.

17

3 Methodological considerations This section discusses key aspects and considerations for the methods used in the presented papers.

Various molecular biology and biochemical techniques were employed on tissue from human patients

and animal models of cardiac disease for in vivo experiments. We also used primary cell cultures and

immortalized human cells for in vitro experiments. Computational methods were used for functional

annotation and construction of protein-protein interaction networks, while biophysical methods were

used to elucidate important mechanisms.

3.1 Human myocardial biopsiesTo quantify and demonstrate the clinical relevance of calpain dysregulation in paper 1, we used

explanted human left ventricle (LV) biopsies in western blot analysis. The LV biopsies were from

patients undergoing elective aortic valve replacement surgery for treatment of severe, symptomatic

aortic stenosis (AS). In AS, calcification of aortic valves results in aortic valve narrowing, which

constricts blood flow into the LV. This constriction increases afterload and ventricular wall stress,

which stimulates hypertrophy of the LV. This hypertrophic remodeling initially restores wall stress,

which preserves the LV function, however, over time, the heart is unable to maintain this high

pressure gradient. Reduced systolic function ensues and progresses to HF (153). Biopsies from AS

hearts therefore serve as a model for hypertrophy, suitable for studying how signaling is altered in

progression to HF. This is advantageous compared to using tissue from patients undergoing heart

transplants, as this usually represents end-stage HF. Obtaining control samples from healthy hearts is

not feasible due to the invasiveness of the procedures and ethical considerations. Instead, as negative

controls, we used biopsies from patients with coronary artery disease (CAD), undergoing coronary

artery bypass graft operation (CABG), as this represent a different pathology from AS. Specifically,

biopsies where taken from non-ischemic areas in CAD patients to ensure viable tissue that has not

progressed to HF. A major limitation with the use of human samples is the low sample numbers.

Informed written consent was obtained from all the patients. The protocol conformed to the

Declaration of Helsinki and was approved by the Regional Committee for Research Ethics in Eastern

Norway (Project 2010/2226).

18

3.2 Animal models Tissue from rat LV was used in all the papers contained in this thesis. Specifically, in papers 1-4,

molecular biology techniques were applied on wild type (WT) rat LV tissue to investigate normal

physiology.

In paper 1-3, the aortic banding (AB) rat model (a model for AS) was used to study molecular

changes that occur during the development of LV hypertrophy and progression to HF. As control we

did not use WT animals, instead we used banded animals, where the silk suture around the ascending

aorta was not tightened (sham-operated control animals). This controls for the effects related to the

surgery (technical control).

There are many advantages of using rats in cardiovascular research: Firstly, its physiology in

pathological conditions is similar to the corresponding human condition (154). Additionally, rat

models offer the opportunity to study molecular changes in cardiomyocytes under relatively controlled

experimental conditions compared to tissue obtained from human patients which may have

comorbidities. From a practical perspective, rats are inexpensive and require little space compared to

larger animals. They also have short life cycles and gestation periods which allow for the generation of

large sample sizes in a short period of time (155). There is good reproducibility of cardiac hypertrophy

with the use of rats, and a low mortality rate after the surgery, compared to for example mice, as rats

have ~10 times larger hearts (156). The large hearts also provide more tissue for biochemical

experiments. The use of rats is, however, not without limitations: One striking difference from humans

is that the heart rate in humans is 60-90 bpm, compared to 250-493 bpm in rats (157). This means that

the rats have a much shorter action potential than humans, indicating differences in the ion transport

during the excitation-contraction coupling. Indeed, more cytosolic Ca2+ removal is mediated by

SERCA2 in rats compared to humans (17,155). Another drawback of using rat models is that they do

not necessarily follow the usual disease progression as it happens in humans. The development of

heart disease in humans is influenced by exposure to various risk factors throughout the individual’s

life, but in animal models, disease states are induced over relatively short periods of time without the

accompanying comorbidities. Also, humans are often taking a cocktail of medicines and are typically

old, whereas young animals are used in experiments, hence the influence of medicines and age related

factors may not be properly simulated by such models.

All animal experiments were approved by the Norwegian Animal Research Committee (FOTS

ID 3820; 7714) and conformed to the Guide for the Care and Use of Laboratory Animals (National

Institutes of Health Publication No. 85-23, revised 1996).

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3.3 In vitro cell models In vitro mammalian cell models are the most commonly used source for protein research as they

minimize the use of live animals. In addition, they allow for the manipulation of cellular physiology in

order to elucidate molecular mechanisms. In the papers contained in this thesis we made use of three

mammalian cell lines: Neonatal and adult rat cardiomyocytes were used as a source of primary culture,

while human embryonic kidney 293 (HEK293) cells was the immortalized cell line used. Primary

cultures are cells that are freshly isolated from organs and consist of a heterogeneous population of

cells. Digestive enzyme (Collagenase type 2) is used to degrade the extracellular matrix of the heart. In

neonatal rat hearts, due to their size the hearts are treated with the enzyme and the tissue is gently

tweezed apart to yield the cells which are maintained in culture for up to five days, based on the

seeding density we employ (3.75 x 105 ml-1). The neonatal cells were isolated from 1-3 day old rats

and these cells were used to investigate the localization of the NCX1-calpain, NCX1-PP1c and NCX1-

PLM interaction, using the fractionation kit. In addition, we also used these cells to investigate calpain

cleavage after Ca2+ stimulation. The main advantage of these cells is that they are easier to culture and

provide a controlled environment for studying the effects of biochemical interventions. One drawback

is that these cells are not terminally differentiated. This means that the contractile machinery is not

developed. Therefore, we also made use of adult cardiomyocytes.

To obtain adult cardiomyocyte cells, the enzyme is perfused through aorta and coronary artery

using Langendorff apparatus. Adult cardiomyocyte cells are more difficult to culture, therefore freshly

isolated adult cardiomyocytes were used in experiments. These cells are differentiated, and subcellular

structures are completely formed. Since cardiomyocyte morphology changes with time, cells were

isolated and used in further assays on same day. Unlike neonatal cardiomyocytes, healthy isolated

adult cardiomyocytes are rod shaped, striated, and do not exhibit spontaneous contractions. However,

the isolation procedure is difficult, and cell quality and yield depend on the mounting of the heart on

the Langendorff perfusion system. There is also variation in the quality of the digestive enzyme

(collagenase) activity from batch to batch. To assess cell quality, we use visual inspection (cells must

be rod shaped, have clear striations, have intact membranes, look viable and not have spontaneous

contractions. One limitation is that results obtained from these cells cannot be extrapolated to tissue or

organ level.

Immortalized cell lines (HEK293) were used in loss-of-function and gain-of-function

experiments. A general advantage of using cell lines is that it allows for the study of cell responses in

isolation, without neurohormonal, mechanical and paracrine effects. Transfection of plasmids with the

Ca2+ method described in paper 2 is used to express a modified protein into the cells. Adult

cardiomyocytes have a low transfection efficiency, therefore HEK293 cells were used. The method

facilitated investigation of NCX1-specific effects, as HEK293 cells have been shown to be devoid of

20

endogenous NCX1 (158). An advantage of using HEK293 cells is their high yield in protein

expression (30-50%) (139), as adult cardiomyocytes have been shown to have a low transfection

efficiency when plasmids are used. They also facilitate reproducibility and are easy to maintain in

culture. HEK293 cells were also used in the functional analysis of NCX1 activity, using the patch

clamp method.

3.4 Biochemical and molecular biology techniquesSeveral molecular biology techniques were used to elucidate the biological function of the novel

NCX1 regulators.

3.4.1 Fractionation

In paper 1-3, we used subcellular fractionation to determine the distribution of NCX1 and its protein

partners. The commercially available compartment protein extraction kit (2145, Merck Millipore

Billerica, MA, USA) separates proteins into their various subcellular locations, based on their

solubility in different buffer-and detergent conditions. Western blot was used to visualize the

subcellular localization of the proteins. This method was employed early in our studies to determine if

the novel partner was in the same subcellular space as NCX1, which is in the membrane.

3.4.2 Co-immunoprecipitation (Co-IP) and pull-down assays

Co-IP’s (paper 1-4) and pull-down assays (paper 1-3) are popular techniques for identifying protein-

protein interactions. In Co-IP’s, specific antibodies are used to capture proteins that are bound to a

specific target protein and the protein complexes are visualized by Western blot. The method identifies

both direct and indirect protein binding partners (Fig. 6). One is more likely to achieve success with

high abundant and stable interactions. Immunoprecipitation is an inexpensive method, however each

interaction must be optimized in order to detect the bound proteins. Nonspecific binding is a problem

often encountered, but can be reduced by changing the ionic strength and detergent conditions of the

wash buffer used. One could also increase or decrease the amount of primary antibody used to capture

the complex. False negative results can be attributed to too stringent washing conditions or the protein

binding site overlaps with the antibody epitope. Antibody heavy and light chain contamination can

also obscure the results, but this interference can be eliminated by using heavy and light chain specific

secondary antibodies which unmask the heavy and light chain regions on polyvinylidene difluoride

(PVDF) membranes. Further, one could covalently couple the antibody to the beads. In all our Co-IP

experiments, species-specific non-relevant antibodies or blocking peptide (block antibody epitope)

was used as negative control. Immunoprecipitation, as it is normally performed, does not provide

quantitative data regarding the affinity or stoichiometry of an interaction, therefore other methods are

required to determine this.

21

Figure 6. Schematic illustration showing the principle of Co-IP. The IP antibody, specific for protein X, is immobilized on protein A/G beads. The latter is a purified A/G recombinant fusion protein that has been covalently immobilized onto agarose beads and enables the capture of antibodies from a wider range of species and isotypes. When the lysate is added to the immobilized antibody, protein X is captured and co-precipitates protein Y by directly binding to protein X (left figure) or binds indirectly through the bridge protein, protein Z (right figure). The protein complexes are visualized by Western blot.

A pull-down assay is an affinity purification method that is similar to immunoprecipitation

and can also be used to detect direct protein/peptide interactions. This method was used to validate the

interactions identified by the Co-IP and peptide array method. This method uses exogenous proteins

tags such as biotin, FLAG, histidine (His) and glutathione S-transferase (GST) as bait (159). In a

typical experiment, the monoclonal anti-biotin beads are incubated with biotinylated peptides and the

recombinant protein of interest (Fig. 7). As negative control the recombinant protein is incubated with

the monoclonal anti-biotin beads however the biotin peptide is not added. The protein complexes are

visualized by Western blot

Figure 7. Schematic illustration showing the principle of a pull-down assay. Anti-biotin beads are co-incubated biotinylated peptides. After which the recombinant proteins is added. Detection of the interaction is visualized by Western blot.

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3.4.3 Peptides

In paper 1-4 we made use of peptide arrays to map antibody epitopes and also to map NCX1 partner

interaction down to amino acid level. Soluble peptides were used to further validate binding by pull-

down assays.

The protein of interest is synthesized as 20-mer overlapping peptides on cellulose membrane

(Fig. 8). To investigate binding, the membrane is then incubated with recombinant protein partner,

biotinylated peptides in solution or an antibody. Binding is visualized by Western blot analysis and is

semi-quantitative.

Two dimensional peptide arrays, where the amino acids in the native peptide sequences were

systemically replaced with every possible amino acid, were used to develop the high affinity NCX1-

PLM disruptor peptide.

Several considerations must be kept in mind in the interpretation of peptide array data. 1)

Signal intensity of the spot is affected by homogeneity of the peptide synthesis. 2) Excess amount of

protein on the membrane might give non-specific binding. 3) Because the protein is synthesized as a

linear sequence, this might create false binding sites as sites which are inaccessible in the folded

protein become accessible.

Figure 8. Schematic representation of a peptide array synthesis. A linearized protein sequence of the protein of interest is synthesized as overlapping peptide fragments on a membrane. Each spot represents the overlapping peptide sequence of certain length e.g. 20 amino acids with a 3 amino acid offset.

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3.4.4 Proximity ligation assay (PLA)

The PLA (Duolink ®) together with confocal microscopy was used in paper 1 and 2 to visualize the

co-localization and interaction of NCX1-calpain (paper 1) and NCX1-PP1c (paper 2). In brief,

species-specific secondary antibodies (from different species), called PLA probes with a short

deoxyribonucleic acid (DNA) strand attached to it, are incubated with the cells after the primary

antibody incubation step. When the PLA probes are within 40 a nanometer range, the DNA strands

hybridize when an enzyme, ligase solution, is added. The signal is amplified via rolling circle

amplification using an amplification solution consisting of polymerase and nucleotides, after which

the cells are fluorescently-labeled for detection. The output signal is visualized as distinct fluorescent

spots (Fig. 9). This method extends the capabilities of the traditional co-localization in that it requires

interacting partners to be within a specified distance (less than or equal to 40 nanometer) in order to

produce a signal. In traditional colocalization, each species emits a signal independently and

irrespective of the distance to the interacting partner, and therefore the signals may appear to overlap

even if the species are far apart. However despite the 40 nanometer range requirement, this distance is

big compared to the resolution we can achieve with recent modern microscopy techniques. Use of

appropriate negative controls is also important. We made use of a blocking peptide which makes the

epitope inaccessible, and also performed the experiment by omitting the secondary antibodies (PLA

probes).

Figure 9. Principle of Duolink. Target antibodies (primary) from different species against the two proteins of interest are co-incubated with secondary antibodies called PLA probes. When the PLA probes are within 40 nanometer range, the DNA strands on them hybridized. The signal is amplified and fluorescently-labeled for detection. The output signal is visualized as a distinct fluorescent spots. Reprinted with permission from Sigma-Aldrich Company Ltd.

3.4.5 Tobacco etch virus (TEV) proteaseThe TEV protease was used in paper 2 and 3 to simulate calpain cleavage of NCX1 without calpain

activation. This tool was used to investigate how cleavage of NCX1 regulates its activity, determined

using the whole-cell patch clamp technique (see section 3.6.2). TEV protease is a 27 kDa catalytic

domain of the ‘nuclear inclusion a’ protein encoded by the tobacco etch virus (TEV).

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The sequence “ENLYFQG/S” was engineered into the residues which we had mapped to be the

calpain cleavage site in NCX1. Using the protease, the cleavage occurs between the conserved Q and

G or Q and S. The TEV sequence specificity is more stringent than other proteases such as thrombin

and factor Xa (159).

3.4.6 Phosphatase activity assay

In paper 2 the colorimetric assay for protein phosphatase activity, using a synthetic phospho-peptide

substrate and the malachite green reagent was used. This method was used to determine what effect

the NCX1-PP1c interaction had on the phosphatase activity. Proteins that interact with PP1 can affect

the activity of the phosphatase by serving as inhibitors, pseudosubstrate, substrates or substrate-

specifiers (102)

peptide substrate (RRApSVA) together with either a range of His-trigger factor-NCX1 cytosolic loop

(His-TF-NCX1cyt) recombinant protein or biotinylated NCX1 peptides in a phosphatase buffer

supplemented with 1 mM MnCl2 for 20 min at 37 °C. After the 20 minutes, 100 ml of malachite green

dye was added to the solution and incubated for 15 min at room temperature. The colored complex

formed upon the release of free phosphate was calculated using a standard curve created with various

concentrations of KH2PO4. A spectrophotometer (absorbance 650 nanometer) was used to detect the

release of free phosphate. We opted for this method, as opposed to the classic method, because it is

non-

mM, with a lower limit of detection of approximately 0.1 nM. Limitations to the method are that there

may be kinetic differences in the dephosphorylation reaction in the native protein versus the short

phosphopeptide (160).

been shown to have a comparable kinetic profile to native PP1. We also used PP1 inhibitor-2 as a

control for the assay.

3.4.7 Affinity-purification coupled to mass spectrometry

In paper 4, we used two different strategies of affinity-purification coupled with mass spectrometry

screens to identify novel NCX1 interacting partners in rat LV lysates. In the first screen we used anti-

NCX1 against endogenous NCX1. In the second screen, we used anti-His with His-TF-NCX1cytrecombinant protein as bait. After the affinity-purification, the samples were submitted for mass

spectrometry analysis.

In brief, mass spectrometry (MS) involves ionization of the material obtained from affinity-

purification, and sorting of the resulting ions according to their mass/charge (m/z) ratio, which allow

individual peptide-ion fragmentation and mass determination of the fragments. Combined with a

database of known proteins and the m/z spectrum of their fragments, statistical algorithms are used to

determine which proteins that are (likely) present in the samples. Mass spectrometry is a high-

throughput method, allowing detection of thousands of proteins in a single experiment. The method’s

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high sensitivity allows for detection of proteins whose expression may not be detectable with

molecular biology methods. A disadvantage is that MS also picks up many contaminants. Also, in

order to be detectable, peptides must be volatile/ionizable and featured in the input database. The

enzyme trypsin was used in our experiments to yield the peptides. If the lysine or arginine (trypsin

cleavage sites) is distributed too closely or too far apart in the sequence, trypsin will produce peptides

that are either too short or too long for proper database identification. For our experiments we used an

Easy nLC1000 (Sunnyvale, CA, USA) system connected to a quadrupole–Orbitrap (QExactive)

(Thermo Electron, Bremen, Germany) mass spectrometer equipped with a nanoelectrospray ion source

with direct junction (Thermo Scientific). The Orbitrap traps ions in an orbital motion around an

electrode, and angular frequency depends on the mass to charge ratio through the formula

where k is a force constant. The trapped ions induce a detectable image current on an

outer electrode, and a Fourier transformation is used to convert this detected signal into mass spectra.

3.5 Computational methodsBioinformatics is a field of research at the intersection of biology, computer science, and mathematics.

Several bioinformatics tools were employed in the thesis. In paper 1, the CaMPDB (Calpain for

Modulatory Proteolysis Database) with PSSM and SVM (RBF Kernel) prediction models were used to

identify putative calpain cleavage sites in the cytosolic region of NCX1.

In paper 2, the Protein Pattern Find-Bioinformatics.org database was used to screen the NCX1

primary sequence for the following PP1 binding motif consensus sequence ([RK].{0,1}[VI].[FW]).

NCX1 sequences were also

by Choy et al. 2014 (108). In the program, the FASTA sequences were imported into the text area and

the [RK].{0,1}[VI].[FW] search pattern was used. The search pattern input seems to be case sensitive

and should be as written here. Other programs such as Scansite.mit.edu software can also be used.

In paper 4, the Database for Annotation, Visualization and Integrated Discovery (DAVID),

version 6.7, was used for functional enrichment analysis of protein partners. This tool is used on large-

scale gene lists to get information about Gene Ontology (GO), including biological process, cell

compartment and molecular function ontology. Additionally the Kyoto Encyclopedia of Genes and

Genomes (KEGG) are used for extracting the pathway information from molecular interaction

networks.

To construct the cardiac NCX1 PPI in vitro map, interactions between proteins were obtained

using the Search Tool for the Retrieval of Interacting Genes (STRING). This database provides

information on experimentally verified and predicted protein interactions by calculating their

combined score.

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Computational methods are useful for predicting e.g. binding sites and important motifs,

however, this data must be verified by complimentary experiments. In the first two methods, a linear

NCX1 sequence was used as input to predict the cleavage site and PP1c binding motifs. These

programs do not take into account the protein’s secondary structure and may predict a site which is

inaccessible in the folded protein. A limitation of the DAVID and STRING database is that not all

proteins are annotated.

3.6 Biophysical methods

3.6.1 Surface plasmon resonance (SPR)

SPR technology was used in paper 2 for the NCX1 and PP1c molecular interaction. The principle of

SPR is highlighted in Fig. 10. The SPR technology is integrated in the microfluidic system of the

Biomolecular Interaction Analysis (BIAcore) instrument. This technology is label free and allows for

real time

(161). The system delivers high quality kinetic, affinity, concentration, specificity, selectivity and

thermodynamic interaction data in real time, with high sensitivity. However, the method is not without

limitations. Extensive optimization is needed for each interaction, where buffer conditions, pH, ligand

immobilization and sensor chip choice are important considerations (162). For our interaction we

could not use the standard BIAcore reagents, and therefore we used IP-buffer, which has an optimized

pH, detergent and salt concentration, supplemented with 1% BSA, to block nonspecific interactions,

for the desorb step. We also used sensor chip CM5 (general purpose use) where the ligand is

covalently coupled to the chip. All data is background subtracted, where a reference chip is used

without immobilizing the ligand and the response is recorded. It must be kept in mind that the

carboxymethylated dextran matrix on the chip may occupy the important binding sites or cause steric

hindrance, resulting in false results about interactions. As such, in cases where information about the

interaction is known, this should be employed in the selection of the sensor chip to be used. SPR is not

suitable for interactions with very fast kinetics as the rate of analyte binding may be limited by the rate

at which analyte can be delivered and alternative methods should be employed.

There are alternative methods which give kinetic information about molecular interactions

such as, Isothermal Titration Calorimetry (ITC), which measures the heat change that occurs when two

molecules interact. Unlike SPR, there is no possibility of steric hindrance as interactions are monitored

in solution. This method gives more information about the interaction, such as enthalpy, entropy and

stoichiometry.

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Figure 10. Principle of SPR. The ligand is immobilized on the sensor surface and sample buffer containing the analyte flows past in a flow cell. A wedge of polarized light is shone on the sensor surface, where some light is absorbed by electrons in the gold layer and some is reflected (the absorbance is dependent on the refractive index of the sensor surface). When analyte binds to the ligand it alters the refractive index, detecting interaction.

3.6.2 Patch clamp

In paper 1-3 we used whole-cell patch clamp technique (Fig. 11) to assess the functional significance

of the novel regulators on NCX1 activity. All electrophysiology experiments were done on NCX1

variants expressed in HEK293 cells, which were plated on glass cover slips 24-48 hours following

transfection. In this method a glass pipette is sealed to the surface of the cell, after which the

membrane enclosed by the pipette tip is broken off. Ionic currents passing over the entire plasma

membrane is recorded. This so-called whole-cell configuration also allows us to manipulation the

intracellular environment, by changing the ionic solution in the pipette. To facilitate the recording of

NCX1 current, we dialyzed the intracellular environment with a Ca2+ buffered cesium solution. The

NCX1 current was analyzed in both forward (negative of reversal potential) and reverse (positive of

reversal potential) mode using a voltage ramp protocol from 120 to -100 mV. The NCX1 current is

sensitive to nickel (Ni2+) and following initial control recordings we applied 5 μM NiCl to the cells.

By subtracting the Ni2+ sensitive current we could obtain the NCX1 current. The principle of the

method is to isolate a patch of membrane electrically from the external solution and to record current

flowing into the patch. This is done by pressing a glass pipette, filled with electrolyte solution, against

the surface of a cell and applying light suction which forms a seal with an electrical resistance. The

ramp protocol employed a membrane potential of 120 to -100 mV. The cell morphology of HEK293