Second-Messenger Gated Ion Channels

Tom Mast

Membrane Biophysics

10/5/07

What is a Second Messenger?

An intracellular signal produced in response to a stimulus

usually when a ligand binds a receptor

ex: cyclic nucleotides (cAMP or cGMP)

calcium

inositol 1,4,5 triphosphate (IP3)

diacylglycerol (DAG)

Classic Physiological Role of Second Messenger: cAMP in Rods

http://openwetware.org/wiki/BIO254:DarkNoise

Things to notice:

Amplification steps Modulation

Change in ion flow

Calcium

Feedback

Ligand binding

Second Messenger Channel Topics

Ion species flux- specific or not, calcium

Structure- subunit architecture

pore and selectivity filter

conformational changes

tetramerization

Ligand-binding- what is the ligand

conformation/ shape changeskinetics

Which Channels?

Cyclic gated nucleotide Channel A 2

Transient Receptor Potential C 2

Inositol 1,4,5 Triphosphate 1

Common Subunit Structure

http://www.ukbf.fu-berlin.de/pharma/agschaefermulti.html

Bosanac et al., BBA vol.1742 December 2004, 89-102

IP3 R1

Common Pore RegionChannel Pore and Selectivity

Sequence

Bovine Rod CNGC RKYVYSLYWSTLTLTTIG..ETPPPV

Catfish Olfactory CGNC FCYVYCFYWSTLTLTTIG..EMPPPV

Bacterial K KSA TYPRALWWSVETATTVGYGDLY.PY

Shaker K SIPDAFWWAVVTMTTVGYGDMT.PV

Mammalian IP3R1 LLMCIVTVLSHGLRSGGGVGDVLRK

Mammalian TRPC2 FNETFQFLFWTMFGMEEHTVVDMP

Other regions within the channels are similar (ie S4)Original channel may have been a 1 TMD Ca++Strong et al., Mol. Bio. Evol. 1993 (10) 221-242

Due to these relatively non-selective pore regions these channels flux cations mainly Na+ and Ca++

TRPC2 and IP3R1 Signaling

C. Badland

The TRP Channels

Notice: weak voltage sensor and ‘TRP’ box

Paper 1: TRPC2

Lucas P, Ukhanov K, Leinders-Zufall T, Zufall F

A Diacylglycerol-Gated Cation Channel in Vomeronasal Neuron Dendrites Is Impaired in TRPC2 Mutant Mice

Neuron. 2003 Oct 30;40(3):551-61.

Primary Question: Are DAG-induced currents present in VNO neurons?

Figure 1

A- sensory neuron in vitroB- inside-out patch: response to DAG analogueC- F I-V relationship of SAG-induced currents note: permeable to several ion species

outward current blockby large cation

Figure 2

inside-out patch: single channel responses to SAGA Low spontaneous opening w/o SAG B. increased openingC-D Frequency histograms of opening E I-V relationship of SAG-induced currents

Figure 3

Ligand specifity of the SAG-induced current

Important data: IP3 does not gate current neither do all fatty acids

Figure 4

Whole-cell currentsA. w/o SAG in WTB. w/ SAG in WTC. W/SAG in WT and bath application of large cationD-F. Same a A-C except in TRPC2 KOG. I-V relationship WTH. I-V relationship KOI. Histogram of SAG induced currents

Important Data: TRPC2 KO neuronsLack the SAG-induced current of WT

Figure 5

A. Whole cell recording in Current-clamp dilute urine activates neuronC-D. This activation has an I-V relationship similar to SAG-induced currents

Figures 6 + 7

Below:Phospholipase C inhibitor (U-73122)blocks the urine-induced current in voltage-clamped neurons

Above:DAG kinase inhibitor induces an inward Current which is abolshed by Phospholipase C inhibitor (U-73122) involtage-clamped neurons

Important data: in neurons pharmacologically increasing or decreasingendogenous DAG produces the predicted result

Conclusions

A urine induced current is non-selective for external cations

It is dependent on PLC

It is abolished by gene-targeted deletion of TRPC2

It closely resembles that of a SAG-induced current

Paper 2: IP3 R1

Hamada K, Terauchi A, Mikoshiba K.

Three-dimensional rearrangements within inositol 1,4,5-trisphosphate receptor by calcium.

J Biol Chem. 2003 Dec 26;278(52):52881-9.

Primary Question:

How do the allosteric factors Ca++ and IP3 effect conformational

changes in the channel?

Simple example of Allostery

Binding of a factor at one site alters other sites could be enzymatic activity, affinity, conformation

http://biology.fullerton.edu/biol302/regulation.html

Figure 1

A . Incubation of IP3R1 with a lysine-protease results in different fragment patterns dependent on Ca++ concentration B. Also dependent on C-term of cytoplasmic domain which is involved in tetramerization

C. Location of epitopes used in western analysis

Figure 2 + 3

Left: Mg++ does not affect proteolysis while Sr++ and (maybe) Ba++Does. IP3 does not affect proteolysis.Right: IP3R1 favors a ‘windmill’ shape in certain Ca++ concentrations

Figure 4

Figure 6

Modeling the 3-DShape of IP3R1Based on TransmissionElectron

micrographs (fig 4)

A. w/o Ca++B. w/ Ca++

Figure 7 + 8

w/o Ca++ w/ Ca++

Conclusions

IP3R1 is sensitive to Ca++

Channel-wide conformational changes are due

to Ca++ binding and not IP3

Tetramerization may play a role in the

conformational changes

Paper 3: CNGA2

Nache V, Schulz E, Zimmer T, Kusch J, Biskup C, Koopmann R, Hagen V, Benndorf K

Activation of olfactory-type cyclic nucleotide-gated channels is highly cooperative

J Physiol. 2005 Nov 15;569(Pt 1):91-102

Primary Question:

What is the allosteric model for cGMP binding to CNGA2?

Summary of the Canonical Cilia Cascade

CNCGs consist of three subunits: A2:A4:β1in a 2:1:1 ratio

A2 is required to detect most odors.

Calculating Cooperative Binding

An allosteric relationshipBinding of the first ligandchanges the affinity for

future ligands at other to other sites

h > 1 = positiveh < 1 = negative H cannot be greater than

the # of binding sites

Figure 1

A. Experimental set-up: inside-out patches exposed to light-sensitive cGMP

B. Light pulse

C. Example current

Figure 2

A. Example of current used for calculations

B-D [cXMP]-response curves with calculated parameters

Figure 3

A. Activation time-courses

B. Plot of time-constants

C. Plot of activation ratios

D. Voltage-effect

Figure 4

Activation w/cAMP is slowerAt a rate consistantW/ binding

Indicates changes in activation over[cGMP] is intrinsic

Figure 5

Channels in nativeRatios (2:1:1) haveSimilar activation whenCompared to CNGA2

Indicates kinetics are intrinsic

Figure 6

CNGA2 channels open spontaneouslyhas implications as to type of allosteric model

Figure 8

Activation kinetics are best fit by model with 3 binding stepsand with both negative and positive cooperativity

Conclusions

CNGA2 channels have a greater affinity for

cGMP

CNGA2 channels display cooperative binding

CNGA2 and hetereomultimer channels are affected by Vm