Voltage-dependent gating of ion channels Prof. Keith S. Elmslie

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1 Voltage-Dependent Gating of Ion Channels Department of Pharmacology A.T. Still University Kirksville College of Osteopathic Medicine 1 The action potential (AP) E Na Threshold The N eur onal AP Sensory functions Motor functions Learning and memory Car diac muscle AP Powering blood flow Skeletal muscle AP Motor output of the CNS 5 ms E K Resting membrane potential 2 Voltage-dependent channels and the AP The action potential Na + & K + conductances Hodgk in and Huxley (1952) J Physiol 117: 500 3

2 Channel gating during the AP Depolariz ation Repolariz ation Threshold Afterhyperpolariz ation 4 Voltage-dependent ion channels Potassium channels (K V ) 40 genes Function: repolarize the action potential Sodium channels (Na V ) 9 genes Function: gener ate the action potential Calcium channels (Ca V ) 10 genes Function: tr anslate electrical signals into cellular action e.g. in the CNS, AP invading the nerve terminals activates Ca V to allow influx of Ca 2+, triggering neurotransmitter release e.g. in cardiac muscles, Ca 2+ influx through Ca V channels triggers Ca 2+ release from intracellular stores, resulting in muscle contraction Yu et al., (2005) Pharmac ol Rev 57: Different channels alter AP Neuronal act ion pot ential Na V K V Na V & K V channels Different channels alter AP Cardiac action pot ential Na V Ca V K V Na V, C a V and multi ple K V channels 6

3 Voltage-dependent ionic current Yarotskyy and Elms lie (2010) Unpublis hed 7 Charge movement triggers gating Hyperpolarized Depolarized Closed Open Voltage sensor Armstrong and Bez anilla (1974) J Gen Physiol 63: Measuring voltage sensor movement Gating current Yarotskyy and Elms lie (2010) Unpublis hed 9

4 Voltage-dependent gating current Yarotskyy and Elms lie (2010) Unpublis hed 10 Gating vs. ionic current What i s the source for g ati ng current & voltag e-dependent channel g ati ng? Yarotskyy and Elms lie (2010) Unpublis hed 11 Protein sequence reveals voltage sensor Human K V 1.2 potassium channel 1 MTVATGDPADEAAALPGHPQDTYDPEADHECCERVVINISGLRFETQLKTLAQFPETLLG 61 DPKKRMRYFDPLRNEYFFDRNRPSFDAILYYYQSGGRLRRPVNVPLDIFSEEIRFYELGE 121 EAMEMFREDEGYIKEEERPLPENEFQRQVWLLFEYPESSGPARIIAIVSVMVILISIVSF 181 CLETLPIFRDENEDMHGSGVTFHTYSNSTIGYQQSTSFTDPFFIVETLCIIWFSFEFLVR 241 FFACPSKAGFFTNIMNIIDIVAIIPYFITLGTELAEKPEDAQQGQQAMSLAILRVIRLVR 301 VFRIFKLSRHSKGLQILGQTLKASMRELGLLIFFLFIGVILFSSAVYFAEADERESQFPS 361 IPDAFWWAVVSMTTVGYGDMVPTTIGGKIVGSLCAIAGVLTIALPVPVIVSNFNYFYHRE 421 TEGEEQAQYLQVTSCPKIPSSPDLKKSRSASTISKSDYMEIQEGVNNSNEDFREENLKTA 481 NCTLANTNYVNITKMLTDV Six transmembrane segments (S1- S6) Positiv ely charged amino acids = Arginine (R) and Lysine (K) NCBI Referenc e Sequenc e: NP_

5 Segregated functions within the protein A single potassium channel α subunit Pongs (1992) Physiol Rev 72: S69 13 K V channels comprised of 4 α subunits Subunit 3 V region Subunit 2 V region Subunit 3 P region Subunit 4 P region Subunit 2 P region Subunit 1 P region Subunit 4 V region Subunit 1 V region Long et al., (2005) Scienc e 309: Na V channels have four domains Each domain is equivalent to one K V channel subunit Catterall et al., (2005) Pharmac ol Rev 57:

6 Ca V channels also have four domains Auxiliary subunits modify CaV channel function Catterall et al., (2005) Pharmac ol Rev 57: S4 homology Human Shaker-related potassium channel K V 1.2 S4 ILRVIRLVRVFRIFKLSRHSK NCBI Reference Sequence: NP_ Human skeletal muscle sodium channel Na V 1.4 S4 DI ALRYFRVLRALKTITVIPGLKT Na V 1.4 S4 DII VLRSFRLLRVFKLAKSWPTLNM Na V 1.4 S4 DIII ELGPIKSLRTLRALRPLRALSR Na V 1.4 S4 DIV LFRVIRLARIGRVLRLIRGAKGIR NCBI Reference Sequence: NP_ Human cardiac muscle calcium channel Ca V 1.2 S4 DI DVKALRAFRVLRPLRLVSGVPS Ca V 1.2 S4 DII GISVLRCVRLLRIFKITRYWSS Ca V 1.2 S4 DIII VVKILRVLRVLRPLRAINRAKG Ca V 1.2 S4 DIV SITFFRLFRVMRLVKLLSRGEG NCBI Reference Sequence: NP_ Evidence for S4 movement S4 DIV LFRVIRLARIGRVLRLIRGAKGIR C C C C C C C C = M ethanethiosulfonate (MTS) - ethyltrimethylammonium (ET) 18

7 Cysteine modification method S4 DIV LFRVIRLARIGRVLRLIRGAKGIR C Yang and Horn (1995) Neuron 15: Cysteine modification method (2) Membrane hyperpolarized Membrane depolarized MTSE T in the external solution Yang and Horn (1995) Neuron 15: S4 moves S4 DIV LFRVIRLARIGRVLRLIRGAKGIR C Hyperpolarized Depolarized Exter nal No effect Effect Inter nal No effect No effect Cysteine buried Cysteine exposed to the external solution Hyperpolarized Depolarized Yang and Horn (1995) Neuron 15:

8 The extent of S4 movement Depolarization S4 DIV LFRVIRLARIGRVLRLIRGAKGIR C C C C C C C C Outside Inside Hyperpolarization S4 DIV LFRVIRLARIGRVLRLIRGAKGIR C C C C C C C C Buried Inside 3 charges/domain * 4 domains = 12 charg es/channel, which matches gating current results (In this slide: Na V 1.4) Yang et al., (1996) Neuron 16: Similar conclusions for K V channels Depolarization S4 ILRVIRLVRVFRIFKLSRHS K Outside Not out Hyperpolarization S4 ILRVIRLVRVFRIFKLSRHS K Buried Inside Bezanilla (2000) Phy siol Rev 80: S4 is exposed to the cytoplasm Domain 3 Domain 2 Domain 4 Domain 1 has been removed Based on Yang et al., (1996) Neur on 16:

9 S4 moves through a gating pore Depolarized Based on Yang et al., (1996) Neur on 16: Pathogenic mutations & the gating pore G + G TTX DII + DIV Sokolov et al., (2007) Nature 446: Different functions within one domain Gate Gat ing pore Volt age sen sor Ionic pore The wor k of Hor n and others showed that S1-3 and S5 for m the structur e that all ows S4 to move acr oss the membrane el ectric fi el d 27

10 The activation/deactivation gate Catterall (2010) Neuron 67: Different models of S4 movement Cell Cell membrane Tombola et al., (2006) Annu Rev Cell Dev Biol 22: Summary Na V, Ca V and K V channels generate the cellular electrical signaling critical for functions as diverse as cardiac contraction & memor y A transmembrane segment called S4 contains positi vely charged amino acids that moves in response to changes in membrane potential Gating current is used to assess the movement of the voltage sensors S4 appears to move within a gating pore that is created by the other transmembrane segments (S1-3 & S5); As a result, mutations can cause an ionic current to flow through the gating pore that can lead to diseases such as Hypokalemic Periodic Paralysis The movement of the voltage sensor appears to alter the position of the S6 segment, which forms the gate, to open and close the ionic channels The exact way that S4 moves to trigger gating is currently an open question that is a ver y important area of research 30

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