Voltage-Gated K+ Channels

MK MathewNCBS, TIFRUAS – GKVK CampusBangalore IBRO Course in Neuroscience

Center for Cognitive Neuroscience & Semantics, University of LatviaRiga, LatviaAugust 21-August 29, 2013

PROPERTIES OF VOLTAGE-GATED ION CHANNELS

Voltage sensitive•Requires voltage-sensing apparatus•Requires transduction machinery

High Throughput: 108 ions/second•Protein supports an aqueous column traversing the membrane

Discriminates between Na+ and K+

•Requires a selectivity filter

1982 Noda … Numa Nature AChR a-subunit cloned from peptide information1983 Noda … Numa Nature All AChR subunits cloned1984 Noda … Numa Nature Na channel a-subunit cloned1987 Stuhmer .. Numa Eur J Biophys Xenopus expression Na channel1989 Stuhmer Conti .. Numa Nature S4 mutations in Na channel

Sodium Channel

1982 Noda … Numa Nature AChR a-subunit cloned from peptide information1983 Noda … Numa Nature All AChR subunits cloned1984 Noda … Numa Nature Na channel a-subunit cloned1987 Stuhmer .. Numa Eur J Biophys Xenopus expression Na channel1989 Stuhmer Conti .. Numa Nature S4 mutations in Na channel

1987 Kamb Iverson Tanouye Cell

1987 Papazian … Jan Science Shaker Cloning

!988 Pongs … Ferrus EMBO J

Xenopus oocytes

Two Electrode Voltage clamp

N H 2 C O O H

++++

T1

++++

N H 2

C O O HT12 0 m s-20 m V

+2 0 m V

+ 6 0 m V

5 0 0 n A

5 0 m s

hKv1.1

hKv1.40.0

0.2

0.4

0.6

0.8

1.0

1.2

P oten tia l (m V )

h K v 1 .1

h K v1 .2h K v 1 .4

Po

ELECTROPHYSIOLOGICAL PROPERTIES OF HUMAN K+ CHANNELS

-40 mV

0 mV

+40 mV

50ms

1A

hKv1.4

+ 40 mV500 nA

50 ms

- 40 mV

0 mV

Doyle et al., Science 1998; 280: 69

KcsA

Doyle et al., Science 1998; 280: 69 Jiang et al., Nature 2002 417; 523

Gary Yellen

Doyle et al., Science 1998; 280: 69

KcsA

Zhou .. MacKinnon (01) Nature

Varma & Rempe (2007) Biophys J 93:1093–1099

Zhou .. MacKinnon (01) Nature

Payandeh .. Catterall (2011) Nature

I II III IV selctivity

Ca E E E E Ca/Na~ 3000

Na D E K A Na/Ca ~ 1000

Ca start E E K E Na/Ca ~ 100

RajGanesh et al (2000) Proteins

Doyle et al., Science 1998; 280: 69

KcsA

Kv 1.2 Crystal Structure

MacKinnon ,2005

… Catterall (2006) PNAS

Pathak .. Isacoff (2007) Neuron

hKv1.1 FRIFKLSRHSKGLQILGQTLKASMRELGLLIFFLFIhKv1.2 FRIFKLSRHSKGLQILGQTLKASMRELGLLIFFLFIhKv1.4 FRIFKLSRHSKGLQILGHTLRASMRELGLLIFFLFIhKv1.5 FRIFKLSRHSKGLQILGKTLQASMRELGLLIFFLFIShaker FRIFKLSRHSKGLQILGRTLKASMRELGLLIFFLFIhKv2.1 LRILKLARHSTGLQSLGFTLRRSYNELGLLILFLAMKvAP LRILLIISRGSKFLSAIADAADKIRFYHLFGAVMLT :**: : :.. : . . *: .:

Sequence Alignment of The S4-S5 Region

S4 S5

Ken (1991) PNAS

hKv1.1 FRIFKLSRHSKGLQILGQTLKASMRELGLLIFFLFIhKv1.2 FRIFKLSRHSKGLQILGQTLKASMRELGLLIFFLFIhKv1.4 FRIFKLSRHSKGLQILGHTLRASMRELGLLIFFLFIhKv1.5 FRIFKLSRHSKGLQILGKTLQASMRELGLLIFFLFIShaker FRIFKLSRHSKGLQILGRTLKASMRELGLLIFFLFIhKv2.1 LRILKLARHSTGLQSLGFTLRRSYNELGLLILFLAMKvAP LRILLIISRGSKFLSAIADAADKIRFYHLFGAVMLT :**: : :.. : . . *: .:

Phenylalanine mutations in hKv 1.4Val/Ala mutations in Shaker

McCormack et al., (1991)Judge et al., (2002)

Mutant V1/2 (mV)

Wt 1±5V1 100±6V2 69±7V3 -5±2V4 -22±3V5 -7±7V2A3 32±5

McCormack et al (91) PNAS

V1/2

L1 L2 L3 L4 L5

Bezanilla (2000) Physiol Rev 80, 555 - 592

Schoppa Sigworth (98) JGP

Gating chargeSensor Movement

Po

Kv 1.2 Crystal Structure

MacKinnon ,2005

L312M-L326V DV1/2 = 0 mVV406L–L312M DV1/2 = -42 mVA305L-L312M DV1/2 = +18 mV

Sanjeev

Sanjeev, Nagarajan J Physiol

OPEN STATE

Sanjeev, Nagarajan

CLOSED STATE

CLOSED OPEN

Sanjeev, Nagarajan J Physiol

Ledwell Aldrich (99) JGP

Gating charge

Po

Ledwell Aldrich (99) JGP

Gating charge

Po

ACTIVATED STATE

CLOSED ACTIVATED OPEN

Sanjeev, Nagarajan J Physiol

Kaczmarek Nature Reviews Neuroscience 7, 761–771 (October 2006) | doi:10.1038/nrn1988

D.V. Ivanov et al./FEBS Letters 539 (2003) 156-160

KCNRG : K (Potassium) / CN (Channel) / RG (Regulator)

++++

T1

S1 S2 S3 S4 S5 p S6

Ball

T1KCNRG (monomer)n ?

0

0.5

1

1.5

hKv 1.4 hKv 1.4+KCNRGNo

rma

lize

d F

lou

res

ce

nc

e

* p < 0.05

*5um

Surface Expression Analysis

KCNRG reduces Surface targeting of hKv 1.4

Hyder (2010)

KS Krishnan 1976

Ken McCormack

YD RamuBaron ChandaAnurag VarshneyKavitha SSanjeev UpadhyayS NagarajanHyder Usman

5um