Dale Sanders

9 March 2009

Module 0220502

Membrane Biogenesis and Transport

Lecture 15

Ion Channels

Aims:By the end of the lecture you should

understand…

• The principles behind the patch clamptechnique;

• What information about ion channels canbe extracted from single channelrecordings;

• What are the main classes of ion channel inbiology, and what they do;

• How structure relates to function in theShaker class of ion channel: permeability,voltage-sensing and inactivation.

Reading• Lodish et al (2008) Molecular Cell Biology, 6th

ed. pp.458-465 & 1006-1013

More detailed, highly readable original papers, all from the lab of RodMacKinnon:

• Doyle, DA et al. (1998) The structure of the potassium channel:molecular basis of K+ conduction and selectivity. Science 405:647

• Jiang, Y. et al. (2003) X-ray structure of a voltage-dependent K+

channel. Nature 423: 33 [See also succeeding article: p. 42.]

• Zhou M. et al. (2001) Potassium channel receptor site for theinactivation gate and quaternary amine inhibitors. Nature 411:657

Relies on Giga-Ohm seal between glass and membrane:

current forced through ion channels

Look at single-channel currents, effects of internal and external

regulators

Not readily applicable to most endomembranes

Patch Clamp – The Primary Technique ForStudying Activity of Single Ion Channels

suck

pullSeal(cell-attached)

(whole cell)

(outside-out)

(inside-out)

pull

What Single Channel Currents Look Like

1 pA = 10-12 A; [Current: I]

Ions/s = I.N/F, where N = Avagadro’s No.; F = Faraday Const.

Here = 1.2 x 107 ions/s

Single channel recording only possible because of the relativelyhigh turnover rate

Conformational change between Open & Closed states known as

Gating

O

C

2pA

K+ channelfromT-lymphocyte

100 ms

Questions we can Answer from SingleChannel Recordings

1. What opens the channel?

Normally one (or more) of 3 factors:

• Voltage: changing membrane voltagein depolarizing (+ve) or hyperpolarizing (- ve)direction opening

• Neurotransmitter: binds and activatesfrom outside cell

• 2nd messenger: binds at cytosolicsurface and activates.

Typical recordings:

Note: No change in open channel current, just in time open:

An increase in open-state probability of the channel

(a) Non-permissiveconditions

(b) Permissiveconditions

openings rare

openings

frequent

O

C

(pA)t (ms)

O

C

2. Which ions flow through the channel? [Ion selectivity]Clamp voltage across membrane patch and measure open channelcurrent as a function of voltage:

Construct “current voltage” (I-V) relationship.

e.g.

Erev = –59 mV

Erev = 0 mV

-60 –40 –20 20 40 60

Conclude K+ ions are flowing through this channel

Measure reversalpotential (Erev)

Erev should correspond to equilibrium potential (Eion) of one of theions in solution

Eion = RT/zF ln([ion]out/[ion]in)

Example 1: [K+]out = 100 mM; [K+]in = 100 mM; EK = 0 mV = Erev

Example 2, [K+]out = 10 mM; [K+]in = 100 mM; EK = -59 mV = Erev

12

I (pA)

V (mV)

8

-8

3. What is the single channel conductance?

Ohm’s law: Voltage = Current . Resistance

V = I . R

Thus I = V. 1/R

Since 1/R = conductance (g),

the Slope of the I-V relationship = conductance

Unit of conductance: Siemens (S = 1/ohm)

e.g. previous slide: Slope = 8 pA/50 mV

= (8.10-12) / (50.10-3)

g = 160 pS

4. What inhibitors block the channel?Can either current when channel opens, or probability thatin open state.

e.g. tetrodotoxin (Na+ channels);

tetraethylammonium (K+ channels)

5. Does the channel inactivate?In continued presence of activating stimulus, openings lessfrequent

O

C

t

stimulus

Inactivation is a negative feedback mechanism to prevent toomuch channel activity

Channel type and channel function: anoverview

A. Voltage–Gated Channels

(i) K+ channels

Physiological role:

Primarily, stabilization of negative membrane potential(Vm)

Vm approaches EK if K+ conductance dominant

e.g. animal cell plasma membrane:

[K+]in = 150 mM, [K+]out = 5 mM

EK = 59 log ([K]out/[K+]in) = -87 mV

(ii) Ca2+ channels

Physiological role:

Ca2+ uptake into cell,

especially in Ca2+ -mediated stimulus-response coupling.

(iii) Na+ channels

Physiological role:

Always, to provide a depolarization:

The thrust of many action potentials

B. Neurotransmitter-gated channels

Neurotransmitter Ionic selectivity Effect of opening Response

Acetyl choline Cations Depolarization Action potential

Glutamate Cations Depolarization Action potential

-aminobutyric acid

(GABA) Cl- Hyperpol Inhibits a.p.

Glycine Cl- Hyperpol Inhibits a.p.

5-hydroxytryptamine

(5-HT) Cl- Hyperpol Inhibits a.p.

Cation channels are relatively non-selective among cations:physiologically carry mainly Na+ and Ca2+

C. Second messenger-gated channels

(i) Ca2+ - activated K+ channels

Restore membrane potential during Ca2+ signalling events

(ii) Cyclic nucleotide-gated (CNG) channels

Cation-selective: depolarizing signals in signal transduction:visual…… cGMP

olfactory… cAMP

(iii) Inositol 1,4,5-trisphosphate receptors (IP3R)

Ca2+-selective: release from ER during signal tranduction

Related to IP3Rs, on SR and ER ……

(iv) Ryanodine receptors RyR1 isoform interacts with p.m. Ca2+

channels in skeletal muscle

Other isoforms widespread: mediate Ca2+ release in cells: Ca2+-activated – give rise to Ca2+-induced Ca2+ release

Structure-Function Relations of IonChannels: Shaker-Type K+ Channels

Shaker class

K+: identified by chromosome walking in DrosophilaShaker mutant

Subsequently, related Ca2+ and Na+ channels purifiedfrom vertebrates by affinity chronatography with tight-binding inhibitors:

Channels are V-gated, and inactivate

Functional analysis: inject cRNA into Xenopusoocytes; measure currents after 3 – 4 days

Overall predicted domain structure of pore-forming (α) subunit from Hydropathy Analysis

K+ channel: 4 α subunits collectively form ion pore

Na+ and Ca2+ channels: each pore-forming subunit comprises 4α-likedomains

operate as monomers

S S S SS S1 2 3 4 5 6

N C

H5 or P loop

Ion Permeability and Selectivity

• The P loop forms the selectivity filter

A β –type 2o structure:

H5H5

H5

H5

S6

S6 S6

S5

S5

S5

S5

S6“Selectivity

filter”

Shaker K+ channel: sequence of P loop or H5 domain:

PD

AF

W

WA

VV

T M TT

VG

YG

DMTP

Evidence:• YG mutants lose ability to select for K+ over Na+

• A related Archeal channel has been crystallized: visualize K+

KcsA channel from Streptomyces lividans:Has only equivalent of S5, P loop, S6…

Drawing 9

“S5” “S6”

Aqueouspore

K+

ions

Ploop

Doyle et al. (1998) Science 405: 647

Pore with relativelyhydrophobic lining

+Helixwith dipole

2 K+ ions,mutually repulsive

K+ ionstabilizedby helicaldipoles

high turnover

Model for KcsA

K+ ions coordinatedby main-chaincarbonyl oxygens:high selectivity overNa+ (like valinomycin)

SF = selectivityfilter

Doyle et al. (1998) Science 405: 647

Voltage gating: voltage sensing is achieved by S4

S4 Sequence 'XXRXXRXXRXX(K/R)XXRXXKXX+ + + +

+

+

O

V

Sliding helix model

+ ve charges interact with – ve provided by other helices. Imposition of Vcases helix to swivel and project opening of gateSome evidence:Substitution of + ve residues changes voltage, but not permeation properties

+

Crystallization of an Archael Channel (KvAP)

has led to the Voltage Sensor Paddle Model

Proposes V-sensor is S4 + part of S3

All moves through membrane, pulling S5,S6 away from

interacting S5,S6 on other subunits

Evidence: Strategically-positioned Cys residues reacted withBiotin in side- & voltage-dependent fashion

Closed Open

Membrane

depol-arisation

Jiang et al. (2003) Nature 423: 33

Inactivation is achieved by a “Ball-and-Chain”

at N terminus

Can be either α or β subunit - depends on channel type

Mimics action of quaternary amine inhibitors

e.g. tetraethylammonium (TEA)

Drawing 12

Aldrich (2001) Nature 411: 643

First 10 residues: always hydrophobic – interact with poreResidues 11 – 21: always hydrophilic with net positive charge;interact with aqueous protein surfaces

Evidence: Some from deletion mutants…

Although 4 ball-and-chains, only one needed for blockage.

Singlechannelcurrents

inactivation

noinactivation

Zagotta et al. (1990) Nature 250: 568

Summary

1. The patch clamp technique is an informative method forstudying the properties of single ion channels.

2. Patch clamp allows study of single channel properties

a. Gating b. Ionic selectivity

c. Conductance d. Pharmacology

e. Inactivation

3. Channels fall into 3 main classes

a. V-gated b. Neurotransmitter-gated

c. Second messenger-gated

4. Shaker-type channels have been structurally characterised

Important function–structure correlates…

a. Ion permeation & selectivity: P loop

b. Voltage-sensing: S4 + part of S3

c. Inactivation: N-terminus ball-and-chain