Lecture 10: Membrane Lecture 10: Membrane potential and ion channelspotential and ion channels

Fain ch 3 end10/5/09

Telomere - protects Telomere - protects chromosome endschromosome ends

Chromosomes degrade w/o Chromosomes degrade w/o telomeretelomere

Telomerase adds Telomerase adds telomerestelomeres

QuestionsQuestions

1. How do you follow membrane potential?

2. What can you learn from evolutionary relationships of ion channels?

Example - Membrane Example - Membrane potential in hair cellspotential in hair cells

Important systemsAuditory VestibularLateral line

Hair cell responds to mechanosensationBending causes electrical response

Bullfrog inner earBullfrog inner ear

Very accessible Use the

sacculusLarge hair cellsResponds to head movement (slow frequency)May respond to sound

Frog sacculusFrog sacculus

Maculus is sensory epithelium (location marked by | | |)

Hudspeth and Corey 1977Hudspeth and Corey 1977

Hair cells of inner ear (bull frog)

BundlesKinociliumStereocilia -

microvilli

HC = hair cellSC = supporting cellArrows point to kinocillium

Hudspeth and Corey 1977Hudspeth and Corey 1977

Remove otolithic membrane (OM) to reveal hair cells

Use stimulus probe (SP) to perturb hair cell

Record intracellular potential with microelectrode (ME)

Hair cell motionHair cell motionTowards kinocilium Depolarize

Inside cell less negative

Away from kinocilium Hyperpolarize

Inside cell more -

Sideways motion had no effect

Depol >> Hyperpol

Fain fig 3.11

How can we explain this How can we explain this result?result?

Are channels opening or closing?

What ions are moving?

Cell membrane contains ion pumps Cell membrane contains ion pumps and channels - create concentration and channels - create concentration

gradientsgradients

Na/K ATPaseOutside cell Inside cell

Na+

K+

15 mM

120 mM

Na+

K+

141 mM

3.3 mM

Pump sends NaPump sends Na++ out outChannel lets NaChannel lets Na+ in+ in

Na/K ATPaseOutside cell Inside cell

Na+

K+

15 mM

120 mM

Na+

K+

141 mM

3.3 mM

NaNa++ pumped out

NaNa++ flows in through open channel

Pump sends KPump sends K++ in inChannel lets KChannel lets K+ + outout

Na/K ATPaseOutside cell Inside cell

Na+

K+

15 mM

120 mM

Na+

K+

141 mM

3.3 mM

KK++ flows out through open channel

KK++ pumped in

Possible mechanismsPossible mechanisms

Motion rel kino

Away Toward

Cell Hyperpol Depol

Channel

Na+

Na+ channel

Possible mechanismsPossible mechanisms

Motion rel kino

Away Toward

Cell Hyperpol Depol

Channel

Close Open

Na+ Pump out Flow in

Na+ channel

Possible mechanismsPossible mechanisms

Motion rel kino

Away Toward

Cell Hyperpol Depol

Channel

Close Open

Na+ Pump out Flow in

Na+ channel

Motion rel kino

Away Towards

Cell Hyperpol Depol

Channel

K+

K+ channel

Possible mechanismsPossible mechanisms

Motion rel kino

Away Toward

Cell Hyperpol Depol

Channel

Close Open

Na+ Pump out Flow in

Na+ channel

Motion rel kino

Away Toward

Cell Hyperpol Depol

Channel

Open Close

K+ Flow out Pump in

K+ channel

Which is it?Which is it?K+

Na+

Voltage clampingVoltage clamping

Hold cell at fixed voltage

Measure current flow across membraneDirectionSize

Fig 3.13

Ohm’s lawOhm’s law V = I R

Voltage = current * resistance

Current = voltage / resistance

I = V / R

But conductance,g is 1/RI = V g

V

R

I

Cell is a resistance / Cell is a resistance / conductanceconductance

Resistance and conductance depend on how many channels are open

Measure current to learn about conductance

Fig 3.13

Voltage clampingVoltage clamping Current flow

Erev is potential at which no current flows Potential which balances ion concentration

gradient

Vm is membrane potential during stimulation

€

E rev =RT

FlnαNao +KoαNai +K i

€

i = g(Vm − E rev )

Calculate ECalculate Erevrev for hair cells for hair cells equally permeable to Naequally permeable to Na++ and and

KK++

Na/K ATPase

Outside cell Inside cell

Na+

K+

15 mM

120 mM

Na+

K+

141 mM

3.3 mM

€

E rev = 59mV ln140 + 3.3mM

15 +120mM= −1mV=1

For hair cells, because For hair cells, because EErevrev~0~0

€

i = gVm

Ion current is proportional to conductance

As stimulate hair cell, conductance changes

Voltage gated current is prop to conductance

€

Δg =ΔimVm

Current flow directionCurrent flow direction

Fig 3.14

Displace toward kinocilliumDepolarization

Vm positive, current is positiveCurrent flows out

Vm negative, current is negativeCurrent flows in

Vm

Hair cell stimulusHair cell stimulus

Conductance change

So movement towards kinocillium increases conductance €

Δg =ΔimVm

=−current

−60mV= positive

Hair cell stimulusHair cell stimulus

Conductance change

So movement towards kinocillium increases conductance Channels openNa channels!

€

Δg =ΔimVm

=−current

−60mV= positive

Evolution of ion channelsEvolution of ion channels

How are different ion channels related?

What are structural similarities?

KK++ channel channel

Simplified 2TM channelSimplified 2TM channel

Roderick Mackinnon used the Streptomyces lividans channel in his Xray crystallography studies

Found it was similar to vertebrate K+ channels because both are blocked by neurotoxins

Only need 2 transmembrane TM regions and the pore region

KK++ ion pore formed from 4 ion pore formed from 4 subunitssubunits

Ion selectivity determined Ion selectivity determined by S5, S6 and poreby S5, S6 and pore

S1-S4 adds channel gatingS1-S4 adds channel gating

How channels are gated by How channels are gated by voltagevoltage

Nature 423 (2003) 42-8

Voltage sensitive paddles - Voltage sensitive paddles - move to open and close move to open and close

channelchannel

Large motion of S4 helix in Large motion of S4 helix in response to charge : response to charge :

Arginines (+)Arginines (+)

Family Family of ion of ion

channelschannels

Label Ion :K, Na, Ca How channel is gated: voltage Ca

Root is Root is likely likely

the 2TM the 2TM channelschannels

TM channelsTM channels

Bacteria group A

Bacteria group B

Bacteria group C

Verts+inverts

Verts+inverts

Gain of S1-S4 enables Gain of S1-S4 enables voltage gatingvoltage gating

Bacteria group A

Bacteria group B

Bacteria group C

Verts+inverts

Verts+inverts

Some Some species species

have have 4x6TM 4x6TM regions regions

Voltage gated sodium channelVoltage gated sodium channelResult of gene duplication and Result of gene duplication and

fusionfusion

Multimeric channelsMultimeric channels

Bacteria

Yeast

Jelly, cnidarians, inverts

Verts+inverts

Verts

Na+

Ca+

Na+

Phylogenies of different Phylogenies of different channelschannels

What would be difficult about building a tree comprised of these kinds of genes?

CNG CNG channels channels

are are importaimportant for nt for vision vision and and

smellsmell

Ion channel summaryIon channel summary

Structure and function reasonably well understood

Domain and gene duplications followed by fusions played role

Diversity of ways to gate channels

Crystal structure of KCrystal structure of Kvv channel in open statechannel in open state