Cellular Neuroscience (207)Ian Parker

Lecture # 2 - Ion channels: electrophysiology

Single ion channels

cytosol

extracellular

Molecular structure Physical structure Functional model Simplified model

Cell

membrane

Single channel kinetics

Transitions from open to shut are instantaneous

Mean open time is a fixed characteristic of the channel

Mean closed time shortens with increasing stimulus(e.g. depolarization or agonist concentration)

Single channel current depends on channel conductanceand electrochemical gradient for ion flow

Channel opening doesnot require energy source(ATP), so channel continuesto work in isolated membrane patch.

Energy for ion flow (current)comes from electrochemicalgradient across membrane

How big are single channel currents?

Amps (log scale)

1

10-3 (mA)

10-6 (A)

10-9 (nA)

10-12 (pA)

10-15 (fA)

100 W light bulb

calculator

action potential at nodeof Ranvier

e.p.s.c. (current evoked by 1 vesicleof neurotransmitter)

Single channel currents

One ion per ms

Limit of conventionalVoltage clamp

Single channel current and conductance• Because single channel current varies with membrane potential and ion gradient, a better measure is the

conductance of the channel (). This is a fixed characteristic (‘fingerprint’) of a given channel.

• = i / (V-Veq)

• (v = membrane potential : Veq = reversal potential for current flow through channel)

• Unit of conductance is the Siemen (S : 1/Ohm) : single channel conductances are expressed in pS

10050-50-100

Membrane potential (mV)

-1

1

Single channel current (pA)

Line in red shows the current/voltageRelationship for a single channel.

What ion(s) likely pass through the channel?

What is its conductance?

Range of channel conductances

Conductance (pS)

500

100

20

10

1Limit of presenttechnology

Maximal conductance of 3 Ao diameteraqueous pore

Ca2+-activated ‘BK’ K+ channel

Nicotinic channel

K+ channel in axon

Na+ channel in axon

Many channels in5-30 pS range

Store-operated Ca 2+ channels

Aqueous pore or carrier ?

Largest channels conduct 108 ions per second

Fastest enzymes and transporters have turnoverRates of 105 per sec (more typically 102-104)

So – ions transport must be by diffusion throughaqueous pore : now confirmed by structural data.

Recording the activity of single channels

‘Patch-clamp’ technique : Neher & Sakmann, 1976, (Nobel Prize1991)

Limitation of voltage-clamp is ‘noise’ generatedby large area of cell membrane. Patch-clampovercomes this by isolating currents from tinypatch of cell membrane. Sensitive circuit thenamplifies current through channel(s) in patch, whileclamping voltage of pipette fixed. Current through a single channel is too small to appreciably alterResting potential of cell, so potential across patch Remains constant.

A commercial patch-clamp amplifier

Patching onto cultured cells under a microscope

How can you see a channel to know to where to patch onto the membrane?

You can’t! It’s a blind fishing expedition, and takes a lot of patience.Sometimes you might catch one channel, sometimes many channelsand sometimes nothing. Getting only one channel is the ideal, as Records with more than one channel in the patch are hard to interpret.

How do you know if you catch more than one channel?

Sometimes you will see ‘double’ openings.

The ‘giga-seal’

Clearly, Rleak must be >> Rp for faithfulrecording. Rp is pretty much fixed (a few M Ohm) by the size of the tip (a few m).

Also, Rleak generates noisefrom thermal motion of ions, which decreases as Rleak increases.

So, the higher Rleak can be made, the better!

By using clean cell membrane (e.g. enzyme treatment to remove connective tissue, or by using cultured cells) the glass of the pipette actually sticks to the lipid membrane, forminga ‘giga-seal’ (Rleak > 1 G Ohm)

Seal formation is accomplished by gently pressing the tip of the patch-pipette againstthe cell membrane, then applying gentle Suction.

Gigaseal recording configurationsAn unexpected, but very useful discovery was that the pipette sticks so tightly after forming a

gigaseal that isolated patches of membrane can be pulled off intact from a cell.

‘cell-attached’ mode

Study single channelsin their intact cellularenvironment.

‘whole-cell clamp’

Voltage-clamp ofwhole cell, but canbe applied to little Cells (e.g. neurons)that are inacessibleto regular voltage-clamp

‘Inside-out’ excisedpatch.

Study single channelsisolated from cell. Cytosolic face is accessibleto bathing fluid, so can readily apply intracellularsecond messengers.

‘Outside-out’ excised patch

Study single channels isolated from cell. Extracellular face is accessible to Bathing fluid, so can readily apply neurotransmitters or other ligands.

What can patch-clamp recordings tell us?

Obtain long recording with hundreds of events (openings and closings), then measure amplitudes, open and closed times for each and plot distribution histograms

Distribution of single channel amplitudes

Current (i) through a channel is about the same everytime it opens (providing voltage is constant). However, measurement noise introduces some variability, sodistributions of channel amplitudes follow a Gaussianwith mean = i.

Mean channel open time is a characteristic of any particular type of channel, but individual openings vary randomly

Short openingLong opening

Random behavior gives rise to exponential distribution of open times (many short openings, few long openings) : analogous to radioactive decay

Time constant of decay ((time to fall to 1/eof anyInitial value) = mean opentimeExponential distribution

of open times on linearplot

Plotting on logarithmicy-axis transforms exponential distributionto linear

[We will talk about distribution of closed times in a future lecture]