The voltage clamp

The voltage clamp technique relies on Ohm ’s law to measure the flow o f ionic currents across the cell membrane.

O hm ’s law: V = IR

W h e re V = p o te n tia l d iffe re n c e o r v o lta g e , I = c u rre n t a n d R = re s is ta n c e

In a voltage clamp experiment the membrane potential (Vm) is kept constant (clamped) and set to a desired membrane potential (Vcmd) by the experimenter. The voltage clamp amplifier uses a negative feedback circuit to instantaneously inject current (I) into the cell, through a micro electrode o f resistance R, and hold the membrane at the set potential ( V h ) .

2.4.1. Two electrode voltage clamp

T h e tw o -e le c tr o d e v o lta g e c la m p (T E V C ) te c h n iq u e is u sed to record large currents p ro d u ced by m u ltip le io n c h a n n e ls in w h o le c e ll r eco r d in g , m o st c o m m o n ly in the

X e n o p i i s o o c y te e x p r e s s io n sy ste m (s e e fig u r e 2 .4 .1 .a.).

Figure 2.4.1.a. The voltage clamp circuit

A2

ME. ME,

Ref

F ig u re 2 .4 .1 .a. A gra p h ica l re p re s e n ta tio n of the TE V C circu it. W h e re A t re p re se n ts the high im p e d a n c e un ity-g a in p re a m p lifie r and A2, th e high g a in c u rre n t in je ctin g a m p lifie r of the A xo n v o lta g e c la m p a m p lifie r; MEv, re cordin g e le ctro d e ; ME, cu rre n t in je ctin g e le ctro d e ; ref, ba th g ro u n d A g /A gC I pe lle t; V i, p o te n tia l m e a su re d b y ME^; Vcmd. cla m p

c o m m a n d p o te n tia l.

T E V C u se s a fast, h ig h g ain current in je c tin g a m p lifie r (A 2 ) to p ro v id e a n e g a tiv e

fe e d b a c k sy ste m to ‘c la m p ’ the c e ll m em b ran e. T h e m em b ran e p o ten tia l (Vm) is

m easu red by the reco rd in g e le c tr o d e in serted in the m em b ran e (M E v o f r e sista n ce R y)

and c o n n e c te d to the h igh im p e d a n c e u n ity -g a in p re a m p lifie r (A1 ) w ith resp ect to

ground (Ref). The high impedance o f A1 ensures that the amplifier draws negligible current from the oocyte and that the potential reaching the input o f A1 ( V i ) is as close to

Vm as possible. A1 then passes on this signal to A2 for comparison with the set holding potential ( V c m d ) - Any change in V m , caused by the net flow o f ions across the

membrane through activated ion channels, triggers A2 to instantaneously inject current (I) into the cell through the current injecting electrode (MEj o f resistance Ri) inserted in another part o f the membrane and clamp the membrane at Vcmd-

The efficiency o f the clamp and the accuracy with which the amplifier reaches Vm = Vcmd for a given ionic current (current flow through activated ion channels) depends on the gain on the amplifier and the access resistance (current injection electrode resistance + cytoplasmic resistance).

For a given ionic current:

V = V ,_{m cmd} R a l l + A I + A

W h e re Vm is th e m e m b ra n e p o te n tia l (m V ), Vcmd is th e c la m p c o m m a n d p o te n tia l s e t on th e a m p lifie r, A is th e g a in o f th e

a m p lifie r, Ra is th e a c c e s s re s is ta n c e an d I is th e io n ic c u rre n t (e q u a tio n fro m T h e P ly m o u th W o rk s h o p H a n d b o o k: M ic ro e le c tro d e T e c h n iq u e s . p19).

In order to increase the accuracy o f the clamp we can increase the gain (A) o f the amplifier; pull electrodes o f lower resistance to reduce the access resistance (Ra) and reduce the expression o f the ion channel to reduce the ionic current (I). However, the access resistance is not eliminated and there will still be an error (see clamp errors section).

The speed at which Vm = Vcmd is reached will be affected by the membrane capacitance

( C m ) . When injecting current into the cell, the amplifier must first charge the membrane

capacitance before Vm = Vcmd with time constant x (see equation 2.3.4.b.).