Voltage clamp

[3] The concept of the voltage clamp is attributed to Kenneth Cole[4] and George Marmont[5] in the spring of 1947.

[6] They inserted an internal electrode into the giant axon of a squid and began to apply a current.

Cole discovered that it was possible to use two electrodes and a feedback circuit to keep the cell's membrane potential at a level set by the experimenter.

Cole developed the voltage clamp technique before the era of microelectrodes, so his two electrodes consisted of fine wires twisted around an insulating rod.

Because this type of electrode could be inserted into only the largest cells, early electrophysiological experiments were conducted almost exclusively on squid axons.

Squids squirt jets of water when they need to move quickly, as when escaping a predator.

The squid giant axon was the first preparation that could be used to voltage clamp a transmembrane current, and it was the basis of Hodgkin and Huxley's pioneering experiments on the properties of the action potential.

[7] Using experiments with the voltage clamp, Hodgkin and Andrew Huxley published 5 papers in the summer of 1952 describing how ionic currents give rise to the action potential.

[8] The final paper proposed the Hodgkin–Huxley model which mathematically describes the action potential.

The use of voltage clamps in their experiments to study and model the action potential in detail has laid the foundation for electrophysiology; for which they shared the 1963 Nobel Prize in Physiology or Medicine.

The feedback circuit passes current into the cell to reduce the error signal to zero.

The two-electrode voltage clamp (TEVC) technique is used to study properties of membrane proteins, especially ion channels.

[9] Researchers use this method most commonly to investigate membrane structures expressed in Xenopus oocytes.

[10] The TEVC method utilizes two low-resistance pipettes, one sensing voltage and the other injecting current.

The microelectrodes are filled with conductive solution and inserted into the cell to artificially control membrane potential.

Current readings can be used to analyze the electrical response of the cell to different applications.

[11] The two-electrode system is also desirable for its fast clamp settling time and low noise.

TEVC microelectrodes can provide only a spatial point source of current that may not uniformly affect all parts of an irregularly shaped cell.

[12] Gap junctions are pores that directly link two cells through which ions and small molecules flow freely.

Discontinuous single-electrode voltage-clamp (SEVC-d) technique is used with penetrating intracellular recording.

SEV-c has the advantage that you can record from small cells that would be impossible to impale with two electrodes.

However: A single-electrode voltage clamp — discontinuous, or SEVC-d, has some advantages over SEVC-c for whole-cell recording.

A SEVC-d amplifier operates on a "time-sharing" basis, so the electrode regularly and frequently switches between passing current and measuring voltage.

At the start of the next cycle, voltage is measured again, a new error signal generated, current passed etc.

The experimenter sets the cycle length, and it is possible to sample with periods as low as about 15 microseconds, corresponding to a 67 kHz switching frequency.

Switching frequencies lower than about 10 kHz are not sufficient when working with action potentials that are less than 1 millisecond wide.

Note that not all discontinuous voltage-clamp amplifier support switching frequencies higher than 10 kHz.

Thus, the frequency of the duty cycle is limited to the speed at which the electrode voltage rises and decays while passing current.

This error can grow with each cycle until the amplifier oscillates out of control (“ringing”); this usually results in the destruction of the cell being recorded.

The investigator wants a short duty cycle to improve temporal resolution; the amplifier has adjustable compensators that will make the electrode voltage decay faster, but, if these are set too high the amplifier will ring, so the investigator is always trying to “tune” the amplifier as close to the edge of uncontrolled oscillation as possible, in which case small changes in recording conditions can cause ringing.