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Voltage Clamp Technique





To understand the classical voltage clamp experiments and to illustrate how these measurements were used to formulate the kinetics of voltage activated ion channels responsible for excitation (Na+, K+, fast gates, slow gates, etc.)


Voltage Clamp protocol:


The voltage clamp technique was used by Hodgkin and Huxley to determine the behavior of the ionic conductances responsible for the generation of the action potential. The basic circuit for the squid axon is shown below.






The voltage clamp apparatus consists of a Feedback Amplifier, a Voltage Amplifier, and an Ammeter. The Voltage Amplifier is connected to a Voltage Electrode implanted inside the neuronal membrane, and to the Feedback Amplifier. The Feedback Amplifier is connected to a Current Electrode (C. E.). Finally, a Ground Electrode completes the feedback and voltage circuits through an ammeter to ground.
The Voltage Amplifier is responsible for monitoring the membrane potential, Vm, and transmitting its value to the Feedback Amplifier. The Feedback Amplifier is responsible for maintaining Vm at the value desired by the experimenter. The ammeter displays the magnitude and direction of current flow through the membrane (Im).


Information about Vm flows in only one direction, from the voltage electrode to the Voltage Amplifier, then to the Feedback Amplifier. Similarly, the clamping voltage is fed into the Feedback Amplifier by the experimenter. In contrast, current can - and will - be sent in both directions through the Im circuit by the Feedback Amplifier.


The first step in conducting a voltage clamp experiment is to set the value at which Vm is to be maintained. This value is termed the clamping voltage, and is entered into the Feedback Amplifier by the experimenter. Usually, the clamping voltage is entered into the feedback amplifier as a change in the membrane's potential (dVm) relative to its resting value, rather than as a specific Vm value.


As soon as the experiment is started by applying the clamping voltage to the axon's membrane, the Feedback Amplifier commences comparison of the actual Vm with the desired clamping voltage. If Vm deviates from the clamping voltage, the Feedback Amplifier uses Ohm's Law to calculate the value of Im required to return Vm to the desired value (recall that V = I×R = I/g), and adjusts Im based on the results of those calculations. Note that both the magnitude and the direction of the current are subject to control by the feedback amplifier, the direction being determined by whether the deviation from clamping Vm is a depolarization or hyperpolarization.


At rest the concentration gradient causing K+ to diffuse out of the cell is balanced by the electrical force (membrane voltage) acting in the opposite direction. Consequently, very little K+ leaks out.


When the voltage clamp is turned on, a small pulse of negative charge is delivered to the external membrane surface and an equivalent positive charge is delivered to the internal surface. This new charge is just sufficient to jump the membrane potential from -65 mV to 0 mV.


This new 0 mV membrane potential tending to force positive charge into the cell is too weak to balance the tendency of K+ to diffuse out of the cell. In addition, the depolarization of the membrane opens more K+ channels. K+ diffusing out of the cell would add positive charge to the outside and change the membrane potential, but the voltage clamp monitors E and prevents any change by adding one negative charge for each K+ that crosses the membrane out of the cell.


The value of the voltage clamp is due to the fact that with modern technology it is not possible to chemically measure the small amounts of K+ that enter or leave the cell within a fraction of a millisecond, but the charge delivered by the voltage clamp can be measured easily. If both Na+ and K+ channels are open then the measured voltage clamp current equals the sum of the individual Na+ and K+ currents.


The sensitivity and response time of the system are such that Vm can be maintained with a few mV (or even nanovolts) of the clamping voltage at all times. The ammeter then records the magnitude and direction of Im, These data allow calculation of time-dependent changes in gm, which is the purpose of the voltage clamp experiment.


The voltage clamp experiment is still being used extensively by researchers in their efforts to understand the function of excitable cells.




Cite this Simulator:

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