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VOLTAGE CLAMPING

Voltage clamping is an experimental method that uses electrodes to alter the membrane potential. This simple concept allows us to test membrane conductance of ions at specific membrane potentials. It also allows us to measure the equilibrium potential of the membrane to certain ions. These voltage clamp tests are usually done on the squid giant axon, whose size facilitates such tests. These tests are usually plotted as a graph with the y-axis as the current (ion flow). Positive values for y indicate a flow of ions out of the cell, while negative values indicate a flow of ions into the cell. It is graphed against time in milliseconds (the x-axis). The graph below represents the flow of ions during an action potential at certain initial membrane potentials. At -20 mV the initial downward deflection represents the inward Na+ currents and the latter upward deflections represent the out flow of potassium. This makes sense if one remembers what happens during an action potential. As the membrane is depolarized the sodium ions pour into the cell, initially at a rapid rate. This rate increases to a maximum at about 0 mV at which point the influx begins to decline in magnitude. Notice in the graph the increase in inward sodium flow from -20 to 0 mV and the decrease from 0 to +50 mV.

At membrane potentials above +50 mV sodium actually exits the nerve cell. At +50 mV there is no sodium flow for a reason; this membrane potential must be at or near the equilibrium potential for sodium. Even though the chemical gradient favors an inward flow of sodium ions, the inside of the cell has become so positive that at the equilibrium potential the electrical gradient is equal and opposite to the chemical gradient. Thus anything above this value causes an efflux of sodium from the cell, while anything below this value will cause an influx of sodium in to the cell. It is important to note that the magnitude of the ion movement, is directly proportional to how far away the membrane potential is from the sodium equilibrium potential.

Voltage clamp analysis also gives key information on the timing of important events in the cell. For example it is evident that sodium influx is at a maximum at about 1 millisecond. Potassium efflux on the other hand is at a maximum at about 3 ms. What one can not tell from the above graph is the time at which the sodium influx ends, or the time at which the potassium efflux begins. This is because these two phenomenon overlap. Such timings can be tested however, and the easiest way is with the use of certain drugs. These drugs block the ion channels in which sodium or potassium diffuse through. Thus if the membrane is artificially excited (via voltage clamp) to induce an action potential, the blocked ions will not diffuse across the membrane. Thus the only deflection will be from the ion of choice. TTX (tetrodotoxin) effectively blocks the sodium channels in nerve cells. This is the same toxin found in pufferfish, that can cause a victim to become completely paralyzed and unable to breath. It does this obviously by blocking the sodium channels and effectively eliminating long distance neural communication and action potential generation. Because of this TTX is the drug of choice when testing the membrane?s conductance and timing as far as potassium is concerned. A drug called TEA (tetraethylammonium) will effectively block potassium flow and allow for the testing of membrane conductance and timing for sodium influx.

It is important to note that voltage clamping experiments were important in confirming and/or identifying many important neurophysiological events. The experiment also illustrates how important maintaining proper membrane potential is to the neuron. Relatively small changes in potential, can effect ion flow, and thus action potential generation.


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