Ionic Currents Across an Axon Membrane Under Voltage Clamp

The membrane currents flowing in a squid giant axon during a maintained depolarization can be studied in an experiment like that shown in Figure 7-4.

Figure 7-4 A diagram of the current injected by a voltage-clamp amplifier into an axon in response to a voltage step from -70 to -20 mV.

In this case, the command voltage to the voltage-clamp amplifier is first set to be equal to the normal resting potential of the axon, which is about - 60 to -70 mV. The command voltage is then suddenly stepped to -20 mV, driving the membrane potential rapidly up to the same depolarized value. A depolarization of this magnitude is well above threshold for eliciting an action potential in the axon; however, the voltage-clamp circuit prevents the membrane potential from undergoing the usual sequence of changes that occur during an action potential. The membrane potential remains clamped at -20 mV.

What current must the voltage-clamp amplifier inject into the axon in order to keep Em at -20 mV? The sodium permeability of the membrane will increase in response to the depolarization and an increased sodium current will enter the axon through the increased membrane conductance to sodium. In the absence of the voltage clamp, this would set up a regenerative depolarization that would drive Em up near ENa, to about +50 mV. In order to counter this further depolarization, the voltage-clamp amplifier must inject a hyperpolarizing current during the strong depolarizing phase of the action potential. With time, however, the sodium permeability of the membrane declines, and the potassium permeability increases in response to the depolarization ofthe membrane. Normally, this would drive Em back down near EK. To counter this tendency and maintain Em at -20 mV, the voltage clamp then must pass a depolarizing current that is maintained as long as potassium permeability remains elevated. Thus, in response to a depolarizing step above threshold, the membrane of an excitable cell would be expected to show a transient inward current followed

Figure 7-5 A diagram of the current injected by a voltage-clamp amplifier into an axon in response to a voltage step from the normal resting membrane potential to the sodium equilibrium potential. The initial sodium current is absent because there is no driving force for sodium current when Em equals ENa.

Figure 7-5 A diagram of the current injected by a voltage-clamp amplifier into an axon in response to a voltage step from the normal resting membrane potential to the sodium equilibrium potential. The initial sodium current is absent because there is no driving force for sodium current when Em equals ENa.

by a maintained outward current. The voltage-clamp records of membrane current illustrating this sequence of changes are shown in Figure 7-4.

What was the nature of the evidence that the initial inward current was carried by sodium ions? This was demonstrated by measuring the membrane current resulting from a series of voltage steps of different amplitudes. As we have seen previously, if the clamped value of membrane potential were equal to the sodium equilibrium potential, there would be no driving force for a net sodium current across the membrane. Therefore, if the initial current is carried by sodium ions, that component of the current should disappear when the command voltage is equal to ENa. A sample of membrane current observed in response to a voltage step to ENa is shown in Figure 7-5. The initial component of inward current disappears in this situation, leaving only the late outward current. Hodgkin and Huxley went one step further and systematically varied ENa by altering the external sodium concentration; they found that the membrane potential at which the early current component disappeared was always ENa. This is strong evidence that the inward component of current in response to a depolarization is carried by sodium ions. This notion also agrees with early observations that the membrane potential reached by the peak of the action potential was strongly influenced by the external sodium concentration.

The two components of membrane current can be separated by comparing the current observed following a voltage step to a particular voltage when that voltage is equal to ENa and when ENa has been moved to another value by altering the external sodium concentration. A specific example is shown in Figure 7-6. In this case, voltage-clamp steps are made to 0 mV in ECF containing normal sodium and in ECF with sodium reduced to be equal with internal sodium concentration. In the normal sodium ECF, ENa will be positive to the command voltage; in the reduced sodium ECF, ENa will equal the command potential and there will be no net sodium current across the membrane. When the observed current in reduced sodium ECF is subtracted from the current in

(c) Subtract current in (b) from current in (a) to isolate sodium current at 0 mV.

0 mV

Command Restin9 £m voltage outward Membrane Î

outward Membrane Î

Figure 7-6 The procedure for isolating the sodium component of membrane current by varying external sodium concentration to alter the sodium equilibrium potential.

Figure 7-6 The procedure for isolating the sodium component of membrane current by varying external sodium concentration to alter the sodium equilibrium potential.

normal ECF, the difference will be the sodium component of membrane current in response to a step depolarization to 0 mV. This isolated sodium current is shown in Figure 7-6c. The membrane currents of Figure 7-6c can be converted to membrane conductance according to Equation (7-2), and the result gives the time-course of the membrane sodium and potassium conductances in response to a voltage-clamp step to 0 mV. This procedure can be repeated for a series of different values of command potential and ENa, generating a full characterization of the sodium and potassium conductance changes as a function of both time and membrane voltage. The increase in sodium conductance in response to depolarization is transient, even if the depolarization is maintained. The increasing phase is called sodium activation, and the delayed fall is called sodium inactivation. We will discuss activation first and return later to the mechanism of inactivation. The onset of the increase in potassium conductance is slower than sodium activation and does not inactivate with maintained depolarization. Thus, at least on the brief time-scale relevant to the action potential, potassium conductance remains high for the duration of the depolarizing voltage step.

This rather involved procedure has been simplified considerably by the discovery of specific drugs that block the voltage-sensitive sodium channels and other drugs that block the voltage-sensitive potassium channels. The sodium channel blockers most commonly used are the biological toxins tetrodotoxin and saxitoxin. Both seem to interact with specific sites within the aqueous pore of the channel and physically plug the channel to prevent sodium movement. Potassium channel blockers include tetraethylammonium (TEA) and 4-aminopyridine (4-AP). Thus, the isolated behavior of the sodium current could be studied by treating an axon with TEA, while the isolated potassium current could be studied in the presence of tetrodotoxin.

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