Sodium Inactivation

Recall that the change in sodium conductance following a maintained depolarizing step is transient. We have so far considered only the first part of that change: the increase in sodium conductance called sodium activation. We will now turn to the delayed decline in sodium conductance following depolarization. This delayed decline in conductance is called sodium inactivation. Following along in the vein used in the analysis of sodium and potassium channel opening, Hodgkin and Huxley assumed that sodium inactivation was caused by a voltage-sensitive gating mechanism. They supposed that the conducting state of the sodium channel was controlled by two gates: the activation gate whose opening we discussed above, and the inactivation gate. A diagram of this arrangement is shown in Figure 7-11. Like the activation gate, the inactivation gate is controlled by a charged gating particle; when the binding site on the gate is occupied, the inactivation gate is open. Unlike the activation gate, however, the inactivation gate is normally open and closes upon depolarization. If we keep the convention of the gating particle being positively charged, this behavior can be modeled by an arrangement with the inactivation gate and its binding site on the inner face of the membrane. Upon depolarization, the probability that a gating particle is on the inner face decreases, and so the probability that the gate closes will increase.

To study the voltage dependence of the sodium-inactivation process, Hodgkin and Huxley performed the type of experiment illustrated in Figure 7-12. They used a fixed depolarizing test step of a particular amplitude and measured the peak amplitude of the increase in sodium conductance that resulted from the test step. The test depolarization was preceded by a long-duration prepulse whose amplitude could be varied. As shown in Figure 7-12, they found that a depolarizing prepulse reduced the amplitude of the response to the test depolarization, while a hyperpolarizing prepulse increased the size of the test response. This implied that the depolarizing prepulses closed the inactivation gates of some portion of the sodium channels, so that those channels did not conduct even when the activation gates were opened by the

Activation gate

OUTSIDE

Resting

Activation gating particle

INSIDE

Activation gating particle

Inactivation gate

Inactivation gating particle on in binding site.

Plasma membrane

Inactivation gate

Soon after depolarization

Later after depolarization

Soon after depolarization

Later after depolarization

Figure 7-11 A diagram of the sodium channel protein, showing the gating particles for both the activation and the inactivation gates.

Figure 7-12 The procedure for measuring the voltage dependence of sodium channel inactivation.

Figure 7-12 The procedure for measuring the voltage dependence of sodium channel inactivation.

Peak gNa

in response

\

to test

\

depolarization

V_

f 0

Resting Em

Membrane potential during prepulse

Figure 7-13 The relation between amplitude of an inactivating prepulse and the peak sodium conductance in response to a subsequent test depolarization.

Figure 7-13 The relation between amplitude of an inactivating prepulse and the peak sodium conductance in response to a subsequent test depolarization.

subsequent depolarization; therefore, there was a smaller increase in sodium conductance during the test step. The finding that hyperpolarizing prepulses increased the test response suggests that the inactivation gates of some portion of the sodium channels are already closed at the normal resting potential; increasing Em causes those gates to open, and the channels are then able to conduct in response to the test depolarization. By varying the amplitude of the prepulse, Hodgkin and Huxley were able to establish the dependence of the inactivation gate on membrane potential. The relation between Em during the prepulse and the peak sodium conductance during the test depolarization is shown in Figure 7-13. Note that all the inactivation gates close when the membrane potential reaches about 0 mV, and that even a small depolarization can cause a significant reduction in the peak change in sodium conductance.

The time-course of sodium inactivation was studied by varying the duration of the prepulse, rather than its amplitude. With short prepulses, there was not much time for the inactivation gates to close, and the response to the test depolarization was only slightly reduced. With longer prepulses, there was a progressively larger effect. This relation between prepulse duration and peak sodium conductance during the test step is shown in Figure 7-14. It was found that the data were described by a single exponential equation, rather than the powers of exponentials that were necessary to describe the kinetics of sodium and potassium activation. Recall from the discussion of the voltage-dependent opening of the sodium channel that a single exponential is what would be expected if the state of the gate is controlled by a single gating particle. Thus, the closing of the inactivation gate seems to occur when a single particle comes off a single binding site on the gating mechanism. An equation analogous to Equations (7-6) and (7-8) can be written to describe the temporal behavior of the inactivation gate:

In this case, however, the parameter h decreases with depolarization; that is, upon depolarization, h declines exponentially from its original value (h0) to its

Figure 7-14 (a) The procedure for measuring the time-course of sodium channel inactivation by varying the duration of depolarizing prepulses. (b) The resulting exponential time-course of the closing of the inactivation gate of the sodium channel.

(b)

Figure 7-14 (a) The procedure for measuring the time-course of sodium channel inactivation by varying the duration of depolarizing prepulses. (b) The resulting exponential time-course of the closing of the inactivation gate of the sodium channel.

Duration of prepulse

final value (hj. The rate of that decline is governed by the rate constants, ah and bh, for movement of the inactivation gating particle through the membrane. As expected from the discussion in Chapter 6, the closing of the inactivation gate is slower than the opening of the activation gate, implying that the inactivation gating particle is less mobile (i.e., the rate constants are smaller).

Is there any reason to suppose that the activation and inactivation gates are separate entities, as drawn in Figure 7-11 and throughout Chapter 6? After all, we could get the same behavior of the channel with a single gate that first opens, then closes upon depolarization. There is evidence, however, that the processes of activation and inactivation of the sodium channel are controlled by distinct and separable parts of the channel protein molecule. If, for example, we apply a proteolytic enzyme, such as trypsin or pronase, to the intracellular membrane face, we can selectively eliminate sodium channel inactivation while leaving activation intact. The sodium current observed in such an experiment is shown in Figure 7-15. As we have seen previously, in the normal situation the sodium current first increases, then decreases after a step depolarization as the channels open and then close with a delay (Figure 7-15a). After applying a protease to the internal face of the membrane (Figure 7-15b), the sodium current increases upon depolarization, as before, but now the current remains on for the duration of the depolarization: the inactivation gate has been

(a) Normal Na current (b) After pronase treatment

inward inward

Figure 7-15 Removal of the inactivation gate by treating the inside of the membrane with a proteolytic enzyme, pronase. (a) Normal sodium current. The current rises (activates), then declines (inactivates) during a maintained depolarization. (b) Sodium current after pronase treatment. The current activates normally, but fails to inactivate during a maintained depolarization.

destroyed but activation is normal. This supports the idea that there are two separate gates controlling access to the sodium channel pore. It also suggests that the inactivation gate is on the intracellular part of the channel protein molecule, because the proteolytic enzyme is ineffective on the outside of the membrane.

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