MOVEMENT OF THE NA+ CHANNEL INACTIVATION GATE DURING INACTIVATION

Phenylalanine 1489 in the inactivation gate of the rat brain IIA sodiu m channel cu subunit is required for stable inactivation. It is propos ed to move into the intracellular mouth of the pore and occlude it dur ing inactivation, but direct evidence for movement of this residue dur ing inactivation has not been presented. We used the substituted cyste ine accessibility method to test the availability of a cysteine residu e substituted at position 1489 to modification by methanethiosulfonate reagents applied from the cytoplasmic side. Mutation of Phe-1489 to C ys results in a small (8%) fraction of noninactivating current, Ag+ an d methanethiosulfonate reagents irreversibly slowed the inactivation r ate and increased the fraction of noninactivating current of F1489C bu t not wild-type channels. Single channel analysis showed that modifica tion slowed inactivation from both closed and open states and destabil ized the inactivated state. Depolarization prevented rapid modificatio n of Cys-1489 by these reagents, and the voltage dependence of their r eaction rate correlated closely with steady-state inactivation, Modifi cation was not detectably voltage-dependent at voltages more negative than channel gating. Our results show that, upon inactivation, Phe-148 9 in the inactivation gate moves from an exposed and modifiable positi on outside the membrane electric field to a buried and inaccessible po sition, perhaps in or near the intracellular mouth of the channel pore .