Abstract
Phenylalanine 1489 in the inactivation gate of the rat brain IIA sodium channel alpha subunit is required for stable inactivation. It is proposed to move into the intracellular mouth of the pore and occlude it during inactivation, but direct evidence for movement of this residue during inactivation has not been presented. We used the substituted cysteine accessibility method to test the availability of a cysteine residue substituted at position 1489 to modification by methanethiosulfonate reagents applied from the cytoplasmic side. Mutation of Phe-1489 to Cys results in a small (8%) fraction of noninactivating current. Ag+ and methanethiosulfonate reagents irreversibly slowed the inactivation rate and increased the fraction of noninactivating current of F1489C but not wild-type channels. Single channel analysis showed that modification slowed inactivation from both closed and open states and destabilized the inactivated state. Depolarization prevented rapid modification of Cys-1489 by these reagents, and the voltage dependence of their reaction rate correlated closely with steady-state inactivation. Modification was not detectably voltage-dependent at voltages more negative than channel gating. Our results show that, upon inactivation, Phe-1489 in the inactivation gate moves from an exposed and modifiable position outside the membrane electric field to a buried and inaccessible position, perhaps in or near the intracellular mouth of the channel pore.
Highlights
Treatment of the intracellular surface of Naϩ channels with proteolytic enzymes blocks inactivation, indicating that intracellular parts of the channel are required for inacti
It has been proposed that the intracellular loop between domains III and IV serves as an inactivation gate, which closes over the intracellular mouth of the pore and binds to a putative inactivation gate receptor via the IFM motif when the channel inactivates [1, 15, 18]
Co-expression of the mutant ␣ subunit F1489C together with the 1 subunit in Xenopus oocytes resulted in Naϩ channels with altered inactivation; 8% of the Naϩ current failed to inactivate by the end of an 11-mslong depolarization compared with 2% for the wild type (WT) channel (Fig. 1A)
Summary
Naϩ Channel Expression—Site-directed mutagenesis was performed as described in Ref. 15. Healthy stage V and VI oocytes were pressure-injected with 50 –100 nl of a solution containing a 1:1 ratio of ␣ and 1 subunits at a concentration of 10 –200 ng/l. Electrophysiological Recording—The excised inside-out configuration of the patch clamp technique [19] was used to obtain both macropatch and single channel data. The bath solution contained 10 mM NaCl, 140 mM KCl, 1 mM MgCl2, 10 mM EGTA, and 10 mM HEPES, pH 7.4. For treatment with Agϩ, the bath solution contained 150 mM potassium aspartate, 1 mM Mg(NO3) mM EGTA, and 10 mM.
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