The selectivity filter of the voltage-gated sodium channel is involved in channel activation

Amino acids located in the outer vestibule of the voltage-gated Na+ channel determine the permeation properties of the channel. Recently, residues lin ing the outer pore have also been implicated in channel gating. The domain (D) IV P-loop residue alanine 1529 forms a part of the putative selectivity filter of the adult rat skeletal muscle (mu1) Na+ channel. Here we report that replacement of alanine 1529 by aspartic acid enhances entry to an ultr aslow inactivated state. Ultra-slow inactivation is characterized by recove ry time constants on the order of similar to 100 s from prolonged depolariz ations and by the fact that entry to this state can be reduced by binding t o the pore of a mutant p-conotoxin GIIIA, suggesting that ultra-slow inacti vation may reflect a structural rearrangement of the outer vestibule. The v oltage dependence of ultra-slow inactivation in DIV-A1529D is U-shaped, wit h a local maximum near -60 mV, whereas activation is maximal only above -20 mV. Furthermore, a train of brief depolarizations produces more ultra-slow inactivation than a single maintained depolarization of the same duration. These data suggest that ultra-slow inactivation emanates from "partially a ctivated" closed states and that the P-loop in DIV may undergo a conformati onal change during channel activation, which is accentuated by DIV-A1529D.