EXTERNAL PORE RESIDUE MEDIATES SLOW INACTIVATION IN MU-1 RAT SKELETAL-MUSCLE SODIUM-CHANNELS

1. Upon depolarization, voltage-gated sodium channels assume non-condu cting inactivated states which may be characterized as 'fast' or 'slow ' depending on the length of the repolarization period needed for reco very. Skeletal muscle Na+ channel a-subunits expressed in Xenopus laev is oocytes display anomalous gating behaviour, with substantial slow i nactivation after brief depolarizations. We exploited this kinetic beh aviour to examine the structural basis for slow inactivation. 2. While fast inactivation in Na+ channels is mediated by cytoplasmic occlusio n of the pore by III-IV linker residues, the structural features of sl ow inactivation are unknown. Since external pore-lining residues modul ate C-type inactivation in potassium channels, we performed serial cys teine mutagenesis in the permeation loop (P-loop) of the rat skeletal muscle Na+ channel (mu 1) to determine whether similarly placed residu es are involved in Na+ channel slow inactivation. 3. Wild-type and mut ant alpha-subunits were heterologously expressed in Xenopus oocytes, a nd Na+ currents were recorded using a two-electrode voltage clamp. Slo w inactivation after brief depolarizations was eliminated by the W402C mutation in domain I. Cysteine substitution of the homologous tryptop han residues in domains II, III and IV did not alter slow inactivation . 4. Analogous to the W402C mutation, coexpression of the wild-type al pha-subunit with rat brain Na+ channel beta(1)-subunit attenuated slow inactivation. However, the W402C mutation imposed a delay on recovery from fast inactivation, while beta(1)-subunit coexpression did not. W e propose that the W402C mutation and the beta(1)-subunit modulate gat ing through distinct mechanisms. 5. Removal of fast inactivation in wi ld-type alpha-subunits with the III-IV linker mutation I1303Q; F1304Q; M1305Q markedly slowed tile development of slow inactivation. We prop ose that slow inactivation in Na+ channels involves conformational cha nges in the external pore. Mutations that affect fast and slow inactiv ation appear to inter act despite their remote positions in the channe l.