The role of the putative inactivation lid in sodium channel gating currentimmobilization

We investigated the contribution of the putative inactivation lid in voltag e-gated sodium channels to gating charge immobilization (i.e., the slow ret urn of gating charge during repolarization) by studying a lid-modified muta nt of the human heart sodium channel (hH1a) that had the phenylalanine at p osition 1485 in the isoleucine, phenylalanine, and methionine (IFM) region of the domain III-IV linker mutated to a cysteine (ICM-hH1a). Residual fast inactivation of ICM-hH1a in fused tsA201 cells was abolished by intracellu lar perfusion with 2.5 mM 2-(trimethylammonium)ethyl methanethiosulfonate ( MTSET). The time constants of gating current relaxations in response to ste p depolarizations and gating charge-voltage relationships were not differen t between wild-type hH1a and ICM-hH1a(MTSET). The time constant of the deve lopment of charge immobilization assayed at - 180 mV after depolarization t o 0 mV was similar to the time constant of inactivation of I-Na at 0 mV for hH1a. By 44 ms, 53% of the gating charge during repolarization returned sl owly; i.e., became immobilized. In ICM-hH1a(MTSET), immobilization occurred with a similar time course, although only 31% of gating charge upon repola rization (OFF charge) immobilized. After modification of hH1a and ICM-hH1a( MTSET) with Anthopleurin-A toxin, a site-3 peptide toxin that inhibits move ment of the domain IV-S4, charge immobilization did not occur for condition ing durations up to 44 ms. OFF charge for both hH1a and ICM-hH1a(MTSET) mod ified with Anthopleurin-A toxin were similar in time course and in magnitud e to the fast component of OFF charge in ICM-hH1a(MTSET) in control. We con clude that movement of domain IV-S4 is the rate-limiting step during repola rization, and it contributes to charge immobilization regardless of whether the inactivation lid is bound. Taken together with previous reports, these data also suggest that S4 in domain III contributes to charge immobilizati on only after binding of the inactivation lid.