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Original Article

^a From the Nora Eccles Harrison Cardiovascular Research & Training Institute and The Department of Internal Medicine, University of Utah, Salt Lake City, Utah 84112
^b Department of Neurobiology, Pharmacology, and Physiology
^c Department of Medicine, University of Chicago, Chicago, Illinois 60637
CVRTI, Bldg. 500, 95 South 2000 East, University of Utah, Salt Lake City, UT 84112.801-581-3128

michael{at}cvrti.utah.edu

We investigated the contribution of the putative inactivation lid in voltage-gated sodium channels to gating charge immobilization (i.e., the slow return of gating charge during repolarization) by studying a lid-modified mutant of the human heart sodium channel (hH1a) that had the phenylalanine at position 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 intracellular perfusion with 2.5 mM 2-(trimethylammonium)ethyl methanethiosulfonate (MTSET). The time constants of gating current relaxations in response to step depolarizations and gating charge–voltage relationships were not different between wild-type hH1a and ICM-hH1a_MTSET. The time constant of the development of charge immobilization assayed at –180 mV after depolarization to 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 slowly; i.e., became immobilized. In ICM-hH1a_MTSET, immobilization occurred with a similar time course, although only 31% of gating charge upon repolarization (OFF charge) immobilized. After modification of hH1a and ICM-hH1a_MTSET with Anthopleurin-A toxin, a site-3 peptide toxin that inhibits movement of the domain IV-S4, charge immobilization did not occur for conditioning durations up to 44 ms. OFF charge for both hH1a and ICM-hH1a_MTSET modified with Anthopleurin-A toxin were similar in time course and in magnitude to the fast component of OFF charge in ICM-hH1a_MTSET in control. We conclude that movement of domain IV-S4 is the rate-limiting step during repolarization, 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 immobilization only after binding of the inactivation lid.

Key Words: sodium channel • gating charge • inactivation • site-3 toxin • immobilization

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