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Original Article

^a Department of Physiology, Jefferson Medical College, Philadelphia, Pennsylvania 19107
^b Department of Applied Physiology and Neurology, University of Ulm, 89081 Ulm, Germany
^c Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-6304
^d Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-6304
Department of Physiology, Jefferson Medical College, 1020 Locust St., Philadelphia, PA 19107.215-503-2073

richard.horn{at}mail.tju.edu

Depolarization of sodium channels initiates at least three gating pathways: activation, fast inactivation, and slow inactivation. Little is known about the voltage sensors for slow inactivation, a process believed to be separate from fast inactivation. Covalent modification of a cysteine substituted for the third arginine (R1454) in the S4 segment of the fourth domain (R3C) with negatively charged methanethiosulfonate-ethylsulfonate (MTSES) or with positively charged methanethiosulfonate-ethyltrimethylammonium (MTSET) produces a marked slowing of the rate of fast inactivation. However, only MTSES modification produces substantial effects on the kinetics of slow inactivation. Rapid trains of depolarizations (2–20 Hz) cause a reduction of the peak current of mutant channels modified by MTSES, an effect not observed for wild-type or unmodified R3C channels, or for mutant channels modified by MTSET. The data suggest that MTSES modification of R3C enhances entry into a slow-inactivated state, and also that the effects on slow inactivation are independent of alterations of either activation or fast inactivation. This effect of MTSES is observed only for cysteine mutants within the middle of this S4 segment, and the data support a helical secondary structure of S4 in this region. Mutation of R1454 to the negatively charged residues aspartate or glutamate cannot reproduce the effects of MTSES modification, indicating that charge alone cannot account for these results. A long-chained derivative of MTSES has similar effects as MTSES, and can produce these effects on a residue that does not show use-dependent current reduction after modification by MTSES, suggesting that the sulfonate moiety can reach a critical site affecting slow inactivation. The effects of MTSES on R3C are partially counteracted by a point mutation (W408A) that inhibits slow inactivation. Our data suggest that a region near the midpoint of the S4 segment of domain 4 plays an important role in slow inactivation.

Key Words: thiol reagents • cysteine modification • S4 segment • site-directed mutagenesis

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