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Originally published as Biophys J. BioFAST on April 22, 2005.
doi:10.1529/biophysj.104.056994
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Biophysical Journal 89:232-242 (2005)
© 2005 The Biophysical Society

The Pore, not Cytoplasmic Domains, Underlies Inactivation in a Prokaryotic Sodium Channel

Evgeny Pavlov, Christopher Bladen, Robert Winkfein, Catherine Diao, Perry Dhaliwal and Robert J. French

Department of Physiology and Biophysics, University of Calgary, Calgary, Alberta, Canada

Correspondence: Address reprint requests to Robert French, Dept. of Physiology and Biophysics, University of Calgary, 3330 Hospital Dr. NW, Calgary, Alberta T2N 4N1, Canada. Tel.: 403-220-6893; Fax: 403-283-8731; E-mail: french{at}ucalgary.ca.

Kinetics and voltage dependence of inactivation of a prokaryotic voltage-gated sodium channel (NaChBac) were investigated in an effort to understand its molecular mechanism. NaChBac inactivation kinetics show strong, bell-shaped voltage dependence with characteristic time constants ranging from ~50 ms at depolarized voltages to a maximum of ~100 s at the inactivation midpoint. Activation and inactivation parameters for four different covalently linked tandem dimer or tandem tetramer constructs were indistinguishable from those of the wild-type channel. Point mutations in the outer part of the pore revealed an important influence of the S195 residue on the process of inactivation. For two mutants (S195D and S195E), the maximal and minimal rates of inactivation observed were increased by ~2.5-fold, and the midpoint of the steady-state inactivation curve was shifted ~20 mV in the hyperpolarizing direction, compared to the wild-type channel. Our data suggest that pore vestibule structure is an important determinant of NaChBac inactivation, whereas the inactivation mechanism is independent of the number of free cytoplasmic N- and C-termini in the functional channel. In these respects, NaChBac inactivation resembles C-type or slow inactivation modes observed in other voltage-gated K and Na channels.




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