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

Abstract

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 approximately 50 ms at depolarized voltages to a maximum of approximately 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 approximately 2.5-fold, and the midpoint of the steady-state inactivation curve was shifted approximately 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.

Comparative Study of the Gating Motif and C-type Inactivation in Prokaryotic Voltage-gated Sodium Channels

“…C-type inactivation has been explained by conformational changes around the selectivity filter of the channel (23)(24)(25). These conformational changes, which cause the selectivity filter to collapse, act as an inactivation gate.…”

“…Although C-type inactivation is common in tetrameric cation channels, its molecular mechanism remains unclear. It has been proposed that C-type inactivation may be related to a collapse of the selectivity filter (23)(24)(25). Prokaryotic Na V s lack an obvious cytoplasmic N-type inactivation peptide, and inactivation may thus only occur through the C-type inactivation mechanism (25,26).…”

“…It has been proposed that C-type inactivation may be related to a collapse of the selectivity filter (23)(24)(25). Prokaryotic Na V s lack an obvious cytoplasmic N-type inactivation peptide, and inactivation may thus only occur through the C-type inactivation mechanism (25,26). Furthermore, comparative studies and mutational analysis of these new Na V homologues provide evidence that the rate of C-type inactivation is affected by the residues of helix S6.…”

Modulation of a voltage-gated Na+channel by sevoflurane involves multiple sites and distinct mechanisms

“…Sevoflurane enters the four equivalent extracellular sites quickly during the anesthetic partitioning phase, and remains there for the duration of the simulations. The extracellular region of NaChBac could influence the conformation of the selectivity filter to regulate P/C type inactivation (28,30). Therefore, sevoflurane interaction at the extracellular site could alter the stability of the filter region.…”

“…Although crystal structures are not yet reported, the bacterial Na + channel NaChBac has been extensively characterized by electrophysiology (22,(28)(29)(30)(31)(32)(33)(34)(35)(36). Additionally NaChBac exhibits conserved slow open channel block in response to local and general anesthetic agents (15, 37).…”