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Batrachotoxin-resistant Na+ channels derived from point mutations in transmembrane segment D4-S6
1Department of Biological Sciences, State University of New York at Albany, Albany, New York 12222, USA.
Biophysical Journal
|June 4, 1999
Summary
Local anesthetics (LAs) and batrachotoxin (BTX) interact with voltage-gated sodium channels. Mutations in the D4-S6 segment confer BTX resistance, suggesting intertwined binding sites for LAs and BTX.
Area of Science:
- Neuroscience
- Molecular Biology
- Pharmacology
Background:
- Local anesthetics (LAs) inhibit voltage-gated sodium channels (Navs) by binding to the D4-S6 segment.
- Batrachotoxin (BTX) paradoxically stabilizes Navs in an open state, with its receptor localized to the D1-S6 segment.
Purpose of the Study:
- To investigate the interplay between LA and BTX binding sites on Navs.
- To determine if mutations within the D4-S6 LA receptor influence BTX binding and channel gating.
Main Methods:
- Site-directed mutagenesis of rat skeletal muscle Nav1.4 (mu1/rSkm1) channels at key D4-S6 residues.
- Electrophysiological assessment of mutant channel function in response to BTX and LA (cocaine) application.
- Analysis of channel gating kinetics, including activation and inactivation.
Main Results:
- Mutations mu1-F1579K and mu1-N1584K rendered Nav channels resistant to BTX.
- The mu1-Y1586K mutation maintained BTX sensitivity, with BTX still eliminating fast and slow inactivation.
- Cocaine exhibited enhanced, time-dependent block of BTX-modified open channels, indicating preferential LA binding to open states.
Conclusions:
- The D4-S6 segment, critical for LA binding, influences BTX interaction, suggesting overlapping or allosteric binding sites.
- Channel opening likely facilitates simultaneous binding of both LAs and BTX by altering the proximity of D1-S6 and D4-S6 segments.
- The BTX receptor is proposed to reside at the D1-S6/D4-S6 interface, adjacent to the LA receptor, with simultaneous binding hindered in the closed state.