期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2021
卷号：118
期号：28
DOI：10.1073/pnas.2102285118
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Significance
Voltage-gated sodium channels initiate electric signals in cell communications. The S4–S5 linker between the voltage-sensing and pore modules transmits depolarization signals to trigger channel activation. The mechanisms of this action, however, remain elusive. By combining biophysical and computational approaches, we identify a critical residue, T140, in the S4–S5 linker of the bacterial sodium channel NaChBac, which plays a pivotal role in channel activation and drug modulation of slow inactivation. Specifically, we discovered conformation-dependent drug binding at this site and unveiled a toggling mode of action by T140, which switches interaction partners with different S6 residues to regulate channel activation and slow inactivation. These observations suggest the possibility of conformation-specific drugs targeting the gating machinery of voltage-gated ion channels.
Voltage-gated sodium (Na
V) channels control excitable cell functions. While structural investigations have revealed conformation details of different functional states, the mechanisms of both activation and slow inactivation remain unclear. Here, we identify residue T140 in the S4–S5 linker of the bacterial voltage-gated sodium channel NaChBac as critical for channel activation and drug effects on inactivation. Mutations at T140 either attenuate activation or render the channel nonfunctional. Propofol, a clinical anesthetic known to inhibit NaChBac by promoting slow inactivation, binds to a pocket between the S4–S5 linker and S6 helix in a conformation-dependent manner. Using
19F-NMR to quantify site-specific binding by saturation transfer differences (STDs), we found strong STDs in inactivated, but not activated, NaChBac. Molecular dynamics simulations show a highly dynamic pocket in the activated conformation, limiting STD buildup. In contrast, drug binding to this pocket promotes and stabilizes the inactivated states. Our results provide direct experimental evidence showing distinctly different associations between the S4–S5 linker and S6 helix in activated and inactivated states. Specifically, an exchange occurs between interaction partners T140 and N234 of the same subunit in activation, and T140 and N225 of the domain-swapped subunit in slow inactivation. The drug action on slow inactivation of prokaryotic Na
V channels seems to have a mechanism similar to the recently proposed “door-wedge” action of the isoleucine-phenylalanine-methionine (IFM) motif on the fast inactivation of eukaryotic Na
V channels. Elucidating this gating mechanism points to a possible direction for conformation-dependent drug development.
关键词：engeneral anesthesia;propofol;NaChBac;NMR;saturation transfer difference
