Abstract
The structural bases of general anesthetic action on a neuronal K(+) channel were investigated using the series of homologous 1-alkanols, electrophysiology, and mutational analysis. Domain swapping between dShaw2 (alkanol-sensitive) and hKv3.4 (alkanol-resistant) and site-directed mutagenesis demonstrated that a 13-amino acid cytoplasmic loop (S4-S5) determines the selective inhibition of native dShaw2 channels by 1-alkanols. The S4-S5 loop may contribute to a receptor for both 1-alkanols and the inactivation particle, because the enhanced 1-alkanol sensitivity of hKv3.4 channels hosting S4-S5 mutations correlates directly with disrupted channel inactivation. Evidence of a discrete protein site was also obtained from the analysis of the relationship between potency and alkyl chain length, which begins to level off after 1-hexanol. Rapid application to the cytoplasmic side of inside-out membrane patches shows that the interaction between dShaw2 channels and 1-alkanols equilibrates in <200 ms. By contrast, the equilibration time is >1000-fold slower when the drug is applied externally to outside-out membrane patches. The data strongly favor a mechanism of inhibition involving a discrete internal site for 1-alkanols in dShaw2 K(+) channels. A new working hypothesis proposes that 1-alkanols lock dShaw2 channels in their closed conformation by a direct interaction at a crevice formed by the S4-S5 loop.
