General anesthetic action at an internal protein site involving the S4-S5 cytoplasmic loop of a neuronal K(+) channel.
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.
Harris
T
T
Department of Pathology, Anatomy and Cell Biology, Jefferson Medical College, Philadelphia, Pennsylvania 19107, USA.
Shahidullah
M
M
Ellingson
J S
JS
Covarrubias
M
M
D020001:1-Butanol / Q000494:pharmacology; D000595:Amino Acid Sequence; D018681:Anesthetics, General / Q000494:pharmacology*; D000818:Animals; D003593:Cytoplasm / Q000187:drug effects* / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism; D015221:Potassium Channels / Q000737:chemistry / Q000187:drug effects*; D024642:Potassium Channels, Voltage-Gated; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000187:drug effects; D017386:Sequence Homology, Amino Acid; D051670:Shaw Potassium Channels; D014981:Xenopus; D029867:Xenopus Proteins
D018681:Anesthetics, General; C492016:KCNC4 protein, Xenopus; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D011994:Recombinant Proteins; D051670:Shaw Potassium Channels; D029867:Xenopus Proteins; D020001:1-Butanol