ALCOHOLS INHIBIT A CLONED POTASSIUM CHANNEL AT A DISCRETE SATURABLE SITE - INSIGHTS INTO THE MOLECULAR-BASIS OF GENERAL-ANESTHESIA

The molecular basis of general anesthetic action on membrane proteins that control ion transport is not yet understood, In a previous report (Covarrubias, M., and Rubin, E. (1993) Proc. Natl. Acad. Sci. 90, 695 7-6960), we found that low concentrations of ethanol (17-170 mM) selec tively inhibited a noninactivating cloned K+ channel encoded by Drosop hila Shaw2. Here, we have conducted equilibrium dose-inhibition experi ments, single channel recording, and mutagenesis in vitro to study the mechanism underlying the inhibition of Shaw2 K+ channels by a homolog ous series of n-alkanols (ethanol to 1-hexanol), The results showed th at: (i) these alcohols inhibited Shaw2 whole-cell currents, the equili brium dose-inhibition relations were hyperbolic, and competition exper iments revealed the presence of a discrete site of action, possibly a hydrophobic pocket; (ii) this pocket may be part of the protein becaus e n-alkanol sensitivity can be transferred to novel hybrid K+ channels composed of Shaw2 subunits and homologous ethanol-insensitive subunit s; (iii) moreover, a hydrophobic point mutation within a cytoplasmic l oop of an ethanol-insensitive K+ channel (human Kv3.4) was sufficient to allow significant inhibition by n-alkanols, with a dose-inhibition relation that closely resembled that of wildtype Shaw2 channels; and ( iv) 1-butanol selectively inhibited long duration single channel openi ngs in a manner consistent with a direct effect on channel gating. The se results strongly suggest that a discrete site within the ion channe l protein is the primary locus of alcohol and general anesthetic actio n.