Abstract
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, 6957-6960), we found that low concentrations of ethanol (17-170mM) selectively inhibited a noninactivating cloned K+ channel encoded by Drosophila Shaw2. Here, we have conducted equilibrium dos-inhibition experiments, single channel recording, and mutagenesis in vitro to study the mechanism underlying the inhibition of Shaw2K+ channels by a homologous series of n-alkanols (ethanol to 1-hexanol). The results showed that: (i) these alcohols inhibited Shaw2 whole-cell currents, the equilibrium dose-inhibition relations were hyperbolic, and competition experiments revealed the presence of a discrete site of action, possibly a hydrophobic pocket; (ii) this pocket may be part of the protein because n-alkanol sensitivity can be transferred to novel hybrid K+ channels composed of Shaw2 subunits and homologous ethanol-insensitive subunits: (iii) moreover, a hydrophobic point mutation within a cytoplasmic loop 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 openings in a manner consistent with a direct effect on channel gating. These results strongly suggest that a discrete site within the ion channel protein is the primary locus of alcohol and general anesthetic action.
Highlights
From the Wepartment of Pathology, Anatomy, and Cell Biology, Jefferson Medical College, Philadelphia, Pennsylvania 19107 and the **Department of Anatomy and Neurobiology, Washington University School of Medicine, St
The results showed that: (i) these alcohols inhibited Shaw2 whole-cell currents, the equilibrium dose-inhibition relations were hyperbolic, and competition experiments revealed the presence of a discrete site of action, possibly a hydrophobic pocket; (ii) this pocket may be part of the protein because n-alkanol sensitivity can be transferred to novel hybrid K+ channels composed of Shaw2 subunits and homologous ethanol-insensitive subunits; (iii) a hydrophobic point mutation within a cytoplasmic loop of an ethanolinsensitive K+ channel was sufficient to allow significant inhibition by n-alkanols, with a doseinhibition relation that closely resembled that of wildtype Shaw2 channels; and (iv) I-butanol selectively inhibited long duration single channel openings in a manner consistent with a direct effect on channel gating
Equilibrium Dose-inhibition Experiments Suggest the Presence of a Saturable Site for n-Alkanols-To investigate the presence of a saturable site for aliphatic alcohols, we examined the equilibrium dose-response relation for various members of the homologous series of n-alkanols (C2-C6)
Summary
From the Wepartment of Pathology, Anatomy, and Cell Biology, Jefferson Medical College, Philadelphia, Pennsylvania 19107 and the **Department of Anatomy and Neurobiology, Washington University School of Medicine, St. The results showed that: (i) these alcohols inhibited Shaw whole-cell currents, the equilibrium dose-inhibition relations were hyperbolic, and competition experiments revealed the presence of a discrete site of action, possibly a hydrophobic pocket; (ii) this pocket may be part of the protein because n-alkanol sensitivity can be transferred to novel hybrid K+ channels composed of Shaw subunits and homologous ethanol-insensitive subunits; (iii) a hydrophobic point mutation within a cytoplasmic loop of an ethanolinsensitive K+ channel (human Kv3.4) was sufficient to allow significant inhibition by n-alkanols, with a doseinhibition relation that closely resembled that of wildtype Shaw channels; and (iv) I-butanol selectively inhibited long duration single channel openings in a manner consistent with a direct effect on channel gating These results strongly suggest that a discrete site within the ion channel protein is the primary locus of alcohol and general anesthetic action. II Supported in part by Hospital de Especialidades, Instituto Mexicano del Seguro Social, Mexico City
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