Molecular features of an alcohol binding site in a neuronal potassium channel.
Aliphatic alcohols (1-alkanols) selectively inhibit the neuronal Shaw2 K(+) channel at an internal binding site. This inhibition is conferred by a sequence of 13 residues that constitutes the S4-S5 loop in the pore-forming subunit. Here, we combined functional and structural approaches to gain insights into the molecular basis of this interaction. To infer the forces that are involved, we employed a fast concentration-clamp method (10-90% exchange time = 800 micros) to examine the kinetics of the interaction of three members of the homologous series of 1-alkanols (ethanol, 1-butanol, and 1-hexanol) with Shaw2 K(+) channels in Xenopus oocyte inside-out patches. As expected for a second-order mechanism involving a receptor site, only the observed association rate constants were linearly dependent on the 1-alkanol concentration. While the alkyl chain length modestly influenced the dissociation rate constants (decreasing only approximately 2-fold between ethanol and 1-hexanol), the second-order association rate constants increased e-fold per carbon atom. Thus, hydrophobic interactions govern the probability of productive collisions at the 1-alkanol binding site, and short-range polar interactions help to stabilize the complex. We also examined the relationship between the energetics of 1-alkanol binding and the structural properties of the S4-S5 loop. Circular dichroism spectroscopy applied to peptides corresponding to the S4-S5 loop of various K(+) channels revealed a correlation between the apparent binding affinity of the 1-alkanol binding site and the alpha-helical propensity of the S4-S5 loop. The data suggest that amphiphilic interactions at the Shaw2 1-alkanol binding site depend on specific structural constraints in the pore-forming subunit of the channel.
D000438:Alcohols / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D002942:Circular Dichroism; D029721:Drosophila Proteins; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009474:Neurons / Q000378:metabolism*; D009865:Oocytes; D018408:Patch-Clamp Techniques / Q000379:methods; D015221:Potassium Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D051670:Shaw Potassium Channels; D013816:Thermodynamics; D014981:Xenopus
D000438:Alcohols; D029721:Drosophila Proteins; D015221:Potassium Channels; D011994:Recombinant Proteins; D051670:Shaw Potassium Channels; C064032:Shaw protein, Drosophila