2Ј-Pyridine ring substituted analogs of epibatidine were assessed for equilibrium binding affinity, functional potency, and efficacy at rat neuronal nicotinic receptors expressed in Xenopus oocytes. Binding affinities were determined in membrane homogenates from oocytes expressing ␣2␤2, ␣2␤4, ␣3␤2, ␣3␤4, ␣4␤2, or ␣4␤4. Efficacy (relative to acetylcholine) and potency were measured electrophysiologically with oocytes expressing ␣3␤4, ␣4␤2, and ␣4␤4. Hydroxy, dimethylamino, and trifluoromethanesulfonate analogs had affinities too low for accurate measurement. The bromo analog had affinities 4-to 55-fold greater at ␤2 than at ␤4-containing receptors, modestly greater efficacy at ␣4␤4 than at ␣4␤2, and 5-to 10-fold greater potency at a4␤4 than at ␣3␤4 or ␣4␤2. The fluoro analog displayed affinities 52-to 875-fold greater at ␤2-than at ␤4-containing receptors, efficacy at ␣4␤4 receptors 3-fold greater than at ␣4␤2 and ␣3␤4, and was equipotent at all receptors tested. The norchloro analog showed affinities 114-to 3500-fold greater at ␤2-than at ␤4-containing receptors, 2-fold greater efficacy at ␣4␤2 and ␣4␤4 than at ␣3␤4, and 4-to 5-fold greater potency at ␣4␤4 and ␣3␤4 than at ␣4␤2. The amino analog displayed affinities 10-to 115-fold greater at ␤2-than at ␤4-containing receptors, 3-fold greater efficacy at ␣3␤4 than at ␣4␤2, and 2-to 4-fold greater potency at ␣3␤4 and ␣4␤4 than at ␣4␤2. Although these compounds displayed a variety of differences in affinity, efficacy, and potency, with one exception (binding affinity and functional potency at ␣4␤4 receptors) there were no significant correlations among these properties.

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Alcohol is a widely used and abused substance. A major unresolved issue in the alcohol research field is determining which of the many alcohol target proteins identified to date is responsible for shaping each specific alcohol-related behavior. The large-conductance, calcium- and voltage-activated potassium channel (BK channel) is a conserved target of ethanol. Genetic manipulation of the highly conserved BK channel influences alcohol-related behaviors across phylogenetically diverse species that include worm, fly, mouse, and man. A pharmacological tool that prevents alcohol's action at a single target, like the BK channel, would complement genetic approaches in the quest to define the behavioral consequences of alcohol at each target. To identify agents that specifically modulate the action of ethanol at the BK channel, we executed a high-throughput phagemid-display screen in combination with a behavioral genetics assay. This screen selected a novel nonapeptide, LS10, which moderated acute ethanol intoxication in a BK channel-humanized strain without altering basal behavior. LS10's action in vivo was dependent upon BK channel functional activity. Single-channel electrophysiological recordings in vitro showed that preincubation with a submicromolar concentration of LS10 restricted ethanol-induced changes in human BK channel gating. In contrast, no substantial changes in basal human BK channel function were observed after LS10 application. The results obtained with the LS10 peptide provide proof-of-concept evidence that a combined phagemid-display/behavioral genetics screening approach can provide novel tools for understanding the action of alcohol at the BK channel and how this, in turn, exerts influence over central nervous system function.

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