Ethanol modulation of calcium-and voltage-gated potassium (slo1) channels alters neuronal excitability, cerebrovascular tone, brain function, and behavior, yet the mechanism of this modulation remains unknown. Using patch-clamp electrophysiology on recombinant BK Ca channels cloned from mouse brain and expressed in Xenopus laevis oocytes, we demonstrate that ethanol, even at concentrations maximally effective to modulate BK Ca channel function (100 mM), fails to gate the channel in absence of activating calcium. Moreover, ethanol does not modify intrinsic, voltage-or physiological magnesium-driven gating. The alcohol works as an adjuvant of calcium by selectively facilitating calcium-driven gating. This facilitation, however, renders differential ethanol effects on channel activity: potentiation at low (Ͻ10 M) and inhibition at high (Ͼ10 M) calcium, this dual pattern remaining largely unmodified by coexpression of brain slo1 channels with the neuronally abundant BK Ca channel ␤ 4 subunit. Calcium recognition by either of the slo1 high-affinity sensors (calcium bowl and RCK1 Asp362/ Asp367) is required for ethanol to amplify channel activation by calcium. The Asp362/Asp367 site, however, is necessary and sufficient to sustain ethanol inhibition. This inhibition also results from ethanol facilitation of calcium action; in this case, ethanol favors channel dwelling in a calcium-driven, lowactivity mode. The agonist-adjuvant mechanism that we advance from the calcium-ethanol interaction on slo1 might be applicable to data of ethanol action on a wide variety of ligandgated channels.

Objective Hypercholesterolemia and alcohol drinking constitute independent risk factors for cerebrovascular disease. Alcohol constricts cerebral arteries in several species, including humans. This action results from inhibition of voltage- and calcium-gated potassium channels (BK) in vascular smooth muscle cells (VSMC). BK activity is also modulated by membrane cholesterol. We investigated whether VSMC cholesterol regulates ethanol actions on BK and cerebral arteries. Methods and Results After myogenic tone development, cholesterol depletion of rat, resistance-size cerebral arteries ablated ethanol-induced constriction, a result that was identical in intact and endothelium-free vessels. Cholesterol depletion reduced ethanol inhibition of BK whether the channel was studied in VSMC or after rat cerebral artery myocyte subunit (cbv1+β1) reconstitution into phospholipid bilayers. Homomeric cbv1 channels reconstituted into bilayers and VSMC BK from β1 KO mice were both resistant to ethanol-induced inhibition. Moreover, arteries from β1 KO mice failed to respond to ethanol even when VSMC cholesterol was kept unmodified. Remarkably, ethanol inhibition of cbv1+β1 in bilayers and wt mouse VSMC BK were drastically blunted by cholesterol depletion. Consistently, cholesterol depletion suppressed ethanol constriction of wt mouse arteries. Conclusion VSMC cholesterol and BK β1 are both required for ethanol inhibition of BK and the resulting cerebral artery constriction, with health-related implications for manipulating cholesterol levels in alcohol-induced cerebrovascular disease.

Bile acids and other steroids modify large conductance, calcium-and voltage-gated potassium (BK) channel activity contributing to non-genomic modulation of myogenic tone. Accessory BK b 1 subunits are necessary for lithocholate (LC) to activate BK channels and vasodilate. The protein regions that sense steroid action, however, remain unknown. Using recombinant channels in 1-palmitoyl-2-oleoyl-phosphatidylethanolamine/ 1-palmitoyl-2-oleoyl-phosphatidylserine bilayers we now demonstrate that complex proteolipid domains and cytoarchitecture are unnecessary for b 1 to mediate LC action; b 1 and a simple phospholipid microenvironment suffice. Since b 1 senses LC but b 4 does not, we made chimeras swapping regions between these subunits and, following channel heterologous expression, demonstrate that b 1 TM2 is a bile acid-recognizing sensor.