Among the nongenomic effects of steroids, control of vasomotion has received increasing attention. Lithocholate (LC) and other physiologically relevant cholane-derived steroids cause vasodilation, yet the molecular targets and mechanisms underlying this action remain largely unknown. We demonstrate that LC (45 M) reversibly increases the diameter of pressurized resistance cerebral arteries by ϳ10%, which would result in ϳ30% increase in cerebral blood flow. LC action is independent of endothelial integrity, prevented by 55 nM iberiotoxin, and unmodified by 0.8 mM 4-aminopyridine, indicating that LC causes vasodilation via myocyte BK channels. Indeed, LC activates BK channels in isolated myocytes through a destabilization of channel long-closed states without modifying unitary conductance. LC channel activation occurs within a wide voltage range and at Ca 2ϩ concentrations reached in the myocyte at rest and during contraction. Channel accessory ␤ 1 subunits, which are predominant in smooth muscle, are necessary for LC to modify channel activity. In contrast, ␤ 4 subunits, which are predominant in neuronal tissues, fail to evoke LC sensitivity. LC activation of cbv1ϩ␤ 1 and native BK channels display identical characteristics, including EC 50 (46 M) and E max (Ϸ300 M) values, strongly suggesting that the cbv1ϩ␤ 1 complex is necessary and sufficient to evoke LC action. Finally, intact arteries from ␤ 1 subunit knockout mice fail to relax in response to LC, although they are able to respond to other vasodilators. This study pinpoints the BK ␤ 1 subunit as the molecule that senses LC, which results in myocyte BK channel activation and, thus, endothelial-independent relaxation of small, resistance-size arteries.

a b s t r a c tEthanol-induced inhibition of myocyte large conductance, calcium-and voltage-gated potassium (BK) current causes cerebrovascular constriction, yet the molecular targets mediating EtOH action remain unknown. Using BK channel-forming (cbv1) subunits from cerebral artery myocytes, we demonstrate that EtOH potentiates and inhibits current at Ca 2þ i lower and higher than $15 lM, respectively. By increasing cbv1's apparent Ca 2þ i -sensitivity, accessory BK b 1 subunits shift the activation-to-inhibition crossover of EtOH action to <3 lM Ca 2þ i , with consequent inhibition of current under conditions found during myocyte contraction. Knocking-down KCNMB1 suppresses EtOHreduction of arterial myocyte BK current and vessel diameter. Therefore, BK b 1 is the molecular effector of alcohol-induced BK current inhibition and cerebrovascular constriction.

The activity (Po) of large-conductance voltage/Ca2+-gated K+ (BK) channels is blunted by cholesterol levels within the range found in natural membranes. We probed BK channel–forming α (cbv1) subunits in phospholipid bilayers with cholesterol and related monohydroxysterols and performed computational dynamics to pinpoint the structural requirements for monohydroxysterols to reduce BK Po and obtain insights into cholesterol’s mechanism of action. Cholesterol, cholestanol, and coprostanol reduced Po by shortening mean open and lengthening mean closed times, whereas epicholesterol, epicholestanol, epicoprostanol, and cholesterol trisnorcholenic acid were ineffective. Thus, channel inhibition by monohydroxysterols requires the β configuration of the C3 hydroxyl and is favored by the hydrophobic nature of the side chain, while having lax requirements on the sterol A/B ring fusion. Destabilization of BK channel open state(s) has been previously interpreted as reflecting increased bilayer lateral stress by cholesterol. Lateral stress is controlled by the sterol molecular area and lipid monolayer lateral tension, the latter being related to the sterol ability to adopt a planar conformation in lipid media. However, we found that the differential efficacies of monohydroxysterols to reduce Po (cholesterol≥coprostanol≥cholestanol>>>epicholesterol) did not follow molecular area rank (coprostanol>>epicholesterol>cholesterol>cholestanol). In addition, computationally predicted energies for cholesterol (effective BK inhibitor) and epicholesterol (ineffective) to adopt a planar conformation were similar. Finally, cholesterol and coprostanol reduced Po, yet these sterols have opposite effects on tight lipid packing and, likely, on lateral stress. Collectively, these findings suggest that an increase in bilayer lateral stress is unlikely to underlie the differential ability of cholesterol and related steroids to inhibit BK channels. Remarkably, ent-cholesterol (cholesterol mirror image) failed to reduce Po, indicating that cholesterol efficacy requires sterol stereospecific recognition by a protein surface. The BK channel phenotype resembled that of α homotetramers. Thus, we hypothesize that a cholesterol-recognizing protein surface resides at the BK α subunit itself.

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.

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Ethanol alters BK (slo1) channel function leading to perturbation of physiology and behavior. Site(s) and mechanism(s) of ethanol-BK channel interaction are unknown. We demonstrate that ethanol docks onto a water-accessible site that is strategically positioned between the slo1 calcium-sensors and gate. Ethanol only accesses this site in presence of calcium, the BK channel's physiological agonist. Within the site, ethanol hydrogen-bonds with K361. Moreover, substitutions that hamper hydrogen bond formation or prevent ethanol from accessing K361 abolish alcohol action without altering basal channel function. Alcohol interacting site dimensions are approximately 10.7 × 8.6 × 7.1 Å, accommodating effective (ethanol-heptanol) but not ineffective (octanol, nonanol) channel activators. This study presents: (i) to our knowledge, the first identification and characterization of an n-alkanol recognition site in a member of the voltage-gated TM6 channel superfamily; (ii) structural insights on ethanol allosteric interactions with ligand-gated ion channels; and (iii) a first step for designing agents that antagonize BK channel-mediated alcohol actions without perturbing basal channel function.is a psychoactive agent that has been overwhelmingly consumed by mankind across cultures and civilizations. Alcohol actions on central nervous system (CNS) physiology and behavior are largely independent of beverage type but due to ethanol itself (1). Ethanol alters cell excitability by modifying function of transmembrane (TM) ion channel proteins, including K + channels. These channels constitute the most heterogeneous and extensive group of ion channels, its members belonging to TM2, TM4, and TM6 protein superfamilies. Within this myriad of proteins, several K + channels have been shown to modify behavior in response to acute exposure to ethanol concentrations that reach the CNS and other excitable tissues during alcohol drinking (2-5). However, with the sole exception of the TM2, G protein-regulated inward rectifier K + (GIRK) channel (6), there is no structural information on ethanol-K + channel protein interacting sites currently available. Voltage/Ca 2+ -gated, large conductance K + channels (BK), which are members of the TM6 voltage-gated ion channel superfamily, constitute major mediators of alcohol actions in excitable tissues. Acute exposure to ethanol levels reached in CNS during alcohol intoxication alters BK-mediated currents and thus, elicits widespread and profound modifications in physiology and behavior. In rodent models, acute ethanol exposure leads to reduced vasopressin, oxytocin and growth hormone release with consequent perturbation in physiology and behavior (7), altered firing rates in nucleus accumbens (8) and dorsal root ganglia neurons (9), and alcohol-induced cerebral artery constriction (10, 11). Moreover, studies in both mammals and invertebrate models demonstrate that ethanol targeting of neuronal BK is involved in development of alcohol tolerance and dependence (12-16). Although the physiological and be...

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.

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