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.

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...

Voltage/Ca2+ i-gated, large conductance K+ (BK) channels result from tetrameric association of α (slo1) subunits. In most tissues, BK protein complexes include regulatory β subunits that contain two transmembrane domains (TM1, TM2), an extracellular loop, and two short intracellular termini. Four BK β types have been identified, each presenting a rather selective tissue-specific expression profile. Thus, BK β modifies current phenotype to suit physiology in a tissue-specific manner. The smooth muscle-abundant BK β1 drastically increases the channel's apparent Ca2+ i sensitivity. The resulting phenotype is critical for BK channel activity to increase in response to Ca2+ levels reached near the channel during depolarization-induced Ca2+ influx and myocyte contraction. The eventual BK channel activation generates outward K+ currents that drive the membrane potential in the negative direction and eventually counteract depolarization-induced Ca2+ influx. The BK β1 regions responsible for the characteristic phenotype of β1-containing BK channels remain to be identified. We used patch-clamp electrophysiology on channels resulting from the combination of smooth muscle slo1 (cbv1) subunits with smooth muscle-abundant β1, neuron-abundant β4, or chimeras constructed by swapping β1 and β4 regions, and determined the contribution of specific β1 regions to the BK phenotype. At Ca2+ levels found near the channel during myocyte contraction (10 µM), channel complexes that included chimeras having both TMs from β1 and the remaining regions (“background”) from β4 showed a phenotype (Vhalf, τact, τdeact) identical to that of complexes containing wt β1. This phenotype could not be evoked by complexes that included chimeras combining either β1 TM1 or β1 TM2 with a β4 background. Likewise, β “halves” (each including β1 TM1 or β1 TM2) resulting from interrupting the continuity of the EC loop failed to render the normal phenotype, indicating that physical connection between β1 TMs via the EC loop is also necessary for proper channel function.

Ethanol levels reached in circulation during moderate-to-heavy alcohol intoxication (50-100 mM) modify Ca 21 -and voltagegated K 1 (BK) channel steady-state activity, eventually altering both physiology and behavior. Ethanol action on BK steadystate activity solely requires the channel-forming subunit slo1 within a bare lipid environment. To identify the protein regions that confer ethanol sensitivity to slo1, we tested the ethanol sensitivity of heterologously expressed slo1 and structurally related channels. Ethanol (50 mM) increased the steady-state activities of mslo1 and Ca 21 -gated MthK, the latter after channel reconstitution into phospholipid bilayers. In contrast, 50-100 mM ethanol failed to alter the steady-state activities of Na 1 /Cl 2 -gated rslo2, H 1 -gated mslo3, and an mslo1/3 chimera engineered by joining the mslo1 region encompassing the N terminus to S6 with the mslo3 cytosolic tail domain (CTD). Collectively, data indicate that the slo family canonical design, which combines a transmembrane 6 (TM6) voltage-gated K 1 channel (K V ) core with CTDs that empower the channel with ion-sensing, does not necessarily render ethanol sensitivity. In addition, the region encompassing the N terminus to the S0-S1 cytosolic loop (missing in MthK) is not necessary for ethanol action. Moreover, incorporation of both this region and an ionsensing CTD to TM6 K V cores (a design common to mslo1, mslo3, and the mslo1/mslo3 chimera) is not sufficient for ethanol sensitivity. Rather, a CTD containing Ca 21 -sensing regulator of conductance for K 1 domains seems to be critical to bestow K V structures, whether of TM2 (MthK) or TM6 (slo1), with sensitivity to intoxicating ethanol levels.

Cholesterol is one of the major lipid components of membranes in mammalian cells. In recent years, cholesterol has emerged as a major regulator of ion channel function. The most common effect of cholesterol on ion channels in general and on inwardly rectifying potassium (Kir) channels in particular is a decrease in activity. In contrast, we have recently shown that native G-protein gated Kir (GIRK or Kir3) channels that underlie atrial KACh currents are up-regulated by cholesterol. Here we unveil the biophysical basis of cholesterol-induced increase in KACh activity. Using planar lipid bilayers we show that cholesterol significantly enhances the channel open frequency of the Kir3.1/Kir3.4 channels, which underlie KACh currents. In contrast, our data indicate that cholesterol does not affect their unitary conductance. Furthermore, using fluorescent and TIRF microscopy as well as surface protein biotinylation, we also show that cholesterol enrichment in vitro has no effect on surface expression of GFP-tagged channels expressed in Xenopus oocytes or transfected into HEK293 cells. Together, these data demonstrate for the first time that cholesterol enhances Kir3-mediated current by increasing the channel open probability.