Guruprasad Kuntamallappanavar

An alcohol-sensing site in the calcium- and voltage-gated, large conductance potassium (BK) channel

Significance Alcohol (ethanol) is one of the most widely consumed psychoactive agents. Ethanol’s targets in the body include voltage/calcium-gated, large conductance potassium (BK) channels. Here we identified and characterized for the first time an alcohol-sensing site in BK channel-forming proteins. Our discovery opens new ground for rational design of tools to counteract the effects of ethanol intoxication that are mediated by BK channels. We anticipate that genetic or epigenetic modifications of the BK channel ethanol-recognition site could explain differential sensitivity to ethanol. Finally, since individuals with low sensitivity to ethanol are prone to developing heavy drinking habits, genetic, epigenetic or other mechanisms acting at the newly identified BK channel ethanol-recognition site might be considered as potential predictors for developing alcohol preference. 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.

Smooth Muscle Cholesterol Enables BK β1 Subunit-Mediated Channel Inhibition and Subsequent Vasoconstriction Evoked by Alcohol

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+&bgr;1) reconstitution into phospholipid bilayers. Homomeric cbv1 channels reconstituted into bilayers and VSMC BK from &bgr;1 knockout mice were both resistant to ethanol-induced inhibition. Moreover, arteries from &bgr;1 knockout mice failed to respond to ethanol even when VSMC cholesterol was kept unmodified. Remarkably, ethanol inhibition of cbv1+&bgr;1 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 &bgr;1 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.

Distinct Sensitivity of Slo1 Channel Proteins to Ethanol

Ethanol levels reached in circulation during moderate-to-heavy alcohol intoxication (50–100 mM) modify Ca2+- and voltage-gated K+ (BK) channel steady-state activity, eventually altering both physiology and behavior. Ethanol action on BK steady-state 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 Ca2+-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+/Cl−-gated rslo2, H+-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+ channel (KV) 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 ion-sensing CTD to TM6 KV cores (a design common to mslo1, mslo3, and the mslo1/mslo3 chimera) is not sufficient for ethanol sensitivity. Rather, a CTD containing Ca2+-sensing regulator of conductance for K+ domains seems to be critical to bestow KV structures, whether of TM2 (MthK) or TM6 (slo1), with sensitivity to intoxicating ethanol levels.

Both transmembrane domains of BK β1 subunits are essential to confer the normal phenotype of β1-containing BK channels

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 channels 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.

Cholesterol increases the open probability of cardiac KACh currents

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.

Modulation of BK Channels by Ethanol

In alcohol-naïve systems, ethanol (<100mM) exposure of calcium-gated BK channels perturbs physiology and behavior. Brief (several minutes) ethanol exposure usually leads to increased BK current, which results from ethanol interaction with a pocket mapped to the BK channel-forming slo1 protein cytosolic tail domain. The importance of this region in ethanol-induced intoxication has been independently supported by an unbiased screen of Caenorhabditis elegans slo1 mutants. However, ethanol-induced BK activation is not universal as refractoriness and inhibition have been reported. The final effect depends on many factors, including intracellular calcium levels, slo1 isoform, BK beta subunit composition, posttranslational modification of BK proteins, channel lipid microenvironment, and type of ethanol administration. Studies in Drosophila melanogaster, C. elegans, and rodents show that protracted/repeated ethanol administration leads to tolerance to ethanol-induced modification of BK-driven physiology and behavior. Unveiling the mechanisms underlying tolerance is of major importance, as tolerance to ethanol has been proposed as predictor of risk for alcoholism.

Endothelial Nitric Oxide Mediates Caffeine Antagonism of Alcohol-Induced Cerebral Artery Constriction

Despite preventive education, the combined consumption of alcohol and caffeine (particularly from “energy drinks”) continues to rise. Physiologic perturbations by separate intake of ethanol and caffeine have been widely documented. However, the biologic actions of the alcohol-caffeine combination and their underlying subcellular mechanisms have been scarcely studied. Using intravital microscopy on a closed-cranial window and isolated, pressurized vessels, we investigated the in vivo and in vitro action of ethanol-caffeine mixtures on cerebral arteries from rats and mice, widely recognized models to address cerebrovascular pathophysiology and pharmacology. Caffeine at concentrations found in human circulation after ingestion of one to two cups of coffee (10 µM) antagonized the endothelium-independent constriction of cerebral arteries evoked by ethanol concentrations found in blood during moderate-heavy alcohol intoxication (40–70 mM). Caffeine antagonism against alcohol was similar whether evaluated in vivo or in vitro, suggesting independence of systemic factors and drug metabolism, but required a functional endothelium. Moreover, caffeine protection against alcohol increased nitric oxide (NO•) levels over those found in the presence of ethanol alone, disappeared upon blocking NO• synthase, and could not be detected in pressurized cerebral arteries from endothelial nitric-oxide synthase knockout (eNOS−/−) mice. Finally, incubation of de-endothelialized cerebral arteries with the NO• donor sodium nitroprusside (10 µM) fully restored the protective effect of caffeine. This study demonstrates for the first time that caffeine antagonizes ethanol-induced cerebral artery constriction and identifies endothelial NO• as the critical caffeine effector on smooth muscle targets. Conceivably, situations that perturb endothelial function and/or NO• availability will critically alter caffeine antagonism of alcohol-induced cerebrovascular constriction without significantly disrupting endothelium-independent, alcohol-induced cerebral artery constriction itself.

Alcohol modulation of BK channel gating depends on β subunit composition

Large conductance K+ channels of the slo1 family are gated by Ca2+ and voltage and either inhibited or potentiated by ethanol. Kuntamallappanavar and Dopico analyze the effects of ethanol in detail and find that the absence or presence of β subunits leads to differential modification of channel gating parameters by intoxicating levels of ethanol.

BK β1 subunit‐dependent facilitation of ethanol inhibition of BK current and cerebral artery constriction is mediated by the β1 transmembrane domain 2

Ethanol at concentrations obtained in the circulation during moderate‐heavy episodic drinking (30–60 mM) causes cerebral artery constriction in several species, including humans. In rodents, ethanol‐induced cerebral artery constriction results from ethanol inhibition of large conductance voltage/Ca2+i‐gated K+ (BK) channels in cerebral artery myocytes. Moreover, the smooth muscle‐abundant BK β1 accessory subunit is required for ethanol to inhibit cerebral artery myocyte BK channels under physiological Ca2+i and voltages and thus constrict cerebral arteries. The molecular bases of these ethanol actions remain unknown. Here, we set to identify the BK β1 region(s) that mediates ethanol‐induced inhibition of cerebral artery myocyte BK channels and eventual arterial constriction.

Activation of human smooth muscle BK channels by hydrochlorothiazide requires cell integrity and the presence of BK β1 subunit

Thiazide-like diuretics are the most commonly used drugs to treat arterial hypertension, with their efficacy being linked to their chronic vasodilatory effect. Previous studies suggest that activation of the large conductance voltage- and Ca2+-dependent K+ (BK) channel (Slo 1, MaxiK channel) is responsible for the thiazide-induced vasodilatory effect. But the direct electrophysiological evidence supporting this claim is lacking. BK channels can be associated with one small accessory β-subunit (β1–β4) that confers specific biophysical and pharmacological characteristics to the current phenotype. The β1-subunit is primarily expressed in smooth muscle cells (SMCs). In this study we investigated the effect of hydrochlorothiazide (HCTZ) on BK channel activity in native SMCs from human umbilical artery (HUASMCs) and HEK293T cells expressing the BK channel (with and without the β1-subunit). Bath application of HCTZ (10 μmol/L) significantly augmented the BK current in HUASMCs when recorded using the whole-cell configurations, but it did not affect the unitary conductance and open probability of the BK channel in HUASMCs evaluated in the inside-out configuration, suggesting an indirect mechanism requiring cell integrity. In HEK293T cells expressing BK channels, HCTZ-augmented BK channel activity was only observed when the β1-subunit was co-expressed, being concentration-dependent with an EC50 of 28.4 μmol/L, whereas membrane potential did not influence the concentration relationship. Moreover, HCTZ did not affect the BK channel current in HEK293T cells evaluated in the inside-out configuration, but significantly increases the open probability in the cell-attached configuration. Our data demonstrate that a β1-subunit-dependent mechanism that requires SMC integrity leads to HCTZ-induced BK channel activation.