Alex M. Dopico

Specificity of cholesterol and analogs to modulate BK channels points to direct sterol–channel protein interactions

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.

Multiple Cholesterol Recognition/Interaction Amino Acid Consensus (CRAC) Motifs in Cytosolic C Tail of Slo1 Subunit Determine Cholesterol Sensitivity of Ca2+- and Voltage-gated K+ (BK) Channels

Background: Cholesterol regulation of large conductance, Ca2+- and voltage-gated K+ (BK) channels has widespread pathophysiological consequences. Results: Cholesterol-channel recognition involves hydrophobic and hydrophilic interactions and several cholesterol recognition/interaction amino acid consensus motifs in the BK channel long C-end. Conclusion: Cholesterol regulation of BK channels involves specific channel protein-sterol recognition. Significance: we provide for the first time the structural basis of BK channel cholesterol sensitivity. Large conductance, Ca2+- and voltage-gated K+ (BK) channel proteins are ubiquitously expressed in cell membranes and control a wide variety of biological processes. Membrane cholesterol regulates the activity of membrane-associated proteins, including BK channels. Cholesterol modulation of BK channels alters action potential firing, colonic ion transport, smooth muscle contractility, endothelial function, and the channel alcohol response. The structural bases underlying cholesterol-BK channel interaction are unknown. Such interaction is determined by strict chemical requirements for the sterol molecule, suggesting cholesterol recognition by a protein surface. Here, we demonstrate that cholesterol action on BK channel-forming Cbv1 proteins is mediated by their cytosolic C tail domain, where we identified seven cholesterol recognition/interaction amino acid consensus motifs (CRAC4 to 10), a distinct feature of BK proteins. Cholesterol sensitivity is provided by the membrane-adjacent CRAC4, where Val-444, Tyr-450, and Lys-453 are required for cholesterol sensing, with hydrogen bonding and hydrophobic interactions participating in cholesterol location and recognition. However, cumulative truncations or Tyr-to-Phe substitutions in CRAC5 to 10 progressively blunt cholesterol sensitivity, documenting involvement of multiple CRACs in cholesterol-BK channel interaction. In conclusion, our study provides for the first time the structural bases of BK channel cholesterol sensitivity; the presence of membrane-adjacent CRAC4 and the long cytosolic C tail domain with several other CRAC motifs, which are not found in other members of the TM6 superfamily of ion channels, very likely explains the unique cholesterol sensitivity of BK channels.

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.

Ethanol modulation of mammalian BK channels in excitable tissues: molecular targets and their possible contribution to alcohol-induced altered behavior.

In most tissues, the function of Ca2+- and voltage-gated K+ (BK) channels is modified in response to ethanol concentrations reached in human blood during alcohol intoxication. In general, modification of BK current from ethanol-naïve preparations in response to brief ethanol exposure results from changes in channel open probability without modification of unitary conductance or change in BK protein levels in the membrane. Protracted and/or repeated ethanol exposure, however, may evoke changes in BK expression. The final ethanol effect on BK open probability leading to either BK current potentiation or BK current reduction is determined by an orchestration of molecular factors, including levels of activating ligand (Ca2+i), BK subunit composition and post-translational modifications, and the channels lipid microenvironment. These factors seem to allosterically regulate a direct interaction between ethanol and a recognition pocket of discrete dimensions recently mapped to the channel-forming (slo1) subunit. Type of ethanol exposure also plays a role in the final BK response to the drug: in several central nervous system regions (e.g., striatum, primary sensory neurons, and supraoptic nucleus), acute exposure to ethanol reduces neuronal excitability by enhancing BK activity. In contrast, protracted or repetitive ethanol administration may alter BK subunit composition and membrane expression, rendering the BK complex insensitive to further ethanol exposure. In neurohypophyseal axon terminals, ethanol potentiation of BK channel activity leads to a reduction in neuropeptide release. In vascular smooth muscle, however, ethanol inhibition of BK current leads to cell contraction and vascular constriction.

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.

Cerebrovascular Dilation via Selective Targeting of the Cholane Steroid-Recognition Site in the BK Channel β1-Subunit by a Novel Nonsteroidal Agent

The Ca2+/voltage-gated K+ large conductance (BK) channel β1 subunit is particularly abundant in vascular smooth muscle. By determining their phenotype, BK β1 allows the BK channels to reduce myogenic tone, facilitating vasodilation. The endogenous steroid lithocholic acid (LCA) dilates cerebral arteries via BK channel activation, which requires recognition by a BK β1 site that includes Thr169. Whether exogenous nonsteroidal agents can access this site to selectively activate β1-containing BK channels and evoke vasodilation remain unknown. We performed a chemical structure database similarity search using LCA as a template, along with a two-step reaction to generate sodium 3-hydroxyolean-12-en-30-oate (HENA). HENA activated the BK (cbv1 + β1) channels cloned from rat cerebral artery myocytes with a potency (EC50 = 53 μM) similar to and an efficacy (×2.5 potentiation) significantly greater than that of LCA. This HENA action was replicated on native channels in rat cerebral artery myocytes. HENA failed to activate the channels made of cbv1 + β2, β3, β4, or β1T169A, indicating that this drug selectively targets β1-containing BK channels via the BK β1 steroid-sensing site. HENA (3–45 μM) dilated the rat and C57BL/6 mouse pressurized cerebral arteries. Consistent with the electrophysiologic results, this effect was larger than that of LCA. HENA failed to dilate the arteries from the KCNMB1 knockout mouse, underscoring BK β1’s role in HENA action. Finally, carotid artery-infusion of HENA (45 μM) dilated the pial cerebral arterioles via selective BK-channel targeting. In conclusion, we have identified for the first time a nonsteroidal agent that selectively activates β1-containing BK channels by targeting the steroid-sensing site in BK β1, rendering vasodilation.

Lipid regulation of BK channel function.

This mini-review focuses on lipid modulation of BK (MaxiK, BKCa) current by a direct interaction between lipid and the BK subunits and/or their immediate lipid environment. Direct lipid-BK protein interactions have been proposed for fatty and epoxyeicosatrienoic acids, phosphoinositides and cholesterol, evidence for such action being less clear for other lipids. BK α (slo1) subunits are sufficient to support current perturbation by fatty and epoxyeicosatrienoic acids, glycerophospholipids and cholesterol, while distinct BK β subunits seem necessary for current modulation by most steroids. Subunit domains or amino acids that participate in lipid action have been identified in a few cases: hslo1 Y318, cerebral artery smooth muscle (cbv1) R334,K335,K336, cbv1 seven cytosolic CRAC domains, slo1 STREX and β1 T169,L172,L173 for docosahexaenoic acid, PIP2, cholesterol, sulfatides, and cholane steroids, respectively. Whether these protein motifs directly bind lipids or rather transmit the energy of lipid binding to other areas and trigger protein conformation change remains unresolved. The impact of direct lipid-BK interaction on physiology is briefly discussed.

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.

Calcium- and Voltage-Gated Potassium (BK) Channel Activators in the 5β-Cholanic Acid-3α-ol Analogue Series with Modifications in the Lateral Chain

Large conductance, calcium‐ and voltage‐gated potassium (BK) channels regulate various physiological processes and represent an attractive target for drug discovery. Numerous BK channel activators are available. However, these agents usually interact with the ubiquitously distributed channel‐forming subunit and thus cannot selectively target a particular tissue. We performed a structure–activity relationship study of lithocholic acid (LCA), a cholane that activates BK channels via the accessory BK β1 subunit. The latter protein is highly abundant in smooth muscle but scarce in most other tissues. Modifications to the LCA lateral chain length and functional group yielded two novel smooth muscle BK channel activators in which the substituent at C24 has a small volume and a net negative charge. Our data provide detailed structural information that will be useful to advance a pharmacophore in search of β1 subunit‐selective BK channel activators. These compounds are expected to evoke smooth muscle relaxation, which would be beneficial in the pharmacotherapy of prevalent human disorders associated with increased smooth muscle contraction, such as systemic hypertension, cerebral or coronary vasospasm, bronchial asthma, bladder hyperactivity, and erectile dysfunction.

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.