Anna N. Bukiya

Direct Regulation of BK Channels by Phosphatidylinositol 4,5-Bisphosphate as a Novel Signaling Pathway

Large conductance, calcium- and voltage-gated potassium (BK) channels are ubiquitous and critical for neuronal function, immunity, and smooth muscle contractility. BK channels are thought to be regulated by phosphatidylinositol 4,5-bisphosphate (PIP2) only through phospholipase C (PLC)–generated PIP2 metabolites that target Ca2+ stores and protein kinase C and, eventually, the BK channel. Here, we report that PIP2 activates BK channels independently of PIP2 metabolites. PIP2 enhances Ca2+-driven gating and alters both open and closed channel distributions without affecting voltage gating and unitary conductance. Recovery from activation was strongly dependent on PIP2 acyl chain length, with channels exposed to water-soluble diC4 and diC8 showing much faster recovery than those exposed to PIP2 (diC16). The PIP2–channel interaction requires negative charge and the inositol moiety in the phospholipid headgroup, and the sequence RKK in the S6–S7 cytosolic linker of the BK channel-forming (cbv1) subunit. PIP2-induced activation is drastically potentiated by accessory β1 (but not β4) channel subunits. Moreover, PIP2 robustly activates BK channels in vascular myocytes, where β1 subunits are abundantly expressed, but not in skeletal myocytes, where these subunits are barely detectable. These data demonstrate that the final PIP2 effect is determined by channel accessory subunits, and such mechanism is subunit specific. In HEK293 cells, cotransfection of cbv1+β1 and PI4-kinaseIIα robustly activates BK channels, suggesting a role for endogenous PIP2 in modulating channel activity. Indeed, in membrane patches excised from vascular myocytes, BK channel activity runs down and Mg-ATP recovers it, this recovery being abolished by PIP2 antibodies applied to the cytosolic membrane surface. Moreover, in intact arterial myocytes under physiological conditions, PLC inhibition on top of blockade of downstream signaling leads to drastic BK channel activation. Finally, pharmacological treatment that raises PIP2 levels and activates BK channels dilates de-endothelized arteries that regulate cerebral blood flow. These data indicate that endogenous PIP2 directly activates vascular myocyte BK channels to control vascular tone.

β1 (KCNMB1) subunits mediate lithocholate activation of large-conductance Ca2+-activated K+ channels and dilation in small, resistance-size arteries

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 μM) reversibly increases the diameter of pressurized resistance cerebral arteries by ∼10%, which would result in ∼30% 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 Ca2+ concentrations reached in the myocyte at rest and during contraction. Channel accessory β1 subunits, which are predominant in smooth muscle, are necessary for LC to modify channel activity. In contrast, β4 subunits, which are predominant in neuronal tissues, fail to evoke LC sensitivity. LC activation of cbv1+β1 and native BK channels display identical characteristics, including EC50 (46 μM) and Emax (≈300 μM) values, strongly suggesting that the cbv1+β1 complex is necessary and sufficient to evoke LC action. Finally, intact arteries from β1 subunit knockout mice fail to relax in response to LC, although they are able to respond to other vasodilators. This study pinpoints the BK β1 subunit as the molecule that senses LC, which results in myocyte BK channel activation and, thus, endothelial-independent relaxation of small, resistance-size arteries.

Sizing up Ethanol‐Induced Plasticity: The Role of Small and Large Conductance Calcium‐Activated Potassium Channels

Small (SK) and large conductance (BK) Ca(2+)-activated K(+) channels contribute to action potential repolarization, shape dendritic Ca(2+)spikes and postsynaptic responses, modulate the release of hormones and neurotransmitters, and contribute to hippocampal-dependent synaptic plasticity. Over the last decade, SK and BK channels have emerged as important targets for the development of acute ethanol tolerance and for altering neuronal excitability following chronic ethanol consumption. In this mini-review, we discuss new evidence implicating SK and BK channels in ethanol tolerance and ethanol-associated homeostatic plasticity. Findings from recent reports demonstrate that chronic ethanol produces a reduction in the function of SK channels in VTA dopaminergic and CA1 pyramidal neurons. It is hypothesized that the reduction in SK channel function increases the propensity for burst firing in VTA neurons and increases the likelihood for aberrant hyperexcitability during ethanol withdrawal in hippocampus. There is also increasing evidence supporting the idea that ethanol sensitivity of native BK channel results from differences in BK subunit composition, the proteolipid microenvironment, and molecular determinants of the channel-forming subunit itself. Moreover, these molecular entities play a substantial role in controlling the temporal component of ethanol-associated neuroadaptations in BK channels. Taken together, these studies suggest that SK and BK channels contribute to ethanol tolerance and adaptive plasticity.

Large conductance, calcium- and voltage-gated potassium (BK) channels: Regulation by cholesterol

Cholesterol (CLR) is an essential component of eukaryotic plasma membranes. CLR regulates the membrane physical state, microdomain formation and the activity of membrane-spanning proteins, including ion channels. Large conductance, voltage- and Ca²⁺-gated K⁺ (BK) channels link membrane potential to cell Ca²⁺ homeostasis. Thus, they control many physiological processes and participate in pathophysiological mechanisms leading to human disease. Because plasmalemma BK channels cluster in CLR-rich membrane microdomains, a major driving force for studying BK channel-CLR interactions is determining how membrane CLR controls the BK current phenotype, including its pharmacology, channel sorting, distribution, and role in cell physiology. Since both BK channels and CLR tissue levels play a pathophysiological role in human disease, identifying functional and structural aspects of the CLR-BK channel interaction may open new avenues for therapeutic intervention. Here, we review the studies documenting membrane CLR-BK channel interactions, dissecting out the many factors that determine the final BK current response to changes in membrane CLR content. We also summarize work in reductionist systems where recombinant BK protein is studied in artificial lipid bilayers, which documents a direct inhibition of BK channel activity by CLR and builds a strong case for a direct interaction between CLR and the BK channel-forming protein. Bilayer lipid-mediated mechanisms in CLR action are also discussed. Finally, we review studies of BK channel function during hypercholesterolemia, and underscore the many consequences that the CLR-BK channel interaction brings to cell physiology and human disease.

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.

The BK channel accessory β1 subunit determines alcohol-induced cerebrovascular constriction

Ethanol‐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 i 2 + lower and higher than ∼15 μM, respectively. By increasing cbv1s apparent Ca i 2 + ‐sensitivity, accessory BK β1 subunits shift the activation‐to‐inhibition crossover of EtOH action to <3 μM Ca i 2 + , with consequent inhibition of current under conditions found during myocyte contraction. Knocking‐down KCNMB1 suppresses EtOH‐reduction of arterial myocyte BK current and vessel diameter. Therefore, BK β1 is the molecular effector of alcohol‐induced BK current inhibition and cerebrovascular constriction.

The second transmembrane domain of the large conductance, voltage- and calcium-gated potassium channel β1 subunit is a lithocholate sensor

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 β 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 β 1 to mediate LC action; β 1 and a simple phospholipid microenvironment suffice. Since β 1 senses LC but β 4 does not, we made chimeras swapping regions between these subunits and, following channel heterologous expression, demonstrate that β 1 TM2 is a bile acid‐recognizing sensor.

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.

The steroid interaction site in transmembrane domain 2 of the large conductance, voltage- and calcium-gated potassium (BK) channel accessory β1 subunit

Large conductance, voltage- and calcium-gated potassium (BK) channels regulate several physiological processes, including myogenic tone and thus, artery diameter. Nongenomic modulation of BK activity by steroids is increasingly recognized, but the precise location of steroid action remains unknown. We have shown that artery dilation by lithocholate (LC) and related cholane steroids is caused by a 2× increase in vascular myocyte BK activity (EC50 = 45 μM), an action that requires β1 but not other (β2–β4) BK accessory subunits. Combining mutagenesis and patch-clamping under physiological conditions of calcium and voltage on BK α- (cbv1) and β1 subunits from rat cerebral artery myocytes, we identify the steroid interaction site from two regions in BK β1 transmembrane domain 2 proposed by computational dynamics: the outer site includes L157, L158, and T165, whereas the inner site includes T169, L172, and L173. As expected from computational modeling, cbv1+rβ1T165A,T169A channels were LC-unresponsive. However, cbv1 + rβ1T165A and cbv1 + rβ1T165A,L157A,L158A were fully sensitive to LC. Data indicate that the transmembrane domain 2 outer site does not contribute to steroid action. Cbv1 + rβ1T169A was LC-insensitive, with rβ1T169S being unable to rescue responsiveness to LC. Moreover, cbv1 + rβ1L172A, and cbv1 + rβ1L173A channels were LC-insensitive. These data and computational modeling indicate that tight hydrogen bonding between T169 and the steroid α-hydroxyl, and hydrophobic interactions between L172,L173 and the steroid rings are both necessary for LC action. Therefore, β1 TM2 T169,L172,L173 provides the interaction area for cholane steroid activation of BK channels. Because this amino acid triplet is unique to BK β1, our study provides a structural basis for advancing β1 subunit–specific pharmacology of BK channels.