BK Β1 Research Articles

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.

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 (30-60mM) 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. We used protein biochemistry, patch-clamp on engineered channel subunits, reversible cDNA permeabilization of KCNMB1 K/O mouse arteries and artery in vitro pressurization. Ethanol inhibition of BK current was facilitated by β1 but not β4 subunits. Furthermore, only BK complexes containing β chimeras with β1 transmembrane (TM) domains on a β4 background or with a β1 TM2 domain on a β4 background displayed ethanol responses identical to those of BK complexes including wild-type β1. Moreover, β1 TM2 itself but not other β regions were necessary for ethanol-induced cerebral artery constriction. BK β1 TM2 is necessary for this subunit to enable ethanol-induced inhibition of myocyte BK channels and cerebral artery constriction at physiological Ca2+ and voltages. Thus, novel agents that target β1 TM2 may be considered to counteract ethanol-induced cerebral artery constriction and associated cerebrovascular conditions.

Abstract 043: Influence of Sex, Hormones and Age on Cerebrovascular Function: Role of Large Conductance Potassium Channel Subunits

Although adult females are protected, the cerebrovascular disease is greater in aged post-menopause females than in age matched males, but the role of vascular function and large conductance potassium (BK) channel is not clear. We hypothesize that “compared to adult males, cerebral vasculature of intact adult female rats displays an attenuated myogenic response and greater intrinsic tone due to differential expression and function of BK channel subunits. Aging or menopause exaggerates myogenic response, diminishes intrinsic tone and thus, vasodilatory capacity in females more than males due to a reduction in the BK subunit function.” Our results suggest that middle cerebral arteries (MCA) of intact adult females had attenuated myogenic response compared to adult males and ovariectomy (OVX) females (% change in diameter: 16±8, -25±4 and -49±7). Development of intrinsic tone was greater in intact adult females than adult males and OVX females (% intrinsic tone: 47±5, 27±4 and 15±5). The ratio of BK β1 to α subunits was less in intact adult females than adult males and OVX females (0.4±0.07, 2±0.3 and 0.6±0.1). Spontaneous transient outward currents (STOC) were higher in smooth muscle cells of intact adult females compared to adult males (pA: 204±27 and 128±7). While aging exaggerated the myogenic response in females, it had no effect in males (fold change 4±1 Vs 0.8±0.5). However, intrinsic tone was decreased in aged females but increased in aged males (% change -43±9 Vs 37± 6). Aging decreased the amplitude of STOCs but with a greater magnitude of effect in females (fold change females: -4.9±1; males: -0.8±0.2). While aging increased BKα subunit, it decreased β1 subunit protein (β1 change: females -52±8; males -37±13). Acute activation of BKα subunit by NS1619, while had no effect in intact adult females (-5±3%), it dilated the MCA of adult males (32±9%). However, acute activation of BKβ1 by estradiol while relaxed MCA of adult females (35±7%) and males (22±2%), it had less effect in OVX females (-7±8%) and aged females (4±9%). In conclusion, a higher incidence of cerebrovascular disease in aged and menopause female may be due to sex-specific changes in vascular properties and BK channels that could result in diminished estradiol-mediated vasodilation.

Membrane depolarization activates BK channels through ROCK-mediated β1 subunit surface trafficking to limit vasoconstriction.

Membrane depolarization of smooth muscle cells (myocytes) in the small arteries that regulate regional organ blood flow leads to vasoconstriction. Membrane depolarization also activates large-conductance calcium (Ca2+)-activated potassium (BK) channels, which limits Ca2+ channel activity that promotes vasoconstriction, thus leading to vasodilation. We showed that in human and rat arterial myocytes, membrane depolarization rapidly increased the cell surface abundance of auxiliary BK β1 subunits but not that of the pore-forming BKα channels. Membrane depolarization stimulated voltage-dependent Ca2+ channels, leading to Ca2+ influx and the activation of Rho kinase (ROCK) 1 and 2. ROCK1/2-mediated activation of Rab11A promoted the delivery of β1 subunits to the plasma membrane by Rab11A-positive recycling endosomes. These additional β1 subunits associated with BKα channels already at the plasma membrane, leading to an increase in apparent Ca2+ sensitivity and activation of the channels in pressurized arterial myocytes and vasodilation. Thus, membrane depolarization activates BK channels through stimulation of ROCK- and Rab11A-dependent trafficking of β1 subunits to the surface of arterial myocytes.

Maternal high salt diet altered Adenosine-mediated vasodilatation via PKA/BK channel pathway in offspring rats.

High salt (HS) diets are related to cardiovascular diseases, and prenatal HS was suggested to increase risks of coronary artery diseases in the offspring. This study tested the hypothesis that prenatal HS may influence Adenosine-induced vasodilatation via protein kinase A (PKA) pathway in coronary arteries. Sprague-Dawley rats were fed with 8% salt diet for gestation, the control was fed with 0.3% salt diet. Coronary arteries from male adult offspring were tested for K+ channels and Adenosine signal pathways. Adenosine-mediated vasodilatation was reduced in coronary arteries in HS. There was no difference in gene expression of A2A receptors between the two groups. After pretreatment with PKA inhibitor, vasodilatation to Adenosine was decreased to a smaller extent in HS than that in control. Forskolin (activator of adenylate cyclase)-mediated vasodilatation was decreased in HS. Iberiotoxin (large-conductance Ca2+ -activated K+ channel [BK channel] inhibitor) attenuated Forskolin-induced vasodilatation in control, not in HS group. Currents of BK channels decreased in coronary artery smooth muscle cells, and PKA-modulated BK channel functions were declined. Protein levels of BK β1 and PKA C-subunits in coronary arteries of HS offspring were reduced. Prenatal HS diets altered Adenosine-mediated coronary artery vasodilatation in the offspring, which was linked to downregulation of cAMP/PKA/BK channel pathway.

Differential Expression of BK Channel Beta1 Subunits in Rat Mesenteric, Coronary and Cerebral Arteries

Large conductance, Ca2+i/voltage-gated K+ (BK) channels regulate several physiological processes (Yang et et al., 2015). In smooth muscle, BK complexes include channel-forming α and auxiliary β1 subunits (Brenner et al., 2000). BK β1 increases the channel's apparent Ca2+ sensitivity (Brenner et al.,2000) and is required for channel activation by lithocholate (LCA)(Bukiya et al., 2009). Here, we studied the expression of BK α and β1 subunits in mesenteric, coronary and cerebral (anterior, middle, posterior and basilar) arteries, and the contribution of such differential expression to LCA-induced artery dilation. Thus, we conducted quantitative real-time PCR, fluorescence confocal imaging and patch-clamp electrophysiology on freshly isolated cerebral artery myocytes, and diameter determination of arteries pressurized in vitro. Data show that: 1) BK α mRNA is higher in cerebral anterior and basilar arteries. In turn, β1 mRNA is higher in basilar and coronary arteries; 2) BK α protein levels at the myocyte membrane surface are similar across the different arteries whereas β1 levels are higher in basilar and coronary arteries; 3) BK channel basal activity is also higher in basilar and coronary artery myocytes; 4) Likewise, BK channels from basilar and coronary artery myocytes are more responsive to LCA; 5) Finally, LCA-induced dilation of basilar and coronary arteries is higher than dilation of other arteries. Our results demonstrate a differential protein expression and functional impact on both basal channel activity and its response to LCA, of BK β1 subunits across arteries from cerebral and systemic vasculatures. In particular, basilar and coronary artery myocytes show an increased biochemical and functional presence of BK β1 subunits, suggesting that these vascular territories are more susceptible to modulation via BK β1. Support: HL104631, R37AA11560 (AMD); AHA Pre-doctoral Fellowship (GK).

Differential distribution and functional impact of BK channel beta1 subunits across mesenteric, coronary, and different cerebral arteries of the rat

Large conductance, Ca2+i- and voltage-gated K+ (BK) channels regulate myogenic tone and, thus, arterial diameter. In smooth muscle (SM), BK channels include channel-forming α and auxiliary β1 subunits. BK β1 increases the channel's Ca2+ sensitivity, allowing BK channels to negatively feedback on depolarization-induced Ca2+ entry, oppose SM contraction and favor vasodilation. Thus, endothelial-independent vasodilation can be evoked though targeting of SM BK β1 by endogenous ligands, including lithocholate (LCA). Here, we investigated the expression of BK β1 across arteries of the cerebral and peripheral circulations, and the contribution of such expression to channel function and BK β1-mediated vasodilation. Data demonstrate that endothelium-independent, BK β1-mediated vasodilation by LCA is larger in coronary (CA) and basilar (BA) arteries than in anterior cerebral (ACA), middle cerebral (MCA), posterior cerebral (PCA), and mesenteric (MA) arteries, all arterial segments having a similar diameter. Thus, differential dilation occurs in extracranial arteries which are subjected to similar vascular pressure (CA vs. MA) and in arteries that irrigate different brain regions (BA vs. ACA, MCA, and PCA). SM BK channels from BA and CA displayed increased basal activity and LCA responses, indicating increased BK β1 functional presence. Indeed, in the absence of detectable changes in BK α, BA and CA myocytes showed an increased location of BK β1 in the plasmalemma/subplasmalemma. Moreover, these myocytes distinctly showed increased BK β1 messenger RNA (mRNA) levels. Supporting a major role of enhanced BK β1 transcripts in artery dilation, LCA-induced dilation of MCA transfected with BK β1 complementary DNA (cDNA) was as high as LCA-induced dilation of untransfected BA or CA.

Distinct mechanisms underlying cholesterol protection against alcohol-induced BK channel inhibition and resulting vasoconstriction

Alcohol (ethanol) at concentrations reached in blood following moderate to heavy drinking (30–80mM) reduces cerebral artery diameter via inhibition of voltage- and calcium-gated potassium channels of large conductance (BK) in cerebral artery smooth muscle. These channels consist of channel-forming α and regulatory β1 subunits. A high-cholesterol diet protects against ethanol-induced constriction via accumulation of cholesterol within the vasculature. The molecular mechanisms of this protection remain unknown. In the present work, we demonstrate that in vitro cholesterol enrichment of rat middle cerebral arteries significantly increased cholesterol within arterial tissues and blunted constriction by 50mM of ethanol. Ethanol-induced BK channel inhibition in inside-out patches excised from freshly isolated cerebral artery myocytes was also abolished by cholesterol enrichment. Enrichment of arteries with enantiomeric cholesterol (ent-cholesterol) also blunted BK channel inhibition and cerebral artery constriction in response to ethanol. The similar protection of cholesterol and ent-cholesterol against ethanol action indicates that this protection does not require protein site(s) that specifically sense natural cholesterol. Cholesterol-driven protection against ethanol-induced BK channel inhibition and vasoconstriction was replicated in myocytes and middle cerebral arteries of C57BL/6 mice. BK β1 subunits are known to regulate vascular diameter and its modification by ethanol. However, blunting of an ethanol effect by in vitro cholesterol enrichment was observed in arteries and myocyte membrane patches from BK β1 (KCNMB1) knockout mice. Thus, BK β1 subunits are not needed for cholesterol protection against ethanol effect on BK channel function and cerebral artery diameter.

Differential Expression of BK Channel Alpha and Beta1 Subunits in Rat Cerebral Arteries

Large conductance, Ca2+i/voltage-gated K+ channels (BK) regulate several physiological processes (Dopico et al., 2012). Native BK complexes consist of channel-forming α and auxiliary β subunits. Four β subunit types were identified, with β1 being highly expressed in smooth muscle (Brenner et al., 2000). BK β1 modifies the channel response to ligands: it increases the apparent Ca2+ sensitivity, mediates channel activation by cholanes (Brenner et al., 2000; Bukiya et al., 2009) and controls cerebral artery tone modification by selective ligands, including lithocholate (Bukiya et al., 2007; 2013). Here, we studied the expression of BK α and β1 subunits across basilar, anterior, middle and posterior cerebral arteries, and the possible contribution of their expression to channel phenotype and cerebral artery diameter response to lithocholate. Methods included quantitative real-time PCR, confocal microscopy, smooth muscle patch-clamp electrophysiology, and diameter determination of in vitro pressurized cerebral arteries. Results demonstrate: 1) BK α and β1 mRNA transcripts are expressed at higher levels in basilar arteries; 2) BK α protein surface levels in myocyte membranes are similar across the different intracranial territories while β1 levels are higher in basilar artery; 3) BK channel activity is also higher in myocytes from basilar arteries; 4) myocyte BK channels in basilar arteries show the highest response to lithocholate; 5) lithocholate-induced dilation of basilar arteries is significantly higher than those measured in other intracranial territories. Collectively, our results demonstrate differential BK β1 expression, as well as its contribution to BK current phenotype and vasodilation by lithocholate, across the major branches of the Willis’ circle. In particular, basilar arteries seem to be highly sensitive to vasoactive agents that act via BK β1 subunits.Support: HL104631 and AA11560 (AMD), and AHA Predoctoral Fellowship (GK).

Age-Dependent Susceptibility to Alcohol-Induced Cerebral Artery Constriction.

BackgroundAge has been recognized as an important contributor into susceptibility to alcohol-driven pathology.PurposeWe aimed at determining whether alcohol-induced constriction of cerebral arteries was age-dependent.Study designWe used rat middle cerebral artery (MCA) in vitro diameter monitoring, patch-clamping and fluorescence labeling of myocytes to study an age-dependent increase in the susceptibility to alcohol in 3 (50 g), 8 (250 g), and 15 (440 g) weeks-old rats.ResultsAn age-dependent increase in alcohol-induced constriction of MCA could be observed in absence of endothelium, which is paralleled by an age-dependent increase in both protein level of the calcium-/voltage-gated potassium channel of large conductance (BK) accessory β1 subunit and basal BK channel activity. Ethanol-induced BK channel inhibition is increased with age.ConclusionsWe demonstrate an increased susceptibility of MCA to ethanol-induced constriction in a period equivalent to adolescence and early adulthood when compared to pre-adolescence. Our work suggests that BK β1 constitutes a significant contributor to age-dependent changes in the susceptibility of cerebral arteries to ethanol.

BK Channel β1 Subunit Contributes to Behavioral Adaptations Elicited by Chronic Intermittent Ethanol Exposure.

Large conductance, calcium- and voltage-activated potassium (BK) channels regulate neuronal excitability and neurotransmission. They can be directly activated by ethanol (EtOH) and they may be implicated in EtOH dependence. In this study, we sought to determine the influence of the auxiliary β1 and β4 subunits on EtOH metabolism, acute sensitivity to EtOH intoxication, acute functional tolerance, chronic tolerance, and handling-induced convulsions during withdrawal. Motor coordination, righting reflex, and body temperature were evaluated in BK β1 and β4 knockout, heterozygous, and wild-type mice following acute EtOH administration. Chronic tolerance and physical dependence were induced by chronic intermittent inhalation of EtOH vapor. Constitutive deficiency in BK β1 or β4 subunits did not alter the clearance rate of EtOH, acute sensitivity to EtOH-induced ataxia, sedation, and hypothermia, nor acute functional tolerance to ataxia. BK β1 deletion reduced chronic tolerance to sedation and abolished chronic tolerance to hypothermia, while BK β4 deletion did not affect these adaptations to chronic EtOH exposure. Finally, the absence of BK β1 accelerated the appearance, while the absence of BK β4 delayed the resolution, of the hyperexcitable state associated with EtOH withdrawal. Altogether, the present findings reveal the critical role of BK β1 in behavioral adaptations to prolonged, repeated EtOH intoxication.

Membrane potential and Ca2+ concentration dependence on pressure and vasoactive agents in arterial smooth muscle: A model.

Arterial smooth muscle (SM) cells respond autonomously to changes in intravascular pressure, adjusting tension to maintain vessel diameter. The values of membrane potential (Vm) and sarcoplasmic Ca(2+) concentration (Ca(in)) within minutes of a change in pressure are the results of two opposing pathways, both of which use Ca(2+) as a signal. This works because the two Ca(2+)-signaling pathways are confined to distinct microdomains in which the Ca(2+) concentrations needed to activate key channels are transiently higher than Ca(in). A mathematical model of an isolated arterial SM cell is presented that incorporates the two types of microdomains. The first type consists of junctions between cisternae of the peripheral sarcoplasmic reticulum (SR), containing ryanodine receptors (RyRs), and the sarcolemma, containing voltage- and Ca(2+)-activated K(+) (BK) channels. These junctional microdomains promote hyperpolarization, reduced Ca(in), and relaxation. The second type is postulated to form around stretch-activated nonspecific cation channels and neighboring Ca(2+)-activated Cl(-) channels, and promotes the opposite (depolarization, increased Ca(in), and contraction). The model includes three additional compartments: the sarcoplasm, the central SR lumen, and the peripheral SR lumen. It incorporates 37 protein components. In addition to pressure, the model accommodates inputs of α- and β-adrenergic agonists, ATP, 11,12-epoxyeicosatrienoic acid, and nitric oxide (NO). The parameters of the equations were adjusted to obtain a close fit to reported Vm and Ca(in) as functions of pressure, which have been determined in cerebral arteries. The simulations were insensitive to ± 10% changes in most of the parameters. The model also simulated the effects of inhibiting RyR, BK, or voltage-activated Ca(2+) channels on Vm and Ca(in). Deletion of BK β1 subunits is known to increase arterial-SM tension. In the model, deletion of β1 raised Ca(in) at all pressures, and these increases were reversed by NO.

Direct Modulation of Calcium- and Voltage-Gated Potassium Channels of Large Conductance by Leukotrienes

Calcium/voltage-gated, large-conductance potassium (BK) channels critically control smooth muscle contractility. Modulation of BK by direct interaction between lipids and channel protein has received increasing attention. Leukotrienes (LTA4-E4) are lipid mediators of inflammation. We performed patch-clamp in Xenopus oocytes that co-expressed BK channel-forming (cbv1; AY330293) and accessory β1 (FJ154955) subunits cloned from rat vascular myocytes. Leukotrienes were applied at 0.1 nM-10 μM to either leaflet of cell-free membranes at a range of [Ca2+]i and transmembrane voltage. LTB4 reversibly increased BK steady-state activity (EC50=1nM; Emax ≈10nM), with physiological [Ca2+]i and voltages favoring this activation. Cbv1 or cbv1+β2 were LTB4-resistant. Computational docking placed LTB4 onto the cholane steroid-sensing region in the BK β1 transmembrane domain 2. Consistent with the idea that cholanes and LTB4 share the docking region, co-application of LTB4 and cholane did not further increase LTB4-induced BK channel activation. LTB4 failed to activate cholane-insensitive cbv1+β1T169A. Moreover, LTB4 failed to activate BK channel containing β1A176S or β1K179I. Ala176 and Lys179 are computationally predicted to participate in LTB4-docking but were proven irrelevant for cholane-sensing. Thus, although cholanes and LTB4 share a docking region, LTB4 fit onto the sensing region is tighter and may explain higher potency of LTB4 to activate BK. Co-application of LTB4 with LTA4,LTC4,LTD4 or LTE4 suppressed LTB4-induced channel activation. Inactive leukotrienes only dock onto a portion of the site likely preventing LTB4's tight docking. In summary, we first document that two endogenous lipids from different chemical families (heterocyclic vs. linear) share their site on a channel protein. Thus, cross-talk between leukotrienes and cholanes might converge on regulation of smooth muscle contractility via BK β1. Moreover, identification of LTB4 as a potent ligand for BK channels may be important for developing β1-specific BK channel activators.

Activation of Calcium- and Voltage-gated Potassium Channels of Large Conductance by Leukotriene B4

Calcium/voltage-gated, large conductance potassium (BK) channels control numerous physiological processes, including myogenic tone. BK channel regulation by direct interaction between lipid and channel protein sites has received increasing attention. Leukotrienes (LTA4, LTB4, LTC4, LTD4, and LTE4) are inflammatory lipid mediators. We performed patch clamp studies in Xenopus oocytes that co-expressed BK channel-forming (cbv1) and accessory β1 subunits cloned from rat cerebral artery myocytes. Leukotrienes were applied at 0.1 nm-10 μm to either leaflet of cell-free membranes at a wide range of [Ca(2+)]i and voltages. Only LTB4 reversibly increased BK steady-state activity (EC50 = 1 nm; Emax reached at 10 nm), with physiological [Ca(2+)]i and voltages favoring this activation. Homomeric cbv1 or cbv1-β2 channels were LTB4-resistant. Computational modeling predicted that LTB4 docked onto the cholane steroid-sensing site in the BK β1 transmembrane domain 2 (TM2). Co-application of LTB4 and cholane steroid did not further increase LTB4-induced activation. LTB4 failed to activate β1 subunit-containing channels when β1 carried T169A, A176S, or K179I within the docking site. Co-application of LTB4 with LTA4, LTC4, LTD4, or LTE4 suppressed LTB4-induced activation. Inactive leukotrienes docked onto a portion of the site, probably preventing tight docking of LTB4. In summary, we document the ability of two endogenous lipids from different chemical families to share their site of action on a channel accessory subunit. Thus, cross-talk between leukotrienes and cholane steroids might converge on regulation of smooth muscle contractility via BK β1. Moreover, the identification of LTB4 as a highly potent ligand for BK channels is critical for the future development of β1-specific BK channel activators.

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 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.

BK β1 Transmembrane Regions Critically Control the Characteristic Phenotype of β1-Containing Bk Channel Currents

Voltage- and Ca2+-gated, large conductance K+ (BK) channels are ubiquitously expressed and play a key role in linking membrane excitability to Ca2+ signaling. In most tissues, BK channel complexes consist of channel-forming α and regulatory β subunits. Each β contains short intracellular N- and C-termini, and two transmembrane domains (TM1, TM2) joined by an extracellular loop. The smooth muscle-abundant BK β1 increases the channel's apparent Ca2+ sensitivity and is required for channel activation by cholane steroids (Brenner et al., 2000; Bukiya et al., 2009). The participation of different β1 regions in regulating channel function has not been fully settled. Here, we used BK channel-forming cbv1 and β1 subunits cloned from rat cerebral artery myocytes, neuron-abundant β4, and chimeras constructed by swapping regions between β1 and β4 to determine the contribution of specific β1 regions to the β1-containing BK phenotype. After co-expressing cbv1 with wt βs or chimeric βs in Xenopus oocytes, macroscopic currents were evoked by 200 ms-long, 10 mV depolarizing steps from −150 to +150 mV (VH=-80 mV). At Ca2+ levels found near the BK channel during smooth muscle cell contraction (10 μM), channel complexes that included chimeras combining both TMs from β1 with remaining regions (EC loop, IC ends) from β4 showed a phenotype (Vhalf, τact, τdeact) identical to that of channel complexes containing wt β1. However, this phenotype could not be evoked by channel complexes that included chimeras combining either β1 TM1 or β1 TM2 with the remaining β4 regions (TM2-EC loop-IC ends and TM1-EC loop-IC ends, respectively). We conclude that at physiological voltages and Ca2+, both TMs from β1 are necessary to confer the characteristic current phenotype of β1-containing BK channels. Support: R37-AA11560, R01-HL10463 (AMD).

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

The Ca(2+)/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.

Abnormal Ca2+ Spark/STOC Coupling in Cerebral Artery Smooth Muscle Cells of Obese Type 2 Diabetic Mice

Diabetes is a major risk factor for stroke. However, the molecular mechanisms involved in cerebral artery dysfunction found in the diabetic patients are not completely elucidated. In cerebral artery smooth muscle cells (CASMCs), spontaneous and local increases of intracellular Ca2+ due to the opening of ryanodine receptors (Ca2+ sparks) activate large conductance Ca2+-activated K+ (BK) channels that generate spontaneous transient outward currents (STOCs). STOCs have a key participation in the control of vascular myogenic tone and blood pressure. Our goal was to investigate whether alterations in Ca2+ spark and STOC activities, measured by confocal microscopy and patch-clamp technique, respectively, occur in isolated CASMCs of an experimental model of type-2 diabetes (db/db mouse). We found that mean Ca2+ spark amplitude, duration, size and rate-of-rise were significantly smaller in Fluo-3 loaded db/db compared to control CASMCs, with a subsequent decrease in the total amount of Ca2+ released through Ca2+ sparks in db/db CASMCs, though Ca2+ spark frequency remained. Interestingly, the frequency of large-amplitude Ca2+ sparks was also significantly reduced in db/db cells. In addition, the frequency and amplitude of STOCs were markedly reduced at all voltages tested (from −50 to 0 mV) in db/db CASMCs. The latter correlates with decreased BK channel β1/α subunit ratio found in db/db vascular tissues. Taken together, Ca2+ spark alterations lead to inappropriate BK channels activation in CASMCs of db/db mice and this condition is aggravated by the decrease in the BK β1 subunit/α subunit ratio which underlies the significant reduction of Ca2+ spark/STOC coupling in CASMCs of diabetic animals.

BK channel β1 and β4 auxiliary subunits exert opposite influences on escalated ethanol drinking in dependent mice

Large conductance calcium-activated potassium (BK) channels play a key role in the control of neuronal activity. Ethanol is a potent activator of BK channel gating, but how this action may impact ethanol drinking still remains poorly understood. Auxiliary β subunits are known to modulate ethanol-induced potentiation of BK currents. In the present study, we investigated whether BK β1 and β4 subunits influence voluntary ethanol consumption using knockout (KO) mice. In a first experiment, mice were first subjected to continuous two-bottle choice (2BC) and were then switched to intermittent 2BC, which progressively increased ethanol intake as previously described in wildtype mice. BK β1 or β4 subunit deficiency did not affect ethanol self-administration under either schedule of access. In a second experiment, mice were first trained to drink ethanol in a limited-access 2BC paradigm. BK β1 or β4 deletion did not affect baseline consumption. Weeks of 2BC were then alternated with weeks of chronic intermittent ethanol (CIE) or air inhalation. As expected, a gradual escalation of ethanol drinking was observed in dependent wildtype mice, while intake remained stable in non-dependent wildtype mice. However, CIE exposure only produced a mild augmentation of ethanol consumption in BK β4 KO mice. Conversely, ethanol drinking increased after fewer CIE cycles in BK β1 KO mice than in wildtype mice. In conclusion, BK β1 or β4 did not influence voluntary ethanol drinking in non-dependent mice, regardless of the pattern of access to ethanol. However, deletion of BK β4 attenuated, while deletion of BK β1 accelerated, the escalation of ethanol drinking during withdrawal from CIE. Our data suggest that BK β1 and β4 subunits have an opposite influence on the negative reinforcing properties of ethanol withdrawal. Modulating the expression, distribution or interactions of BK channel auxiliary subunits may therefore represent a novel avenue for the treatment of alcoholism.

Structural Requirements in the Steroid Lateral Chain for the Activation of β1 Subunit-Containing BK Channels by 5β-Cholanic Acid-3α-Ol Analogues

BK channels regulate many physiological processes and thus constitute an attractive target for drug discovery. Numerous BK channel activators are available. However, these agents usually interact with the ubiquitously distributed channel-forming α subunit and cannot selectively target a particular tissue. Here, we performed a structure-activity relationship study of lithocholic acid (LCA), a cholane that was previously shown to activate BK channels via a steroid site in the accessory BK β1 subunit (Bukiya et al., 2011). This protein is highly abundant in smooth muscle but scarce in most other tissues (Brenner et al., 2000). Modifications to the LCA lateral chain length and functional group yielded a total of 14 compounds. Bath application of these LCA analogs (1-300 μM) to the cytosolic side of Xenopus oocyte membrane patches co-expressing BK channel-forming cbv1 and β1 subunits demonstrates that channel activation (EC50∼50 μM) requires the combination of a critical lateral chain length (optimal at C24), rather small volume of its functional group (65-92 A3), and net negative charge (−0.1 to −0.9 [e]). Thus, analogs having cyano or nitro groups substituting for the carboxyl at C24 in the LCA molecule constitute two novel BK channel activators that satisfy the aforementioned criteria. Data provide detailed structural information that helps us to better understand ligand recognition by the cholane steroid site present in the BK β1 protein, and 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.Support: R01-HL104631; R37-AA011560 (AMD).