BK Alpha Research Articles

Discovery of agonist–antagonist pairs for the modulation of Ca [2]+ and voltage-gated K+ channels of large conductance that contain beta1 subunits

Large conductance, calcium/voltage-gated potassium channels (BK) regulate critical body processes, including neuronal, secretory and smooth muscle (SM) function. While BK-forming alpha subunits are ubiquitous, accessory beta1 subunits are highly expressed in SM. This makes beta1 an attractive target for pharmaceutical development to treat SM disorders, such as hypertension or cerebrovascular spasm. Compounds activating BK via beta1 have been identified, yet they exhibit low potency and off-target effects while antagonists that limit agonist activity via beta 1 remain unexplored. Beta1-dependent BK ligand-based pharmacophore modeling and ZINC database searches identified 15 commercially available hits. Concentration-response curves on BK alpha + beta1 subunit-mediated currents were obtained in CHO cells. One potent (EC50 = 20 nM) and highly efficacious activator (maximal activation = ×10.3 of control) was identified along with a potent antagonist (KB = 3.02 nM), both of which were dependent on beta1. Our study provides the first proof-of-principle that an agonist/antagonist pair can be used to control beta1-containing BK activity.

Loss of Ca2+ spark‐to‐BK channel coupling impairs cerebral autoregulation in hypertension

IntroductionA reduction in blood flow to the brain in hypertension leads to vascular dementia, but what drives this is unknown. In vascular smooth muscle cells (VSMCs) the close spatial coupling (~20 nm) between Ca2+ release events from the sarcoplasmic reticulum (SR) (Ca2+ sparks) and the vasodilatory large‐conductance Ca2+ activated K+ (BK) channels, buffers pressure induced constriction and ensures correct cerebral perfusion.HypothesisImpaired BK channel activation was responsible for the impaired cerebral autoregulation in hypertension‐induced dementia.MethodsMale hypertensive mice (BPH/2) and the normotensive control strain (BPN/3) were euthanized in adhered to the UK Home Office Guidance on the Operation of the Animals (Scientific Procedures) Act 1986. Experiments were carried out using pressure myography, confocal microscopy and electrophysiology techniques.ResultsPressure‐induced contraction was studied at a range of intraluminal pressures (10‐120 mmHg), and cerebral arteries from BPH/2 mice showed a more contractile phenotype compared to BPN/3 controls, confirming impaired autoregulation. BK channel activation by Ca2+ sparks was recorded using perforated patch electrophysiology to record spontaneous transient outward currents (STOCs). STOCs recorded from VSMCs from BPH/2 cerebral arteries, showed a significant reduction in both frequency and amplitude. To determine the mechanism behind this loss of BK activity, we first studied Ca2+ sparks in pressurised cerebral arteries loaded with Fluro‐4‐AM and found no difference between BPH/2 and BPN/3 mice. Next, we looked at BK alpha subunit expression, by recording whole cell BK currents with a fixed [Ca2+] in the pipette solution. Paradoxically, there was an increase in BK current density in the VSMC from the BPH/2 mice compare to BPN/3 mice. We then looked at the Ca2+ sensitivity of the BK channel using single channel electrophysiology, but again there were no differences between the BPH/2 and BPN/3 groups. Subsequently we used live‐cell fluorescent membrane dyes to look at the coupling between the SR (ER‐Tracker) and the plasma membrane (CellMask) in freshly isolated VSMCs. 3D reconstruction of the deconvolved images showed a reduction in both the coupling site volume and frequency in cells isolated from BPH/2 mice.ConclusionsThis data suggests that an increased spatial distance between the SR and the plasma membrane reduces [Ca2+] around the BK channel resulting in increased constriction of the arteries. Opportunities to restore the spark‐to‐BK coupling could be a novel approach to prevent vascular dementia caused by hypertension.

Large conductance voltage- and calcium-gated potassium channels (BK) in cerebral artery myocytes of perinatal fetal primates share several major characteristics with the adult phenotype.

Large conductance voltage- and calcium-gated channels (BK) control fundamental processes, including smooth muscle contractility and artery diameter. We used a baboon (Papio spp) model of pregnancy that is similar to that of humans to characterize BK channels in the middle cerebral artery and its branches in near-term (165 dGa) primate fetuses and corresponding pregnant mothers. In cell-attached patches (K+pipette = 135 mM) on freshly isolated fetal cerebral artery myocytes, BK currents were identified by large conductance, and voltage- and paxilline-sensitive effects. Their calcium sensitivity was confirmed by a lower Vhalf (transmembrane voltage needed to reach half-maximal current) in inside-out patches at 30 versus 3 μM [Ca2+]free. Immunostaining against the BK channel-forming alpha subunit revealed qualitatively similar levels of BK alpha protein-corresponding fluorescence in fetal and maternal myocytes. Fetal and maternal BK currents recorded at 3 μM [Ca2+]free from excised membrane patches had similar unitary current amplitude, and Vhalf. However, subtle differences between fetal and maternal BK channel phenotypes were detected in macroscopic current activation kinetics. To assess BK function at the organ level, fetal and maternal artery branches were pressurized in vitro at 30 mmHg and probed with the selective BK channel blocker paxilline (1 μM). The degree of paxilline-induced constriction was similar in fetal and maternal arteries, yet the constriction of maternal arteries was achieved sooner. In conclusion, we present a first identification and characterization of fetal cerebral artery BK channels in myocytes from primates. Although differences in BK channels between fetal and maternal arteries exist, the similarities reported herein advance the idea that vascular myocyte BK channels are functional near-term, and thus may serve as pharmacological targets during the perinatal-neonatal period.

Voltage Dependence of BK Channels Gating Ring Motion Studied by State Dependent FRET

Large conductance voltage- and calcium-dependent potassium channels (BK) open probability is regulated by changes in transmembrane voltage and intracellular divalent ions (Ca2+ and Mg2+) concentration. The gating ring, formed by the two RCK (Regulator of Conductance of Potassium) domains present in the C-terminal of each subunit of the tetramer, contains the divalent ion binding sites. We previously have showed that large rearrangements of the gating ring occur in a Ca2+ and voltage dependent manner. This movement of the gating ring is not directly related with the opening of the channel. It appears that a change in the voltage dependence of the open probability of the channel does not modify the voltage dependence of the movement of the gating ring. However, the voltage dependence of the movement of the gating ring is shifted toward a more negative potential if we modify the voltage dependence of the voltage sensor by co-expression of the BK alpha subunit with the Beta1 subunit or by mutations. These results indicate that the voltage dependent movement of the gating ring appears to be related to the voltage dependence of the voltage sensor and not directly related with the opening of the gate.

Activation of galanin receptor 2 stimulates large conductance Ca2+-dependent K+ (BK) channels through the IP3 pathway in human embryonic kidney (HEK293) cells

The large conductance Ca2+-activated K+ (BK) channels are widely distributed in the brain, and act as intracellular calcium sensors in neurons. They play an important feedback role in controlling Ca2+ flux and Ca2+-dependent processes, including neurotransmitter release and cellular excitability. In this study, the effects of the neuropeptide galanin on BK channels were examined by determining the whole-cell currents and single-channel activities in human embryonic kidney (HEK293) cells co-expressing GalR2 and the BK alpha subunit. Galanin enhanced the currents of BK channels, in a concentration-dependent and PTX-independent manner, with an ED50 value of 71.8±16.9nM. This activation was mediated by GalR2, since its agonist AR-M1896 mimicked the effect of galanin, and since galanin did not facilitate BK currents in cells co-expressing cDNAs of BK and GalR1 or GalR3. The galanin-induced BK current persisted after replacement with Ca2+-free solution, suggesting that extracellular Ca2+ is not essential. Chelating intracellular Ca2+ by either the slow Ca2+ buffer EGTA or the fast Ca2+ buffer BAPTA abolished galanin-mediated activation of BK channels, indicating the important role of intracellular Ca2+. The role of Ca2+ efflux from the sarcoplasmic reticulum/endoplasmic reticulum (SR/ER) was confirmed by application of thapsigargin, an irreversible inhibitor that depletes Ca2+ from SR/ER. Moreover, the inositol-1,4,5-triphosphate receptor (IP3R) was identified as the mediator responsible for increased intracellular Ca2+ activating BK channels. Taken together, activation of GalR2 leads to elevation of intracellular Ca2+ is due to Ca2+ efflux from ER through IP3R sequentially opening BK channels.

Reduction of large‐conductance Ca2+‐activated K+ channel with compensatory increase of nitric oxide in insulin resistant rats

Cardiovascular disease prevalence and mortality are both increased by insulin resistance, hypertension, and atherosclerosis. The large-conductance Ca(2+)-activated K(+) channel (BK(Ca)) plays a pivotal role in the diastolic function of vascular smooth muscle cells. However, the role of this channel in insulin resistance remains unknown. Male Sprague-Dawley rats were randomly divided into an insulin resistant group and control group. We investigated the BK(Ca) current and subunit expression in myocytes from aortas and mesenteric arteries by Western blot, real-time PCR and the whole-cell patch-clamp methods. BK(Ca) current was decreased in smooth muscle cells in insulin resistant rats, compared with that in control group. Peak BK(Ca) current at + 60 mV was significantly decreased after iberiotoxin (IBTX) perfusion at 100 nmol/L (64.2 ± 4.7 versus 20.3 ± 3.5% in thoracic aortas and 65.6 ± 6.2 versus 29.3 ± 3.9% in mesenteric arteries, both p < 0.01). However, there was no significant difference in BK(Ca) alpha subunit between the two groups, both at the level of mRNA and protein. BK(Ca) beta 1 subunit expression in aortas and mesenteric arteries from the insulin resistant group was lower than in those from control group. The plasma level of nitric oxide was higher in the insulin resistant group than in the control group. Our results demonstrated that the BK(Ca) channel is decreased both in macrovessels and microvessels in insulin resistant rats. These impairments may be related to the down-regulation of β1 subunit expression and compensatory increase in plasma nitric oxide levels.

Stereoselective epoxidation of the last double bond of polyunsaturated fatty acids by human cytochromes P450

Cytochromes P450 (CYPs) metabolize polyunsaturated long-chain fatty acids (PUFA-LC) to several classes of oxygenated metabolites. Through use of human recombinant CYPs, we recently showed that CYP1A1, -2C19, -2D6, -2E1, and -3A4 are mainly hydroxylases, whereas CYP1A2, -2C8, -2C9, and -2J2 are mainly epoxygenases of arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), respectively. It is worth noting that the last double bond of these PUFAs, i.e., omega6 in AA or omega3 in EPA and DHA, respectively, was preferentially epoxidized. In this study, we have characterized the stereoselectivity of this epoxidation reaction by comparison with the PUFA-LC epoxide stereoisomers obtained from the enantioselective bacterial CYP102A1 F87V. The stereoselectivity of the epoxidation of the last olefin of AA (omega6), EPA (omega3), or DHA (omega3) differed between the CYP isoforms but was similar for EPA and DHA. These data give additional insight into the PUFA-LC epoxide enantiomers generated by the hepatic CYPs.

Expression of BK‐type calcium‐activated potassium channel splice variants during chick cochlear development

The appearance of large-conductance, calcium-activated potassium (BK) current is a hallmark of functional maturation in auditory hair cells. Acquisition of this fast-activating current enables high-frequency, graded receptor potentials in all vertebrates and an electrical tuning mechanism in nonmammals. The gene encoding BK alpha subunits is highly alternatively spliced, and the resulting variations in channel isoforms may contribute to functional diversity at the onset of hearing. We examined the tissue specificity of nine BK alpha alternative exons and investigated changes in expression during chick cochlear development using quantitative polymerase chain reaction (qPCR). Each alternative was widely expressed in several tissues except for an insert near the C-terminus Ca(2+) sensing domain, which appeared brain-specific. The only alternative form in the membrane-bound core of the channel was expressed in brain and muscle but was undetected in cochlea. Of the remaining variants, three increased in expression prior to the onset of hearing and acquisition of BK currents. These three variants cause decreased Ca(2+) sensitivity or increased intracellular retention, traits that would not easily explain the advent of calcium-sensitive currents at embryonic day (E)18-19. Expression levels of other variants were mature and stable by E15, days before currents were acquired. Surface expression of C-terminal isoforms was examined using patch-clamp electrophysiology and immunocytochemistry. C-terminal variants that exhibit robust surface expression appeared in the membrane at E18, even though transcripts were unchanged during development starting from E12. These results indicate that delays in protein synthesis and trafficking/scaffolding of channel subunits underlie the late acquisition of BK currents in cochlear hair cells.

Beta Subunits Bridge Two Alpha Subunits Within the BK Channel Tetramer

BK potassium channels contain four pore-forming alpha subunits and four modulatory beta subunits. Transmembrane (TM) helices S1-S6 of BK alpha are homologous to S1-S6 of other V-gated potassium channels; however, BK alpha contains a unique seventh TM helix, S0, N terminal to S1. The beta subunits contain two TM helices, TM1 and TM2. From the extent of endogenous disulfide crosslinking between Cys substituted for the four residues just flanking the extracellular ends of alpha S0-S6 and of beta1 TM1 and TM2, we previously inferred that the flank of S0 was closest to the four-residue loop between S3 and S4 and also contacted the flanks of S1 and S2. Furthermore, the flank of beta1 TM1 was closest to the flanks of S1 and S2, and the flank of TM2 was closest to the flank of S0. We have now extended this analysis to the membrane doman. We find that Cys in the first helical turn of S0 within the membrane forms disulfides with Cys subsituted in the first helical turns of S3 and S4 but not with similarly located Cys in S1 or S2. Thus, in the membrane, S0 is next to S3 and S4 but not to S1 and S2, although the flank of S0 reaches the flanks of S1 and S2. Furthermore, co-expression of the double-Cys mutant of alpha, W23C in the first helical turn of S0 and F144C in the S2 flank, and the double-Cys mutant of beta1, Y42C in the TM1 flank and L157C in the first helical turn of TM2, resulted in the crosslinking of two alphas through one beta1, S0 to TM2 and TM1 to S2. Thus, TM1 and TM2 of each beta subunit lie between the voltage-sensing domains (S0-S4) of adjacent alpha subunits.

Mechanism of Differential Cardiovascular Response to Propofol in Dahl Salt-Sensitive, Brown Norway, and Chromosome 13-Substituted Consomic Rat Strains: Role of Large Conductance Ca2+ and Voltage-Activated Potassium Channels

Cardiovascular sensitivity to general anesthetics is highly variable among individuals in both human and animal models, but little is known about the genetic determinants of drug response to anesthetics. Recently, we reported that propofol (2,6-diisopropylphenol) causes circulatory instability in Dahl salt-sensitive SS/JRHsdMcwi (SS) rats but not in Brown Norway BN/NHsdMcwi (BN) rats and that these effects are related to genes on chromosome 13. Based on the hypothesis that propofol does target mesenteric circulation, we investigated propofol modulation of mesenteric arterial smooth muscle cells (MASMC) in SS and BN rats. The role of chromosome 13 was tested using SS-13(BN)/Mcwi and BN-13(SS)/Mcwi consomic strains with chromosome 13 substitution. Propofol (5 microM) produced a greater in situ hyperpolarization of MASMC membrane potential in SS than BN rats, and this effect was abrogated by iberiotoxin, a voltage-activated potassium (BK) channel blocker. In inside-out patches, the BK channel number, P(o), and apparent Ca(2+) sensitivity, and propofol sensitivity all were significantly greater in MASMC of SS rats. The density of whole-cell BK current was increased by propofol more in SS than BN myocytes. Immunolabeling confirmed higher expression of BK alpha subunit in MASMC of SS rats. Furthermore, the hyperpolarization produced by propofol, the BK channel properties, and propofol sensitivity were modified in MASMC of SS-13(BN)/Mcwi and BN-13(SS)/Mcwi strains toward the values observed in the background SS and BN strains. We conclude that differential function and expression of BK channels, resulting from genetic variation within chromosome 13, contribute to the enhanced propofol sensitivity in SS and BN-13(SS)/Mcwi versus BN and SS-13(BN)/Mcwi strains.

Disulfide Crosslinking Between BK Channel Alpha And Beta1 Subunits In The Membrane Domain

The large-conductance, Ca2+- and voltage-activated potassium channel (BK) alpha subunit is modulated by one of four types of beta subunits, each imparting unique electrophysiological properties. The beta1 subunit is expressed in smooth muscle, where it renders the BK channel sensitive to cytoplasmic calcium in a voltage range near the smooth-muscle resting potential and slows activation and deactivation. Previously, we inferred from the extents of endogenous disulfide bond formation between cysteines substituted in the extracellular flanks of the transmembrane (TM) helices of alpha and of beta1, that the extracellular flank of S0, the unique seventh TM helix of BK alpha, is surrounded on three sides by the extracellular flanks of S1 and S2 and the four-residue, extracellular loop between S3 and S4. We also found that the extracellular flanks of beta1 TM2 and alpha S0 are close and that that the extracellular end of beta1 TM1 is close to the extracellular flanks of both alpha S1 and S2. Within the membrane domain, where presumably there is less flexibility than in the extracellular flanks, we also see endogenous (no added reagents) crosslinking one to two helical turns into the membrane domain. Within the same alpha subunit, Cys-substituted residues in S0 readily form disulfides with Cys-substituted residues in S4, and to a lesser extent with Cys-substituted residues in S3. Also within the membrane domain, we find that cysteines in alpha S0 readily form disulfides with cysteines in beta TM2. Thus, the positions of alpha S0 and of beta1 TM2 relative to alpha S0 are similar in the membrane domain and in the extracellular flanks of these TM helices.Supported by HL081172 and NS054946

Locations Of The Beta2 Transmembrane Helices In The BK Potassium Channel

The large-conductance, Ca2+- and voltage-activated potassium channel (BK) alpha subunit is modulated by one of four types of beta subunits, each imparting unique electrophysiological properties. Beta2, which is expressed in the brain, adrenal chromaffin cells, pancreas, and ovaries, increases Ca2+ sensitivity of BK alpha, by shifting voltage-dependent activation to more negative voltages, and causes N-type inactivation. We explored the contacts between alpha and beta2 subunits by determining the extent of endogenous disulfide bond formation between cysteines substituted in the extracellular flanks of the two beta2 transmembrane (TM) helices, TM1 and TM2, and in the extracellular flanks of each of the seven alpha TM helices, S0-S6. We found that the extracellular ends of beta2 TM2 and alpha S0 are close and that beta2 TM1 is close to both S1 and S2. At their extracellular ends, TM1 and TM2 are not close to S3, S4, S5 or S6. Beta2 TM1 and TM2 are like pincers on either side of the alpha voltage-sensor domain, S0-S4. In all tested pairs of cysteine-substituted alpha and beta2, we found that disulfide crosslinks favored the closed state, shifting the conductance-voltage curves toward more positive potentials and slowing the kinetics of activation. N-type inactivation, involving three specific beta2 residues in its cytoplasmic, N-terminal segment preceding TM1, was not affected by any of the crosslinking. This is consistent with the above locations of TM1 and TM2 because a minimum of 12 residues, spanning up to 40 Å, allows the three N-terminal inactivating residues to reach the pore (Xia et al., 2003 J. Gen. Physiol. 121:125). The positions of the beta2 TM helices are similar to the locations that we previously reported for beta1 TM1 and TM2 (Liu et al., 2008 PNAS 105:10727). Supported by NIH NS054946.

Activation of big conductance Ca2+-activated K+ channels (BK) protects the heart against ischemia–reperfusion injury

Activation of the large-conductance Ca(2+)-activated K(+) channel (BK) in the cardiac inner mitochondrial membrane has been suggested to protect the heart against ischemic injury. However, these findings are limited by the low selectivity profile and potency of the BK channel activator (NS1619) used. In the present study, we address the cardioprotective role of BK channels using a novel, potent, selective, and chemically unrelated BK channel activator, NS11021. Using electrophysiological recordings of heterologously expressed channels, NS11021 was found to activate BK alpha + beta1 channel complexes, while producing no effect on cardiac K(ATP) channels. The cardioprotective effects of NS11021-induced BK channel activation were studied in isolated, perfused rat hearts subjected to 35 min of global ischemia followed by 120 min of reperfusion. 3 microM NS11021 applied prior to ischemia or at the onset of reperfusion significantly reduced the infarct size [control: 44.6 +/- 2.0%; NS11021: 11.4 +/- 2.0%; NS11021 at reperfusion: 19.8 +/- 3.3% (p < 0.001 for both treatments compared to control)] and promoted recovery of myocardial performance. Co-administration of the BK-channel inhibitor paxilline (3 microM) antagonized the protective effect. These findings suggest that tissue damage induced by ischemia and reperfusion can be reduced by activation of cardiac BK channels.

Aldosterone increases KCa1.1 (BK) channel‐mediated colonic K+ secretion

Mammalian K(+) homeostasis results from highly regulated renal and intestinal absorption and secretion, which balances the unregulated K(+) intake. Aldosterone is known to enhance both renal and colonic K(+) secretion. In mouse distal colon K(+) secretion occurs exclusively via luminal K(Ca)1.1 (BK) channels. Here we investigate if aldosterone stimulates colonic K(+) secretion via BK channels. Luminal Ba(2+) and iberiotoxin (IBTX)-sensitive electrogenic K(+) secretion was measured in Ussing chambers. In vivo aldosterone was augmented via a high K(+) diet. High K(+) diet led to a 2-fold increase of luminal Ba(2+) and IBTX-sensitive short-circuit current in distal mouse colonic mucosa. This effect was absent in BK alpha-subunit-deficient (BK(-/-)) mice. The resting and diet-induced K(+) secretion was stimulated by luminal ionomycin. In BK(-/-) mice luminal ionomycin did not stimulate K(+) secretion. In vitro addition of aldosterone likewise triggered a 2-fold increase in K(+) secretion, which was inhibited by the mineralocorticoid receptor antagonist spironolactone and the BK channel blocker IBTX. Semi-quantification of mRNA from colonic crypts showed up-regulation of BK alpha- and beta(2)-subunits in high K(+) diet mice. The BK channel could be detected luminally in colonic crypt cells by immunohistochemistry. The expression level of the channel in the luminal membrane was strongly up-regulated in K(+)-loaded animals. Taken together, these data strongly suggest that aldosterone-induced K(+) secretion occurs via increased expression of luminal BK channels.

Carbon monoxide targets the pore‐forming BK alpha subunit in vascular smooth muscle Ca2+‐activated large‐conductance K+ channels

Carbon monoxide (CO) induces vasodilation by activating arterial smooth muscle Ca2+‐activated large‐conductance potassium (BK) channels. BK channels are composed of the pore‐forming BK alpha and auxiliary BK beta1 subunits that confer an increased sensitivity for changes in membrane potential and calcium to BK channels. To elucidate the mechanism of BK channel activation, we performed whole‐cell perforated‐patch clamp experiments in fresh tibial artery vascular smooth muscle cells (VSMC) from BK beta1 gene‐deficient (−/−) and wild‐type (+/+) mice. BK currents were measured at physiological K+ gradients. Application of hemin (3 to 10 μmol/L), a substrate of heme oxygenase, induced a 4‐fold increase in current amplitude that was blocked by iberiotoxin (100 nmol/L) in +/+ cells. The effect was similar to that observed in cells from −/− mice. We next tested the vasodilator effects of hemin. Hemin (10 μmol/L) induced similar relaxations (~ 30%) of the serotonin (300 nmol/L) preconstricted VSMC from −/− and +/+ mice. This vasorelaxation was largely inhibited by iberiotoxin (100 nmol/L). This study pinpoints the BK alpha subunit as the molecule that senses CO, which results in myocyte BK channel activation and thereby endothelial‐independent relaxation. Our notion agrees with previous studies suggesting that CO modifies the binding of reduced heme to the heme‐binding domain of the channel alpha subunit C terminus.

The vasodilator 17,18‐epoxyeicosatetraenoic acid targets the pore‐forming BK α channel subunit in rodents

17,18-Epoxyeicosatetraenoic acid (17,18-EETeTr) stimulates vascular large-conductance K(+) (BK) channels. BK channels are composed of the pore-forming BK alpha and auxiliary BK beta1 subunits that confer an increased sensitivity for changes in membrane potential and calcium to BK channels. Ryanodine-sensitive calcium-release channels (RyR3) in the sarcoplasmic reticulum (SR) control the process. To elucidate the mechanism of BK channel activation, we performed whole-cell and perforated-patch clamp experiments in freshly isolated cerebral and mesenteric artery vascular smooth muscle cells (VSMC) from Sprague-Dawley rats, BK beta1 gene-deficient (-/-), BK alpha (-/-), RyR3 (-/-) and wild-type mice. The 17,18-EETeTr (100 nm) increased tetraethylammonium (1 mm)-sensitive outward K(+) currents in VSMC from wild-type rats and wild-type mice. The effects were not inhibited by the epoxyeicosatrienoic acid (EET) antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (10 mum). BK channel currents were increased 3.5-fold in VSMC from BK beta1 (-/-) mice, whereas a 2.9-fold stimulation was observed in VSMC from RyR3 (-/-) mice (at membrane voltage 60 mV). The effects were similar compared with those observed in cells from wild-type mice. The BK current increase was neither influenced by strong internal calcium buffering (Ca(2)(+), 100 nm), nor by external calcium influx. The 17,18-EETeTr did not induce outward currents in VSMC BK alpha (-/-) cells. We next tested the vasodilator effects of 17,18-EETeTr on isolated arteries of BK alpha-deficient mice. Vasodilatation was largely inhibited in cerebral and mesenteric arteries isolated from BK alpha (-/-) mice compared with that observed in wild-type and BK beta1 (-/-) arteries. We conclude that 17,18-EETeTr represents an endogenous BK channel agonist and vasodilator. Since 17,18-EETeTr is active in small arteries lacking BK beta1, the data further suggest that BK alpha represents the molecular target for the principal action of 17,18-EETeTr. Finally, the action of 17,18-EETeTr is not mediated by changes of the internal global calcium concentration or local SR calcium release events.

A‐272651, a nonpeptidic blocker of large‐conductance Ca2+‐activated K+ channels, modulates bladder smooth muscle contractility and neuronal action potentials

The large-conductance Ca(2+)-activated K(+) channel (BK(Ca), K(Ca)1.1) links membrane excitability with intracellular Ca(2+) signaling and plays important roles in smooth muscle contraction, neuronal firing, and neuroendocrine secretion. This study reports the characterization of a novel BK(Ca) channel blocker, 2,4-dimethoxy-N-naphthalen-2-yl-benzamide (A-272651). (86)Rb(+) efflux in HEK-293 cells expressing BK(Ca) was measured. Effects of A-272651 on BK(Ca) alpha- and BK(Ca) alphabeta1-mediated currents were evaluated by patch-clamp. Effects on contractility were assessed using low-frequency electrical field stimulated pig detrusor and spontaneously contracting guinea pig detrusor. Effects of A-272651 on neuronal activity were determined in rat small diameter dorsal root ganglia (DRG). A-272651 (10 microM) inhibited (86)Rb(+) efflux evoked by NS-1608 in HEK-293 cells expressing BK(Ca) currents. A-272651 concentration-dependently inhibited BK(Ca) currents with IC(50) values of 4.59 microM (Hill coefficient 1.04, measured at +40 mV), and 2.82 microM (Hill coefficient 0.89), respectively, for BK(Ca) alpha and BK(Ca) alphabeta1-mediated currents. Like iberiotoxin, A-272651 enhanced field stimulated twitch responses in pig detrusor and spontaneous contractions in guinea pig detrusor with EC(50) values of 4.05+/-0.05 and 37.95+/-0.12 microM, respectively. In capsaicin-sensitive DRG neurons, application of A-272651 increased action potential firing and prolonged action potential duration. These data demonstrate that A-272651 modulates smooth muscle contractility and neuronal firing properties. Unlike previously reported peptide BK(Ca) blockers, A-272651 represents one of the first small molecule BK(Ca) channel blockers that could serve as a useful tool for further characterization of BK(Ca) channels in physiological and pathological states.

Altered Expression of BK Channel β 1 Subunit in Vascular Tissues from Spontaneously Hypertensive Rats

Correlation of blood pressure (BP) with expression levels of large-conductance, voltage- and Ca2+-activated K+ (BK) channel beta1 subunit in vascular tissues from spontaneously hypertensive rats (SHR), Wistar-Kyoto rats (WKY), and Sprague-Dawley rats (SD) at different ages was investigated. Systolic BP and BK beta1 expression in mesenteric arteries at either mRNA or protein levels were not different among 4-week-old SHR, WKY, and SD. With hypertension developed at 7 weeks and reached plateau at 12 weeks, expression levels of BK beta1 mRNA in mesenteric arteries and aortae from SHR during this period of time were significantly higher than in age-matched normotensive WKY. The BK beta1 protein expression was significantly higher in mesenteric arteries from 12-week-old but not 7-week-old SHR when compared with age-matched WKY and SD. The BK beta1 protein levels in aortae were not different among 7-week-old SHR, WKY, and SD but were significantly lower in 12-week-old WKY than in age-matched SHR and SD. Captopril treatment normalized BP of 12-week-old SHR. This treatment downregulated BK beta1 protein in mesenteric arteries but upregulated it in aortae. No significant difference in BK alpha subunit expression was detected in mesenteric arteries from three strains of rats as well as the captopril-treated SHR. It appears that expression patterns of BK beta1 in vascular tissues vary depending on tissue types, animal age, and animal strains. Expression of BK beta1 in mesenteric arteries is closely correlated with BP in SHR. Increased BK beta1 expression in mesenteric arteries may represent a compensatory reaction to limit the development of hypertension.

Activation of large-conductance, Ca2+-activated K+ channels by cannabinoids

We have examined the effects of the cannabinoid anandamide (AEA) and its stable analog, methanandamide (methAEA), on large-conductance, Ca2+-activated K+ (BK) channels using human embryonic kidney (HEK)-293 cells, in which the alpha-subunit of the BK channel (BK-alpha), both alpha- and beta1-subunits (BK-alphabeta1), or both alpha- and beta4-subunits (BK-alphabeta4) were heterologously expressed. In a whole cell voltage-clamp configuration, each cannabinoid activated BK-alphabeta1 within a similar concentration range. Because methAEA could potentiate BK-alpha, BK-alphabeta1, and BK-alphabeta4 with similar efficacy, the beta-subunits may not be involved at the site of action for cannabinoids. Under cell-attached patch-clamp conditions, application of methAEA to the bathing solution increased BK channel activity; however, methAEA did not alter channel activity in the excised inside-out patch mode even when ATP was present on the cytoplasmic side of the membrane. Application of methAEA to HEK-BK-alpha and HEK-BK-alphabeta1 did not change intracellular Ca2+ concentration. Moreover, methAEA-induced potentiation of BK channel currents was not affected by pretreatment with a CB1 antagonist (AM251), modulators of G proteins (cholera and pertussis toxins) or by application of a selective CB2 agonist (JWH133). Inhibitors of CaM, PKG, and MAPKs (W7, KT5823, and PD-98059) did not affect the potentiation. Application of methAEA to mouse aortic myocytes significantly increased BK channel currents. This study provides the first direct evidence that unknown factors in the cytoplasm mediate the ability of endogenous cannabinoids to activate BK channel currents. Cannabinoids may be hyperpolarizing factors in cells, such as arterial myocytes, in which BK channels are highly expressed.

Involvement of BK α subunit tyrosine phosphorylation in vascular hyporesponsiveness of superior mesenteric artery following hemorrhagic shock in rats

Vascular hyporesponsiveness is a major complication following severe trauma and shock. It plays important roles in the development of shock and seriously interferes with the treatment of shock. The mechanism responsible for the occurrence of vascular hyporesponsiveness has not been fully understood. The purpose of this study was to determine whether the alpha subunit tyrosine sites of large conductance calcium-activated potassium channel (BK(Ca)) could be phosphorylated and whether the phosphorylation of BK(Ca) was closely associated with the activation of BK(Ca) and the development of vascular hyporesponsiveness following hemorrhagic shock in rats. A hemorrhagic shock (30 mm Hg for 0.5, 2, 4 h) model of Wistar rats was established. Phosphorylation of tyrosine residues of the BK(Ca) alpha subunit from vascular smooth muscle cells (VSMC) in superior mesenteric arteries (SMA) was detected by immunoprecipitation and Western blotting. BK(Ca) activity was evaluated by cell-attached patch clamping. The vascular responsiveness of SMA to norepinephrine was measured with an isolated organ perfusion system. The level of BK(Ca) alpha subunit tyrosine phosphorylation was increased in a time-dependent manner following hemorrhagic shock, which was mediated by protein tyrosine kinases (PTK) and protein tyrosine phosphatases (PTP). The activation of VSMC BK(Ca) following hemorrhagic shock was inhibited by genistein (2x10(-5) mol/L), the permeable isoflavone PTK inhibitor, and was potentiated by the PTP inhibitor sodium orthovanadate (Na(3)VO(4), 10(-3) mol/L). The decreased vasoresponsiveness following hemorrhagic shock was partly restored by genistein (10(-5) mol/L) or by the BK(Ca)-selective inhibitor tetrabutylammonium chloride (0.1 mmol/L), while it was further decreased by Na(3)VO(4) (10(-5) mol/L). The tyrosine residues of BK(Ca) alpha subunit of SMA were phosphorylated following hemorrhagic shock, which was regulated by PTK and PTP and appeared to be related to the activation of BK(Ca) and the development of vascular hyporesponsiveness following hemorrhagic shock.