BK Currents Research Articles

Modulation of BK channel activities by calcium-sensing receptor in rat bronchopulmonary sensory neurons

This study was carried out to investigate the expression of large-conductance Ca2+-activated potassium (BK) channels and to explore the possible modulation of BK channel activities by calcium-sensing receptors (CaSR) in rat bronchopulmonary sensory neurons. The expression of BK channels was demonstrated by immunohistochemistry and RT-PCR. Results from whole-cell patch-clamp recordings demonstrated that activation of CaSR with its agonist spermine or NPS R-568 showed a dual regulating effect on BK channel activities: it potentiated BK currents in cells exhibiting low baseline BK activity while slightly inhibited BK currents in cells with high baseline BK activity. Blocking CaSR with its antagonist NPS 2143 significantly inhibited BK currents. Our results further showed that the modulation of BK currents by CaSR activation or blockade was completely abolished when the intracellular Ca2+ was chelated by BAPTA-AM. In summary, our data suggest that CaSR plays an integrative role in bronchopulmonary afferent signaling, at least partially through the regulation of BK channel activities.

The Angiotensin II Type 1 Receptor (AT1R) Closely Interacts with Large Conductance Voltage- and Ca2+-activated K+ (BK) Channels and Inhibits Their Activity Independent of G-protein Activation

Angiotensin II (ANG-II) and BK channels play important roles in the regulation of blood pressure. In arterial smooth muscle, ANG-II inhibits BK channels, but the underlying molecular mechanisms are unknown. Here, we first investigated whether ANG-II utilizes its type 1 receptor (AT1R) to modulate BK activity. Pharmacological, biochemical, and molecular evidence supports a role for AT1R. In renal arterial myocytes, the AT1R antagonist losartan (10 μM) abolished the ANG-II (1 μM)-induced reduction of whole cell BK currents, and BK channels and ANG-II receptors were found to co-localize at the cell periphery. We also found that BK inhibition via ANG-II-activated AT1R was independent of G-protein activation (assessed with 500 μM GDPβS). In BK-expressing HEK293T cells, ANG-II (1 μM) also induced a reduction of BK currents, which was contingent on AT1R expression. The molecular mechanisms of AT1R and BK channel coupling were investigated in co-transfected cells. Co-immunoprecipitation showed formation of a macromolecular complex, and live immunolabeling demonstrated that both proteins co-localized at the plasma membrane with high proximity indexes as in arterial myocytes. Consistent with a close association, we discovered that the sole AT1R expression could decrease BK channel voltage sensitivity. Truncated BK proteins revealed that the voltage-sensing conduction cassette is sufficient for BK-AT1R association. Finally, C-terminal yellow and cyan fluorescent fusion proteins, AT1R-YFP and BK-CFP, displayed robust co-localized Förster resonance energy transfer, demonstrating intermolecular interactions at their C termini. Overall, our results strongly suggest that AT1R regulates BK channels through a close protein-protein interaction involving multiple BK regions and independent of G-protein activation.

Mg2+‐Dependent Modulation of BKCa Channels by Genistein in Rat Arteriolar Smooth Muscle Cells

Genistein, a protein tyrosine kinase (PTK) inhibitor, regulates ion channel activities. However, the mechanism of action of genistein on large-conductance calcium-activated potassium (BK(Ca)) channels is unclear. This study aimed to investigate whether the mechanism of Mg(2+)-dependent modulation of BK(Ca) channel activity in vascular smooth muscle cells involved inhibition of phosphorylation by genistein or direct interaction between genistein and BK(Ca) channels. The whole-cell and inside-out patch-clamp techniques were used to measure BK(Ca) currents and the effects of genistein on BK(Ca) channel activities in rat mesenteric arteriolar smooth muscle cells. We found that the effects of genistein on BK(Ca) currents were Mg(2+)-dependent. Genistein (50 μM) inhibited BK(Ca) currents if the intracellular free magnesium concentration ([Mg(2+)]i) was 2 μM or 20 μM, but amplified BK(Ca) currents if [Mg(2+)]i was 200 μM or 2000 μM. The inhibitory effect of genistein on BK(Ca) currents was reversed by the protein tyrosine phosphatase inhibitor sodium orthovanadate (0.5 mM). Daidzein (50 μM), an inactive analogue of genistein, also amplified BK(Ca) currents, and its amplification was insensitive to orthovanadate. Another PTK inhibitor, tyrphostin 23 (50 μM), reduced the open probability of BK(Ca) channels. This inhibitory effect was weaker at 200 μM [Mg(2+)]i than at 2 μM [Mg(2+) ]i, and was countered by orthovanadate. Our results suggest that genistein amplifies BK(Ca) currents at a high [Mg(2+)]i, but inhibits BK(Ca) currents at a low [Mg(2+)]i. The mechanism of this biphasic effects involves PTK-independent amplification and [Mg(2+)]i -PTK-dependent inhibition.

Prostaglandin E2 excitatory effects on guinea pig urinary bladder smooth muscle: A novel regulatory mechanism mediated by large-conductance voltage- and Ca2+-activated K+ channels

Prostaglandin E2 (PGE2) is an essential signaling molecule involved in the regulation of detrusor smooth muscle (DSM) function. However, the underlying regulatory mechanism by which PGE2 augments DSM cell excitability and contractility is not well understood. Here, we investigated whether PGE2 inhibits the large conductance voltage- and Ca2+-activated K+ (BK) channels in guinea pig DSM, thereby increasing DSM excitability and contractility. We used a multidisciplinary experimental approach including amphotericin–B perforated patch-clamp electrophysiology and live-cell Ca2+ imaging in native freshly-isolated DSM cells, isometric tension recordings of intact DSM strips, and pharmacological tools to investigate BK channel regulation by PGE2 in guinea pig DSM. PGE2 increased the spontaneous phasic contractions of isolated DSM strips in a concentration-dependent manner (10nM–10µM). BK channel inhibition with paxilline (1µM) attenuated the PGE2-induced DSM phasic contractions, suggesting that BK channels are involved in the mechanism of PGE2-induced DSM contractions. PGE2 (10µM) increased the intracellular Ca2+ levels in freshly-isolated DSM cells. PGE2 (10µM) also caused an inhibition of the amplitude and frequency of spontaneous transient BK currents in DSM cells. Moreover, PGE2 (10µM) did not affect the amplitude of whole cell steady-state BK currents in DSM cells. Our findings provide strong experimental evidence that PGE2 leads to an inhibition of the spontaneous transient BK currents, elevation of intracellular Ca2+ levels in freshly-isolated DSM cells, and augmentation of DSM phasic contractions. Thus, we have revealed a novel mechanism that BK channels mediate PGE2-induced contractions in guinea pig DSM.

Hydrogen sulfide impairs shear stress-induced vasodilation in mouse coronary arteries.

Hydrogen sulfide has emerged as an important endothelium-dependent vasodilator, but its role in shear stress-mediated dilation of coronary arteries is unclear. We examined the role of H2S on shear stress-mediated dilation of isolated mouse coronary arteries. In these vessels, Na2S produced concentration-dependent dilation, which was significantly inhibited by iberiotoxin and by 4-aminopyridine. In addition, BK and Kv currents in mouse coronary smooth muscle cells were directly activated by Na2S, suggesting that H2S produced vasodilation through BK and Kv channel activation. Using a pressure servo controller system, freshly isolated mouse coronary arteries were subjected to physiological levels of shear stress (1 to 25 dynes/cm(2)) and produced graded dilatory responses, but such effects were diminished in the presence of 100 μM Na2S. Pre-incubation with the cystathionine γ-lyase inhibitor, D,L-propargylglycine (PPG), resulted in a paradoxical augmentation of shear stress-mediated vasodilation. However, in the presence of L-NAME or in coronary arteries from eNOS knockout mice, PPG inhibited shear stress-mediated vasodilation, suggesting an interaction between NO and H2S signaling. Na2S inhibited eNOS activity in cultured mouse aortic endothelial cells and reduced the level of phospho-eNOS(serine 1177). These results suggest that both NO and H2S are important shear stress-mediated vasodilators in mouse coronary arteries but there is a complex interaction between these two signaling pathways that results in paradoxical vasoconstrictive effects of H2S through inhibition of NO generation.

Role of Ion Channels and Subcellular Ca2+Signaling in Arachidonic Acid–Induced Dilation of Pressurized Retinal Arterioles

To investigate the mechanisms responsible for the dilatation of rat retinal arterioles in response to arachidonic acid (AA). Changes in the diameter of isolated, pressurized rat retinal arterioles were measured in the presence of AA alone and following pre-incubation with pharmacologic agents inhibiting Ca(2+) sparks and oscillations and K(+) channels. Subcellular Ca(2+) signals were recorded in arteriolar myocytes using Fluo-4-based confocal imaging. The effects of AA on membrane currents of retinal arteriolar myocytes were studied using whole-cell perforated patch clamp recording. Arachidonic acid dilated pressurized retinal arterioles under conditions of myogenic tone. Eicosatetraynoic acid (ETYA) exerted a similar effect, but unlike AA, its effects were rapidly reversible. Arachidonic acid-induced dilation was associated with an inhibition of subcellular Ca(2+) signals. Interventions known to block Ca(2+) sparks and oscillations in retinal arterioles caused dilatation and inhibited AA-induced vasodilator responses. Arachidonic acid accelerated the rate of inactivation of the A-type Kv current and the voltage dependence of inactivation was shifted to more negative membrane potentials. It also enhanced voltage-activated and spontaneous large-conductance calcium-activated K(+) (BK) currents, but only at positive membrane potentials. Pharmacologic inhibition of A-type Kv and BK currents failed to block AA-induced vasodilator responses. Arachidonic acid suppressed L-type Ca(2+) currents. These results suggest that AA induces retinal arteriolar vasodilation by inhibiting subcellular Ca(2+)-signaling activity in retinal arteriolar myocytes, most likely through a mechanism involving the inhibition of L-type Ca(2+)-channel activity. Arachidonic acid actions on K(+) currents are inconsistent with a model in which K(+) channels contribute to the vasodilator effects of AA.

Gentamicin blocks the ACh-induced BK current in guinea pig type II vestibular hair cells by competing with Ca²⁺ at the L-type calcium channel.

Type II vestibular hair cells (VHCs II) contain big-conductance Ca2+-dependent K+ channels (BK) and l-type calcium channels. Our previous studies in guinea pig VHCs II indicated that acetylcholine (ACh) evoked the BK current by triggering the influx of Ca2+ ions through l-type Ca2+ channels, which was mediated by M2 muscarinic ACh receptor (mAChRs). Aminoglycoside antibiotics, such as gentamicin (GM), are known to have vestibulotoxicity, including damaging effects on the efferent nerve endings on VHCs II. This study used the whole-cell patch clamp technique to determine whether GM affects the vestibular efferent system at postsynaptic M2-mAChRs or the membrane ion channels. We found that GM could block the ACh-induced BK current and that inhibition was reversible, voltage-independent, and dose-dependent with an IC50 value of 36.3 ± 7.8 μM. Increasing the ACh concentration had little influence on GM blocking effect, but increasing the extracellular Ca2+ concentration ([Ca2+]o) could antagonize it. Moreover, 50 μM GM potently blocked Ca2+ currents activated by (−)-Bay-K8644, but did not block BK currents induced by NS1619. These observations indicate that GM most likely blocks the M2 mAChR-mediated response by competing with Ca2+ at the l-type calcium channel. These results provide insights into the vestibulotoxicity of aminoglycoside antibiotics on mammalian VHCs II.

Pituitary adenylate cyclase activating polypeptide (PACAP) dilates cerebellar arteries through activation of large-conductance Ca(2+)-activated (BK) and ATP-sensitive (K ATP) K (+) channels.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a potent vasodilator of numerous vascular beds, including cerebral arteries. Although PACAP-induced cerebral artery dilation is suggested to be cyclic AMP (cAMP)-dependent, the downstream intracellular signaling pathways are still not fully understood. In this study, we examined the role of smooth muscle K(+) channels and hypothesized that PACAP-mediated increases in cAMP levels and protein kinase A (PKA) activity result in the coordinate activation of ATP-sensitive K(+) (KATP) and large-conductance Ca(2+)-activated K(+) (BK) channels for cerebral artery dilation. Using patch-clamp electrophysiology, we observed that PACAP enhanced whole-cell KATP channel activity and transient BK channel currents in freshly isolated rat cerebellar artery myocytes. The increased frequency of transient BK currents following PACAP treatment is indicative of increased intracellular Ca(2+) release events termed Ca(2+) sparks. Consistent with the electrophysiology data, the PACAP-induced vasodilations of cannulated cerebellar artery preparations were attenuated by approximately 50% in the presence of glibenclamide (a KATP channel blocker) or paxilline (a BK channel blocker). Further, in the presence of both blockers, PACAP failed to cause vasodilation. In conclusion, our results indicate that PACAP causes cerebellar artery dilation through two mechanisms: (1) KATP channel activation and (2) enhanced BK channel activity, likely through increased Ca(2+) spark frequency.

Novel BK channel regulatory mechanism by protein kinase C in guinea pig urinary bladder smooth muscle (865.1)

Large conductance Ca2+‐activated K+ (BK) channels are critical regulators of urinary bladder smooth muscle (UBSM) function. Here, we provide a novel mechanistic insight into BK channel regulation by protein kinase C (PKC) in UBSM. Voltage‐clamp experiments showed that pharmacological activation of PKC with phorbol 12‐myristate 13‐acetate (PMA), inhibited the spontaneous transient BK currents (TBKCs) in native freshly‐isolated guinea pig UBSM cells. Current‐clamp recordings revealed that PMA significantly depolarized UBSM membrane potential, and reduced the spontaneous transient hyperpolarizations in UBSM cells. The PMA inhibitory effects on UBSM membrane potential were abolished by the selective BK channel inhibitor paxilline and were not observed with its inactive analog, 4‐alpha‐PMA. PMA did not affect the amplitude of the whole cell steady‐state BK current or single BK channel open probability (recorded in cell‐attached mode) upon inhibition of all major Ca2+ sources for BK channel activation with thapsigargin, ryanodine, and nifedipine. PMA elevated the intracellular Ca2+ levels in UBSM cells, and increased spontaneous phasic and nerve‐evoked contractions of UBSM isolated strips. The results support the concept that PKC activation leads to a reduction in BK channel activity in UBSM via a Ca2+‐dependent mechanism, thus increasing UBSM contractility.Grant Funding Source: Supported by R01 DK084284 to G. V. Petkov

Prostaglandin E2 inhibits the large‐conductance voltage‐ and Ca2+‐activated K+ channels in guinea pig urinary bladder smooth muscle (865.7)

Prostaglandin E2 (PGE2) is an important signaling molecule that has been shown to be involved in the regulation of urinary bladder smooth muscle (UBSM) contractility in both rodents and humans. However, the underlying mechanism by which PGE2 evokes UBSM contractions is not well understood. Here, we investigated whether large conductance voltage‐ and Ca2+‐activated K+ (BK) channels mediate the stimulatory effects induced by PGE2 on guinea pig UBSM contractility. We used a combined experimental approach including patch‐clamp electrophysiology, live‐cell Ca2+ imaging, and isometric UBSM tension recordings to investigate BK channel regulation by PGE2 at both cellular and tissue levels. PGE2 increased the spontaneous phasic contractions of UBSM isolated strips in a concentration‐dependent manner (10 nM‐10 µM). BK channel inhibition with paxilline attenuated the PGE2‐induced UBSM phasic contractions, suggesting that BK channels are among the targets of PGE2. Our data also indicate that PGE2 (10 µM) caused an inhibition of the amplitude and frequency of spontaneous transient BK currents and increased the intracellular Ca2+ levels in freshly‐isolated UBSM cells. Thus, for the first time we have revealed a novel mechanism by which BK channels mediate the PGE2 stimulatory effects on guinea pig UBSM contractions.Grant Funding Source: Supported by NIH R01 DK084284 grant to Georgi V. Petkov.

Role of phosphodiesterase‐1 in muscarinic receptor‐induced human urinary bladder smooth muscle excitability and contractility (865.4)

Role of phosphodiesterase‐1 in muscarinic receptor‐induced human urinary bladder smooth muscle excitability and contractility Wenkuan Xin1, Ning Li1, Eric S. Rovner2, Qiuping Cheng1, and Georgi V. Petkov1,2 1Drug Discovery & Biomedical Sciences, South Carolina College of Pharmacy, University of South Carolina, Columbia, SC 29208 and 2Medical University of South Carolina, Charleston, SC 29425 Activation of muscarinic M3 receptors (M3Rs) leads to an inhibition of the large conductance Ca2+‐activated K+ (BK) channels and an increase in human urinary bladder smooth muscle (UBSM) excitability. M3R‐mediated Ca2+ influx activates phosphodiesterase‐1 (PDE1) and reduces cellular cAMP levels. We investigated the mechanism by which the integrated PDE1 and MR signaling pathways stimulate human UBSM excitability and contractility. Pharmacological activation of M3Rs with carbachol (1 µM) decreased the frequency and amplitude of transient BK currents (TBKCs) in UBSM cells and subsequent inhibition of PDE1 with 8MM‐IBMX (10 µM) recovered TBKC activity. In the absence of M3R stimulation, inhibition of PDE1 increased TBKCs frequency, and subsequent inhibition of protein kinase A (PKA) with H‐89 (10 µM) reduced TBKCs frequency. 8MM‐IBMX (10 µM) hyperpolarized UBSM membrane potential, an effect which was blocked upon PKA inhibition. 8MM‐IBMX suppressed spontaneous phasic contractions and nerve‐evoked contractions of UBSM isolated strips. In conclusion, PDE1 enhances the M3R‐mediated inhibition of BK channels. Inhibition of PDE1 attenuates UBSM excitability by increasing BK channel activity via a PKA‐dependent mechanism. A combination of a PDE1 inhibitor and a MR antagonist can be an effective novel approach for the treatment of bladder overactivity.Key words: phosphodiesterase, muscarinic receptor, detrusorGrant Funding Source: NIH R01 DK084284 to Georgi V. Petkov

Expression and function of BK and Kv1.5 channels in aortic smooth muscle from lean and obese Zucker rats (1077.5)

We tested the hypothesis that metabolic syndrome is associated with reduced expression and function of K+ channels in smooth muscle. Aortic smooth muscle from male lean and obese Zucker rats was used as a model. Whole‐cell patch clamp experiments indicated that Ca2+‐activated (BK) and voltage‐dependent (KV) K+ currents dominated membrane conductance. Penitrem A (1 uM) and DPO‐1 (10 uM), selective antagonists of BK and KV1.5 channels were used to determine the expression of these channels. In lean rats, penitrem A inhibited 37 ± 6% of current at +100 mV, whereas subsequent addition of DPO‐1 inhibited 74 ± 4% of the remaining current. In obese rats, penitrem A inhibited 19 ± 6% of current at +100 mV, whereas subsequent addition of DPO‐1 inhibited 78 ± 3% of the remaining current. The penitrem A‐sensitive BK current was 2.7 ± 0.7 fold greater in lean rats. In contrast, and surprisingly, the DPO‐1‐sensitive KV1.5 current was 1.5 ± 0.2 fold greater in obese rats. Western blots supported the patch clamp studies, as BK channel protein was 1.4 ± 0.1 fold greater in lean rats, while KV1.5 protein was 2.5 ± 0.4 fold greater in obese rats. These data indicate that metabolic syndrome decreases the expression of BK channels, but simultaneously increases KV1.5 channel expression. We suggest this opposite change in K+ channel expression may represent a compensatory stage of metabolic disease that serves to preserve cardiovascular function.Grant Funding Source: Supported by T32HL090610

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.

Cysteine String Protein Limits Expression of the Large Conductance, Calcium-Activated K+ (BK) Channel

Large-conductance, calcium-activated K+ (BK) channels are widely distributed throughout the nervous system and play an essential role in regulation of action potential duration and firing frequency, along with neurotransmitter release at the presynaptic terminal. We have previously demonstrated that select mutations in cysteine string protein (CSPα), a presynaptic J-protein and co-chaperone, increase BK channel expression. This observation raised the possibility that wild-type CSPα normally functions to limit neuronal BK channel expression. Here we show by Western blot analysis of transfected neuroblastoma cells that when BK channels are present at elevated levels, CSPα acts to reduce expression. Moreover, we demonstrate that the accessory subunits, BKβ4 and BKβ1 do not alter CSPα-mediated reduction of expressed BKα subunits. Structure-function analysis reveals that the N-terminal J-domain of CSPα is critical for the observed regulation of BK channels levels. Finally, we demonstrate that CSPα limits BK current amplitude, while the loss-of-function homologue CSPαHPD-AAA increases BK current. Our observations indicate that CSPα has a role in regulating synaptic excitability and neurotransmission by limiting expression of BK channels.

BK Channel-Mediated Relaxation of Urinary Bladder Smooth Muscle: A Novel Paradigm for Phosphodiesterase Type 4 Regulation of Bladder Function

Elevation of intracellular cAMP and activation of protein kinase A (PKA) lead to activation of large conductance voltage- and Ca(2+)-activated K(+) (BK) channels, thus attenuation of detrusor smooth muscle (DSM) contractility. In this study, we investigated the mechanism by which pharmacological inhibition of cAMP-specific phosphodiesterase 4 (PDE4) with rolipram or Ro-20-1724 (C(15)H(22)N(2)O(3)) suppresses guinea pig DSM excitability and contractility. We used high-speed line-scanning confocal microscopy, ratiometric fluorescence Ca(2+) imaging, and perforated whole-cell patch-clamp techniques on freshly isolated DSM cells, along with isometric tension recordings of DSM isolated strips. Rolipram caused an increase in the frequency of Ca(2+) sparks and the spontaneous transient BK currents (TBKCs), hyperpolarized the cell membrane potential (MP), and decreased the intracellular Ca(2+) levels. Blocking BK channels with paxilline reversed the hyperpolarizing effect of rolipram and depolarized the MP back to the control levels. In the presence of H-89 [N-[2-[[3-(4-bromophenyl)-2-propenyl]amino]ethyl]-5-isoquinolinesulfonamide dihydrochloride], a PKA inhibitor, rolipram did not cause MP hyperpolarization. Rolipram or Ro-20-1724 reduced DSM spontaneous and carbachol-induced phasic contraction amplitude, muscle force, duration, and frequency, and electrical field stimulation-induced contraction amplitude, muscle force, and tone. Paxilline recovered DSM contractility, which was suppressed by pretreatment with PDE4 inhibitors. Rolipram had reduced inhibitory effects on DSM contractility in DSM strips pretreated with paxilline. This study revealed a novel cellular mechanism whereby pharmacological inhibition of PDE4 leads to suppression of guinea pig DSM contractility by increasing the frequency of Ca(2+) sparks and the functionally coupled TBKCs, consequently hyperpolarizing DSM cell MP. Collectively, this decreases the global intracellular Ca(2+) levels and DSM contractility in a BK channel-dependent manner.

Phosphorylation of a Constitutive Serine (S642) in BK Channel Variants Containing the Alternate Exon ‘SRKR’ Alters Current Properties

Kcnma1, the gene encoding the pore forming subunit of the BK Ca2+-activated K+ channel, undergoes extensive alternative splicing across tissues, tuning BK current properties. Phosphorylation and β subunit association further contribute to diverse BK current properties, but the interplay of these mechanisms have not been well-investigated in BK variants cloned from native tissues. We used RT-PCR to identify two Kcnma1 splice variants in the mouse suprachiasmatic nucleus (BK0 and BKSRKR). Both variants contain an alternate translation start (MANG) compared to the canonical start (MDAL), no insert at splice site 2 (STREX), an insert at site 3 (near the Ca2+ bowl), as well as an alternate C-terminus (VYR). However, they differ at splice site 1, containing a four amino acid SRKR alternate exon located near the end of the RCK1 domain. Voltage-clamp recordings from the variants expressed in HEK293 cells revealed that BKSRKR channels produced BK currents with distinct properties from the insertless BK0 variant, with significantly right-shifted G-Vs, slower activation, and faster deactivation kinetics at 0, 1, and 100 μM Ca2+. Co-expression of the β4 subunit enhanced these differences in a Ca2+-dependent manner. Inclusion of SRKR resulted in two predicted phosphorylation sites in BKSRKR, S642 (a constitutive residue present in both variants) and S644 (within the SRKR exon). Alkaline phosphatase treatment and mutations at both positions revealed that phosphorylation of S642 is both necessary and sufficient to produce the observed differences between BK0 and BKSRKR current properties. These data demonstrate that phosphorylation of S642 underlies the differences in current properties between BKSRKR and BK0, and suggests the alternate exon SRKR serves to enhance S642 phosphorylation in native tissues.

Large-conductance voltage- and Ca2+-activated K+ channel regulation by protein kinase C in guinea pig urinary bladder smooth muscle

Large-conductance voltage- and Ca(2+)-activated K(+) (BK) channels are critical regulators of detrusor smooth muscle (DSM) excitability and contractility. PKC modulates the contraction of DSM and BK channel activity in non-DSM cells; however, the cellular mechanism regulating the PKC-BK channel interaction in DSM remains unknown. We provide a novel mechanistic insight into BK channel regulation by PKC in DSM. We used patch-clamp electrophysiology, live-cell Ca(2+) imaging, and functional studies of DSM contractility to elucidate BK channel regulation by PKC at cellular and tissue levels. Voltage-clamp experiments showed that pharmacological activation of PKC with PMA inhibited the spontaneous transient BK currents in native freshly isolated guinea pig DSM cells. Current-clamp recordings revealed that PMA significantly depolarized DSM membrane potential and inhibited the spontaneous transient hyperpolarizations in DSM cells. The PMA inhibitory effects on DSM membrane potential were completely abolished by the selective BK channel inhibitor paxilline. Activation of PKC with PMA did not affect the amplitude of the voltage-step-induced whole cell steady-state BK current or the single BK channel open probability (recorded in cell-attached mode) upon inhibition of all major Ca(2+) sources for BK channel activation with thapsigargin, ryanodine, and nifedipine. PKC activation with PMA elevated intracellular Ca(2+) levels in DSM cells and increased spontaneous phasic and nerve-evoked contractions of DSM isolated strips. Our results support the concept that PKC activation leads to a reduction of BK channel activity in DSM via a Ca(2+)-dependent mechanism, thus increasing DSM contractility.

Phosphorylation of a constitutive serine inhibits BK channel variants containing the alternate exon “SRKR”

BK Ca2+-activated K+ currents exhibit diverse properties across tissues. The functional variation in voltage- and Ca2+-dependent gating underlying this diversity arises from multiple mechanisms, including alternate splicing of Kcnma1, the gene encoding the pore-forming (α) subunit of the BK channel, phosphorylation of α subunits, and inclusion of β subunits in channel complexes. To address the interplay of these mechanisms in the regulation of BK currents, two native splice variants, BK0 and BKSRKR, were cloned from a tissue that exhibits dynamic daily expression of BK channel, the central circadian pacemaker in the suprachiasmatic nucleus (SCN) of mouse hypothalamus. The BK0 and BKSRKR variants differed by the inclusion of a four–amino acid alternate exon at splice site 1 (SRKR), which showed increased expression during the day. The functional properties of the variants were investigated in HEK293 cells using standard voltage-clamp protocols. Compared with BK0, BKSRKR currents had a significantly right-shifted conductance–voltage (G-V) relationship across a range of Ca2+ concentrations, slower activation, and faster deactivation. These effects were dependent on the phosphorylation state of S642, a serine residue within the constitutive exon immediately preceding the SRKR insert. Coexpression of the neuronal β4 subunit slowed gating kinetics and shifted the G-V relationship in a Ca2+-dependent manner, enhancing the functional differences between the variants. Next, using native action potential (AP) command waveforms recorded from SCN to elicit BK currents, we found that these splice variant differences persist under dynamic activation conditions in physiological ionic concentrations. AP-induced currents from BKSRKR channels were significantly reduced compared with BK0, an effect that was maintained with coexpression of the β4 subunit but abolished by the mutation of S642. These results demonstrate a novel mechanism for reducing BK current activation under reconstituted physiological conditions, and further suggest that S642 is selectively phosphorylated in the presence of SRKR.

Ethanol-mediated relaxation of guinea pig urinary bladder smooth muscle: involvement of BK and L-type Ca2+ channels

Mechanisms underlying ethanol (EtOH)-induced detrusor smooth muscle (DSM) relaxation and increased urinary bladder capacity remain unknown. We investigated whether the large conductance Ca(2+)-activated K(+) (BK) channels or L-type voltage-dependent Ca(2+) channels (VDCCs), major regulators of DSM excitability and contractility, are targets for EtOH by patch-clamp electrophysiology (conventional and perforated whole cell and excised patch single channel) and isometric tension recordings using guinea pig DSM cells and isolated tissue strips, respectively. EtOH at 0.3% vol/vol (~50 mM) enhanced whole cell BK currents at +30 mV and above, determined by the selective BK channel blocker paxilline. In excised patches recorded at +40 mV and ~300 nM intracellular Ca(2+) concentration ([Ca(2+)]), EtOH (0.1-0.3%) affected single BK channels (mean conductance ~210 pS and blocked by paxilline) by increasing the open channel probability, number of open channel events, and open dwell-time constants. The amplitude of single BK channel currents and unitary conductance were not altered by EtOH. Conversely, at ~10 μM but not ~2 μM intracellular [Ca(2+)], EtOH (0.3%) decreased the single BK channel activity. EtOH (0.3%) affected transient BK currents (TBKCs) by either increasing frequency or decreasing amplitude, depending on the basal level of TBKC frequency. In isolated DSM strips, EtOH (0.1-1%) reduced the amplitude and muscle force of spontaneous phasic contractions. The EtOH-induced DSM relaxation, except at 1%, was attenuated by paxilline. EtOH (1%) inhibited L-type VDCC currents in DSM cells. In summary, we reveal the involvement of BK channels and L-type VDCCs in mediating EtOH-induced urinary bladder relaxation accommodating alcohol-induced diuresis.

Molecular heterogeneity of large-conductance calcium-activated potassium channels in canine intracardiac ganglia

Large conductance calcium-activated potassium (BK) channels are widely expressed in the nervous system. We have recently shown that principal neurons from canine intracardiac ganglia (ICG) express a paxilline- and TEA-sensitive BK current, which increases neuronal excitability. In the present work, we further explore the molecular constituents of the BK current in canine ICG. We found that the β1 and β4 regulatory subunits are expressed in ICG. Single channel voltage-dependence at different calcium concentrations suggested that association of the BKα with a particular β subunit was not enough to explain the channel activity in this tissue. Indeed, we detected the presence of several splice variants of the BKα subunit. In conclusion, BK channels in canine ICG may result from the arrangement of different BKα splice variants, plus accessory β subunits. The particular combinations expressed in canine IC neurons likely rule the excitatory role of BK current in this tissue.