Micromolar Ca2+ from sparks activates Ca2+-sensitive K+ channels in rat cerebral artery smooth muscle

2001 ◽  
Vol 281 (6) ◽  
pp. C1769-C1775 ◽  
Author(s):  
Guillermo J. Pérez ◽  
Adrian D. Bonev ◽  
Mark T. Nelson
Keyword(s):  
Smooth Muscle ◽  
Laser Scanning ◽  
Cerebral Arteries ◽  
Bk Channels ◽  
Bk Channel ◽  
Hill Coefficient ◽  
Channel Activity ◽  
Acetoxymethyl Ester ◽  
Open Probability ◽  
Apparent Dissociation Constant

The goal of the present study was to test the hypothesis that local Ca2+ release events (Ca2+ sparks) deliver high local Ca2+concentration to activate nearby Ca2+-sensitive K+ (BK) channels in the cell membrane of arterial smooth muscle cells. Ca2+ sparks and BK channels were examined in isolated myocytes from rat cerebral arteries with laser scanning confocal microscopy and patch-clamp techniques. BK channels had an apparent dissociation constant for Ca2+ of 19 μM and a Hill coefficient of 2.9 at −40 mV. At near-physiological intracellular Ca2+ concentration ([Ca2+]i; 100 nM) and membrane potential (−40 mV), the open probability of a single BK channel was low (1.2 × 10−6). A Ca2+spark increased BK channel activity to 18. Assuming that 1–100% of the BK channels are activated by a single Ca2+ spark, BK channel activity increases 6 × 105-fold to 6 × 103-fold, which corresponds to ∼30 μM to 4 μM spark Ca2+ concentration. 1,2-bis(2-aminophenoxy)ethane- N,N,N′,N′-tetraacetic acid acetoxymethyl ester caused the disappearance of all Ca2+sparks while leaving the transient BK currents unchanged. Our results support the idea that Ca2+ spark sites are in close proximity to the BK channels and that local [Ca2+]i reaches micromolar levels to activate BK channels.

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

2014 ◽  
Vol 306 (5) ◽  
pp. C460-C470 ◽  
Author(s):  
Kiril L. Hristov ◽  
Amy C. Smith ◽  
Shankar P. Parajuli ◽  
John Malysz ◽  
Georgi V. Petkov
Keyword(s):  
Smooth Muscle ◽  
Membrane Potential ◽  
Guinea Pig ◽  
Bk Channels ◽  
Bk Channel ◽  
K Channel ◽  
Channel Activity ◽  
Open Probability ◽  
Channel Regulation ◽  
Pkc Activation

Large-conductance voltage- and Ca2+-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 Ca2+ 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 Ca2+ sources for BK channel activation with thapsigargin, ryanodine, and nifedipine. PKC activation with PMA elevated intracellular Ca2+ 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 Ca2+-dependent mechanism, thus increasing DSM contractility.

scholarly journals Reduced Ca2+ Spark Activity after Subarachnoid Hemorrhage Disables BK Channel Control of Cerebral Artery Tone

2010 ◽  
Vol 31 (1) ◽  
pp. 3-16 ◽  
Author(s):  
Masayo Koide ◽  
Matthew A Nystoriak ◽  
Gayathri Krishnamoorthy ◽  
Kevin P O'Connor ◽  
Adrian D Bonev ◽  
...  
Keyword(s):  
Subarachnoid Hemorrhage ◽  
Cerebral Artery ◽  
Laser Scanning ◽  
Single Channel ◽  
Cerebral Arteries ◽  
Laser Scanning Confocal Microscopy ◽  
Bk Channels ◽  
Bk Channel ◽  
Channel Activity ◽  
Similar Reduction

Intracellular Ca2+ release events (‘Ca2+ sparks’) and transient activation of large-conductance Ca2+-activated potassium (BK) channels represent an important vasodilator pathway in the cerebral vasculature. Considering the frequent occurrence of cerebral artery constriction after subarachnoid hemorrhage (SAH), our objective was to determine whether Ca2+ spark and BK channel activity were reduced in cerebral artery myocytes from SAH model rabbits. Using laser scanning confocal microscopy, we observed ∼50% reduction in Ca2+ spark activity, reflecting a decrease in the number of functional Ca2+ spark discharge sites. Patch-clamp electrophysiology showed a similar reduction in Ca2+ spark-induced transient BK currents, without change in BK channel density or single-channel properties. Consistent with a reduction in active Ca2+ spark sites, quantitative real-time PCR and western blotting revealed decreased expression of ryanodine receptor type 2 (RyR-2) and increased expression of the RyR-2-stabilizing protein, FKBP12.6, in the cerebral arteries from SAH animals. Furthermore, inhibitors of Ca2+ sparks (ryanodine) or BK channels (paxilline) constricted arteries from control, but not from SAH animals. This study shows that SAH-induced decreased subcellular Ca2+ signaling events disable BK channel activity, leading to cerebral artery constriction. This phenomenon may contribute to decreased cerebral blood flow and poor outcome after aneurysmal SAH.

Peroxynitrite reversibly inhibits Ca2+-activated K+ channels in rat cerebral artery smooth muscle cells

2000 ◽  
Vol 278 (6) ◽  
pp. H1883-H1890 ◽  
Author(s):  
Anna K. Brzezinska ◽  
Debebe Gebremedhin ◽  
William M. Chilian ◽  
Balaraman Kalyanaraman ◽  
Stephen J. Elliott
Keyword(s):  
Single Channel ◽  
Cerebral Arteries ◽  
Ionic Current ◽  
Inhibitory Effect ◽  
Bk Channels ◽  
Bk Channel ◽  
Channel Activity ◽  
Open Probability ◽  
Whole Cell ◽  
Cell Current

Peroxynitrite (ONOO−) is a contractile agonist of rat middle cerebral arteries. To determine the mechanism responsible for this component of ONOO−bioactivity, the present study examined the effect of ONOO− on ionic current and channel activity in rat cerebral arteries. Whole cell recordings of voltage-clamped cells were made under conditions designed to optimize K+ current. The effects of iberiotoxin, a selective inhibitor of large-conductance Ca2+-activated K+ (BK) channels, and ONOO− (10–100 μM) were determined. At a pipette potential of +50 mV, ONOO− inhibited 39% of iberiotoxin-sensitive current. ONOO− was selective for iberiotoxin-sensitive current, whereas decomposed ONOO− had no effect. In excised, inside-out membrane patches, channel activity was recorded using symmetrical K+solutions. Unitary currents were sensitive to increases in internal Ca2+ concentration, consistent with activity due to BK channels. Internal ONOO− dose dependently inhibited channel activity by decreasing open probability and mean open times. The inhibitory effect of ONOO− could be overcome by reduced glutathione. Glutathione, added after ONOO−, restored whole cell current amplitude to control levels and reverted single-channel gating to control behavior. The inhibitory effect of ONOO− on membrane K+ current is consistent with its contractile effects in isolated cerebral arteries and single myocytes. Taken together, our data suggest that ONOO− has the potential to alter cerebral vascular tone by inhibiting BK channel activity.

scholarly journals Intracellular Ca2+ Inhibits Smooth Muscle L-Type Ca2+ Channels by Activation of Protein Phosphatase Type 2B and by Direct Interaction with the Channel

1997 ◽  
Vol 110 (5) ◽  
pp. 503-513 ◽  
Author(s):  
Klaus Schuhmann ◽  
Christoph Romanin ◽  
Werner Baumgartner ◽  
Klaus Groschner
Keyword(s):  
Smooth Muscle ◽  
Protein Phosphatase ◽  
Hill Coefficient ◽  
Channel Activity ◽  
Open Probability ◽  
Intact Cells ◽  
Protein Phosphatase 2B ◽  
Dependent Inhibition ◽  
Inside Out ◽  
Ca2 Channels

Modulation of L-type Ca2+ channels by tonic elevation of cytoplasmic Ca2+ was investigated in intact cells and inside-out patches from human umbilical vein smooth muscle. Ba2+ was used as charge carrier, and run down of Ca2+ channel activity in inside-out patches was prevented with calpastatin plus ATP. Increasing cytoplasmic Ca2+ in intact cells by elevation of extracellular Ca2+ in the presence of the ionophore A23187 inhibited the activity of L-type Ca2+ channels in cell-attached patches. Measurement of the actual level of intracellular free Ca2+ with fura-2 revealed a 50% inhibitory concentration (IC50) of 260 nM and a Hill coefficient close to 4 for Ca2+- dependent inhibition. Ca2+-induced inhibition of Ca2+ channel activity in intact cells was due to a reduction of channel open probability and availability. Ca2+-induced inhibition was not affected by the protein kinase inhibitor H-7 (10 μM) or the cytoskeleton disruptive agent cytochalasin B (20 μM), but prevented by cyclosporin A (1 μg/ ml), an inhibitor of protein phosphatase 2B (calcineurin). Elevation of Ca2+ at the cytoplasmic side of inside-out patches inhibited Ca2+ channels with an IC50 of 2 μM and a Hill coefficient close to unity. Direct Ca2+-dependent inhibition in cell-free patches was due to a reduction of open probability, whereas availability was barely affected. Application of purified protein phosphatase 2B (12 U/ml) to the cytoplasmic side of inside-out patches at a free Ca2+ concentration of 1 μM inhibited Ca2+ channel open probability and availability. Elevation of cytoplasmic Ca2+ in the presence of PP2B, suppressed channel activity in inside-out patches with an IC50 of ∼380 nM and a Hill coefficient of ∼3; i.e., characteristics reminiscent of the Ca2+ sensitivity of Ca2+ channels in intact cells. Our results suggest that L-type Ca2+ channels of smooth muscle are controlled by two Ca2+-dependent negative feedback mechanisms. These mechanisms are based on (a) a protein phosphatase 2B-mediated dephosphorylation process, and (b) the interaction of intracellular Ca2+ with a single membrane-associated site that may reside on the channel protein itself.

scholarly journals G protein pathway suppressor 2 enhanced the renal large-conductance Ca2+-activated potassium channel expression via inhibiting ERK1/2 signaling pathway

2018 ◽  
Vol 315 (3) ◽  
pp. F503-F511 ◽  
Author(s):  
Zhizhi Zhuang ◽  
Jia Xiao ◽  
Xinxin Chen ◽  
Xiaohan Hu ◽  
Ruidian Li ◽  
...  
Keyword(s):  
Protein Expression ◽  
Signaling Pathway ◽  
G Protein ◽  
Mapk Signaling ◽  
Bk Channels ◽  
Bk Channel ◽  
Mapk Signaling Pathway ◽  
Dependent Manner ◽  
Channel Activity ◽  
Open Probability

G protein pathway suppressor 2 (GPS2) is a multifunctional protein and transcriptional regulation factor that is involved in the G protein MAPK signaling pathway. It has been shown that the MAPK signaling pathway plays an important role in the regulation of renal large-conductance Ca2+-activated potassium (BK) channels. In this study, we investigated the effects of GPS2 on BK channel activity and protein expression. In human embryonic kidney (HEK) BK stably expressing cells transfected with either GPS2 or its vector control, a single-cell recording showed that GPS2 significantly increased BK channel activity ( NPo), increasing BK open probability ( Po), and channel number ( N) compared with the control. In Cos-7 cells and HEK 293 T cells, GPS2 overexpression significantly enhanced the total protein expression of BK in a dose-dependent manner. Knockdown of GPS2 expression significantly decreased BK protein expression, while increasing ERK1/2 phosphorylation. Knockdown of ERK1/2 expression reversed the GPS2 siRNA-mediated inhibition of BK protein expression in Cos-7 cells. Pretreatments of Cos-7 cells with either the lysosomal inhibitor bafilomycin A1 or the proteasomal inhibitor MG132 partially reversed the inhibitory effects of GPS2 siRNA on BK protein expression. In addition, feeding a high-potassium diet significantly increased both GPS2 and BK protein abundance in mice. These data suggest that GPS2 enhances BK channel activity and its protein expression by reducing ERK1/2 signaling-mediated degradation of the channel.

scholarly journals Molecular mechanism of BK channel activation by the smooth muscle relaxant NS11021

2020 ◽  
Vol 152 (6) ◽  
Author(s):  
Michael E. Rockman ◽  
Alexandre G. Vouga ◽  
Brad S. Rothberg
Keyword(s):  
Smooth Muscle ◽  
Muscle Relaxant ◽  
Time Course ◽  
Bk Channels ◽  
Bk Channel ◽  
Neurological Deficits ◽  
Open Probability ◽  
Channel Activation ◽  
Voltage Sensors ◽  
Smooth Muscle Relaxant

Large-conductance Ca2+-activated K+ channels (BK channels) are activated by cytosolic calcium and depolarized membrane potential under physiological conditions. Thus, these channels control electrical excitability in neurons and smooth muscle by gating K+ efflux and hyperpolarizing the membrane in response to Ca2+ signaling. Altered BK channel function has been linked to epilepsy, dyskinesia, and other neurological deficits in humans, making these channels a key target for drug therapies. To gain insight into mechanisms underlying pharmacological modulation of BK channel gating, here we studied mechanisms underlying activation of BK channels by the biarylthiourea derivative, NS11021, which acts as a smooth muscle relaxant. We observe that increasing NS11021 shifts the half-maximal activation voltage for BK channels toward more hyperpolarized voltages, in both the presence and nominal absence of Ca2+, suggesting that NS11021 facilitates BK channel activation primarily by a mechanism that is distinct from Ca2+ activation. 30 µM NS11021 slows the time course of BK channel deactivation at −200 mV by ∼10-fold compared with 0 µM NS11021, while having little effect on the time course of activation. This action is most pronounced at negative voltages, at which the BK channel voltage sensors are at rest. Single-channel kinetic analysis further shows that 30 µM NS11021 increases open probability by 62-fold and increases mean open time from 0.15 to 0.52 ms in the nominal absence of Ca2+ at voltages less than −60 mV, conditions in which BK voltage sensors are largely in the resting state. We could therefore account for the major activating effects of NS11021 by a scheme in which the drug primarily shifts the pore-gate equilibrium toward the open state.

scholarly journals 14-3-3γ, a novel regulator of the large-conductance Ca2+-activated K+ channel

2020 ◽  
Vol 319 (1) ◽  
pp. F52-F62
Author(s):  
Shan Chen ◽  
Xiuyan Feng ◽  
Xinxin Chen ◽  
Zhizhi Zhuang ◽  
Jia Xiao ◽  
...  
Keyword(s):  
Protein Expression ◽  
Specific Inhibitor ◽  
Bk Channels ◽  
Bk Channel ◽  
K Channel ◽  
Channel Activity ◽  
Open Probability ◽  
Protein Ubiquitination ◽  
293 Cells

14-3-3γ is a small protein regulating its target proteins through binding to phosphorylated serine/threonine residues. Sequence analysis of large-conductance Ca2+-activated K+ (BK) channels revealed a putative 14-3-3 binding site in the COOH-terminal region. Our previous data showed that 14-3-3γ is widely expressed in the mouse kidney. Therefore, we hypothesized that 14-3-3γ has a novel role in the regulation of BK channel activity and protein expression. We used electrophysiology, Western blot analysis, and coimmunoprecipitation to examine the effects of 14-3-3γ on BK channels both in vitro and in vivo. We demonstrated the interaction of 14-3-3γ with BK α-subunits (BKα) by coimmunoprecipitation. In human embryonic kidney-293 cells stably expressing BKα, overexpression of 14-3-3γ significantly decreased BK channel activity and channel open probability. 14-3-3γ inhibited both total and cell surface BKα protein expression while enhancing ERK1/2 phosphorylation in Cos-7 cells cotransfected with flag-14-3-3γ and myc-BK. Knockdown of 14-3-3γ by siRNA transfection markedly increased BKα expression. Blockade of the ERK1/2 pathway by incubation with the MEK-specific inhibitor U0126 partially abolished 14-3-3γ-mediated inhibition of BK protein expression. Similarly, pretreatment of the lysosomal inhibitor bafilomycin A1 reversed the inhibitory effects of 14-3-3γ on BK protein expression. Furthermore, overexpression of 14-3-3γ significantly increased BK protein ubiquitination in embryonic kidney-293 cells stably expressing BKα. Additionally, 3 days of dietary K+ challenge reduced 14-3-3γ expression and ERK1/2 phosphorylation while enhancing renal BK protein expression and K+ excretion. These data suggest that 14-3-3γ modulates BK channel activity and protein expression through an ERK1/2-mediated ubiquitin-lysosomal pathway.

scholarly journals Opposing roles of smooth muscle BK channels and ryanodine receptors in the regulation of nerve-evoked constriction of mesenteric resistance arteries

2014 ◽  
Vol 306 (7) ◽  
pp. H981-H988 ◽  
Author(s):  
Gayathri Krishnamoorthy ◽  
Swapnil K. Sonkusare ◽  
Thomas J. Heppner ◽  
Mark T. Nelson
Keyword(s):  
Smooth Muscle ◽  
Cerebral Arteries ◽  
Ryanodine Receptors ◽  
Electrical Field Stimulation ◽  
Bk Channels ◽  
Bk Channel ◽  
Mesenteric Arteries ◽  
Resistance Arteries ◽  
Sympathetic Nerve Activation ◽  
Neurogenic Vasoconstriction

In depolarized smooth muscle cells of pressurized cerebral arteries, ryanodine receptors (RyRs) generate “Ca2+ sparks” that activate large-conductance, Ca2+-, and voltage-sensitive potassium (BK) channels to oppose pressure-induced (myogenic) constriction. Here, we show that BK channels and RyRs have opposing roles in the regulation of arterial tone in response to sympathetic nerve activation by electrical field stimulation. Inhibition of BK channels with paxilline increased both myogenic and nerve-induced constrictions of pressurized, resistance-sized mesenteric arteries from mice. Inhibition of RyRs with ryanodine increased myogenic constriction, but it decreased nerve-evoked constriction along with a reduction in the amplitude of nerve-evoked increases in global intracellular Ca2+. In the presence of L-type voltage-dependent Ca2+ channel (VDCC) antagonists, nerve stimulation failed to evoke a change in arterial diameter, and BK channel and RyR inhibitors were without effect, suggesting that nerve- induced constriction is dependent on activation of VDCCs. Collectively, these results indicate that BK channels and RyRs have different roles in the regulation of myogenic versus neurogenic tone: whereas BK channels and RyRs act in concert to oppose myogenic vasoconstriction, BK channels oppose neurogenic vasoconstriction and RyRs augment it. A scheme for neurogenic vasoregulation is proposed in which RyRs act in conjunction with VDCCs to regulate nerve-evoked constriction in mesenteric resistance arteries.

Abstract 1368: BK Channel β1 Subunits Are Specific Effectors Of Cholane-induced Channel Activation

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Anna Bukiya ◽  
Ligia Toro ◽  
Alejandro M Dopico
Keyword(s):  
Smooth Muscle ◽  
Cerebral Artery ◽  
Vascular Tone ◽  
Lithocholic Acid ◽  
Cerebral Arteries ◽  
Bk Channels ◽  
Bk Channel ◽  
Α Subunit ◽  
Steady State Activity ◽  
Inside Out

The activity of large conductance, Ca 2+ - and voltage-gated potassium (BK) channels in smooth muscle critically controls vascular tone. Depolarization-induced Ca 2+ -entry in the myocyte activates BK channels, which generate outward positive current that tends to repolarize the membrane, limit Ca 2+ entry and, thus, oppose contraction. Cholane-derived steroids (e.g., lithocholic acid, LC) reduce vascular tone in isolated, resistance-size rat cerebral arteries by selective activation of myocyte BK channels. In most tissues, native BK channels consist of pore-forming α (encoded by KCNMA1 or Slo1 ) and accessory β1–4 (encoded by KCNMB1–4 ) subunits. Remarkably, KCNMB expression is tissue-specific: while KCNMB1 is highly predominant in smooth muscle, KCNMB2–4 are not. Thus, agents that target BK β1 subunits may be used to selectively modulate myocyte BK channel function. After cloning the BK α subunit from rat cerebral artery myocytes (termed “cbv1”, AY330293 ), we demonstrated that homomeric cbv1 channel steady-state activity (NPo) was not affected by acute LC application. In contrast, heteromeric cbv1+β1 channel NPo was reversibly increased by LC (+290% of control at EC max ~150 μM; EC 50 =46 μM). Whether the other BK β subunits (2–4) can substitute for β1 to evoke LC-sensitivity in the BK channel remains unknown. To test this, we applied 150 μM LC to the intracellular side of inside-out patches excised from Xenopus laevis oocytes expressing cbv1 alone or cbv1 with a given BK β subunit subtype (1–4). Currents were evoked with the membrane clamped at ±20mV and free Ca 2+ i set to 10 μM, a concentration found in the cerebral artery myocyte during contraction. As previously found, LC consistently failed to increase homomeric cbv1 NPo, while drastically enhancing heteromeric cbv1+β1 channel NPo. Remarkably, LC failed to activate cbv1+β2, cbv1+β3 and cbv1+β4 heteromeric channels. In conclusion, the BK β1 (smooth muscle-type) subunit serves as a unique sensor for cholane-derived steroids. Thus, these compounds provide a platform for designing therapeutic agents to treat cardiovascular disease where reduction of vascular tone is required.

scholarly journals Enhanced large conductance K+ channel activity contributes to the impaired myogenic response in the cerebral vasculature of Fawn Hooded Hypertensive rats

2014 ◽  
Vol 306 (7) ◽  
pp. H989-H1000 ◽  
Author(s):  
Mallikarjuna R. Pabbidi ◽  
Olga Mazur ◽  
Fan Fan ◽  
Jerry M. Farley ◽  
Debebe Gebremedhin ◽  
...  
Keyword(s):  
Congenic Strain ◽  
Cerebral Arteries ◽  
Bk Channel ◽  
Chromosome 1 ◽  
Brown Norway ◽  
K Channel ◽  
Myogenic Response ◽  
Voltage Sensitivity ◽  
Channel Activity ◽  
Open Probability

Recent studies have indicated that the myogenic response (MR) in cerebral arteries is impaired in Fawn Hooded Hypertensive (FHH) rats and that transfer of a 2.4 megabase pair region of chromosome 1 (RNO1) containing 15 genes from the Brown Norway rat into the FHH genetic background restores MR in a FHH.1BN congenic strain. However, the mechanisms involved remain to be determined. The present study examined the role of the large conductance calcium-activated potassium (BK) channel in impairing the MR in FHH rats. Whole-cell patch-clamp studies of cerebral vascular smooth muscle cells (VSMCs) revealed that iberiotoxin (IBTX; BK inhibitor)-sensitive outward potassium (K+) channel current densities are four- to fivefold greater in FHH than in FHH.1BN congenic strain. Inside-out patches indicated that the BK channel open probability ( NP o) is 10-fold higher and IBTX reduced NP o to a greater extent in VSMCs isolated from FHH than in FHH.1BN rats. Voltage sensitivity of the BK channel is enhanced in FHH as compared with FHH.1BN rats. The frequency and amplitude of spontaneous transient outward currents are significantly greater in VSMCs isolated from FHH than in FHH.1BN rats. However, the expression of the BK-α and -β-subunit proteins in cerebral vessels as determined by Western blot is similar between the two groups. Middle cerebral arteries (MCAs) isolated from FHH rats exhibited an impaired MR, and administration of IBTX restored this response. These results indicate that there is a gene on RNO1 that impairs MR in the MCAs of FHH rats by enhancing BK channel activity.

Sign in / Sign up

Export Citation Format

Share Document