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Jiusheng Yan - One of the best experts on this subject based on the ideXlab platform.
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Glutamate-activated BK Channel complexes formed with NMDA receptors.
Proceedings of the National Academy of Sciences of the United States of America, 2018Co-Authors: Jiyuan Zhang, Andrea L Meredith, Xin Guan, Hui Lin Pan, Jiusheng YanAbstract:The large-conductance calcium- and voltage-activated K + (BK) Channel has a requirement of high intracellular free Ca 2+ concentrations for its activation in neurons under physiological conditions. The Ca 2+ sources for BK Channel activation are not well understood. In this study, we showed by coimmunopurification and colocalization analyses that BK Channels form complexes with NMDA receptors (NMDARs) in both rodent brains and a heterologous expression system. The BK–NMDAR complexes are broadly present in different brain regions. The complex formation occurs between the obligatory BKα and GluN1 subunits likely via a direct physical interaction of the former’s intracellular S0–S1 loop with the latter’s cytosolic regions. By patch-clamp recording on mouse brain slices, we observed BK Channel activation by NMDAR-mediated Ca 2+ influx in dentate gyrus granule cells. BK Channels modulate excitatory synaptic transmission via functional coupling with NMDARs at postsynaptic sites of medial perforant path-dentate gyrus granule cell synapses. A synthesized peptide of the BKα S0–S1 loop region, when loaded intracellularly via recording pipette, abolished the NMDAR-mediated BK Channel activation and effect on synaptic transmission. These findings reveal the broad expression of the BK–NMDAR complexes in brain, the potential mechanism underlying the complex formation, and the NMDAR-mediated activation and function of postsynaptic BK Channels in neurons.
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Relationship between auxiliary gamma subunits and mallotoxin on BK Channel modulation.
Scientific reports, 2017Co-Authors: Xin Guan, Jiusheng YanAbstract:The large-conductance, calcium- and voltage-activated K+(BK) Channel consists of the pore-forming α subunits (BKα) and auxiliary subunits. The auxiliary γ1-3 subunits potently modulate the BK Channel by shifting its voltage-dependence of Channel activation toward the hyperpolarizing direction by approximately 145 mV (γ1), 100 mV (γ2), and 50 mV (γ3). Mallotoxin is a potent small-molecule BK Channel activator. We analyzed the relationship between mallotoxin and the γ subunits in their BK Channel-activating effects in membrane patches excised from HEK-293 cells. We found that mallotoxin, when applied extracellularly, shifted the half-activation voltage (V1/2) of BKα Channels by −72 mV. The Channel-activating effect of mallotoxin was greatly attenuated in the presence of the γ1, γ2, or γ3 subunit, with resultant ΔV1/2 (+/− mallotoxin) values of −9, −28, or −15 mV, respectively. Most examined γ1 mutant subunits antagonized mallotoxin’s Channel-activating effect in a manner that was largely dependent on its own modulatory function. However, mallotoxin caused an irreversible functional and structural disengagement of the γ1-F273S mutant from BK Channels. We infer that the auxiliary γ subunit effectively interferes with mallotoxin on BK Channel modulation via either a direct steric competition or an indirect allosteric influence on mallotoxin’s binding and action on BKα.
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modulation of BK Channel function by auxiliary beta and gamma subunits
International Review of Neurobiology, 2016Co-Authors: Jiusheng YanAbstract:The large-conductance, Ca(2+)- and voltage-activated K(+) (BK) Channel is ubiquitously expressed in mammalian tissues and displays diverse biophysical or pharmacological characteristics. This diversity is in part conferred by Channel modulation with different regulatory auxiliary subunits. To date, two distinct classes of BK Channel auxiliary subunits have been identified: β subunits and γ subunits. Modulation of BK Channels by the four auxiliary β (β1-β4) subunits has been well established and intensively investigated over the past two decades. The auxiliary γ subunits, however, were identified only very recently, which adds a new dimension to BK Channel regulation and improves our understanding of the physiological functions of BK Channels in various tissues and cell types. This chapter will review the current understanding of BK Channel modulation by auxiliary β and γ subunits, especially the latest findings.
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A Shared Mechanism of BK Channel Activation by Mallotoxin and Auxiliary γ1 Subunit
Biophysical Journal, 2016Co-Authors: Xin Guan, Jiusheng YanAbstract:The large-conductance, voltage- and calcium-activated K+ (BK) Channels consist of the pore-forming, voltage- and Ca2+ -sensing α-subunits (BKα) and the regulatory β and γ subunits. The auxiliary γ1 subunit is so far the most potent activator of BK Channels which drastically shifts the voltage-dependence of Channel activation by ∼ 140 mV towards the hyperpolarizing potential direction. Mallotoxin, also called rottlerin, is a potent extracellular small-molecule activator of BK Channels, which was reported to shift the voltage-dependence of Channel activation by ∼ 100 mV in whole-cell patch-clamp recording condition through a yet largely unknown mechanism. Here, we investigated the effects of mallotoxin on BK Channels in excised membrane patches in the absence and presence of the auxiliary γ subunit. Mallotoxin exerted significant activating effect on BK Channels formed by BK alone in excised membrane patches by a ∼ −80 mV shift in voltage-dependence of Channel activation. However, the presence of γ1 and γ3 subunits nearly abolished and γ2 subunit greatly attenuated the activating effect of mallotoxin. Most mutations in γ1 subunit that caused a loss of its modulatory function on BK Channels also resulted in a loss of its influence on mallotoxin. Importantly, we identified a point mutation in the middle of the γ1 subunit's transmembrane domain that had little effect on the γ1 subunit's modulatory function on BK Channels in the absence of mallotoxin but caused an irreversible loss of the γ1 subunit's binding and modulatory function on BK Channel upon a brief exposure to mallotoxin. Therefore, we conclude that γ1 subunit competitively blocks the mallotoxin's binding or action on BK Channel, which can be fully reversed by a point mutation in the middle of the γ1 transmembrane region.
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molecular basis for differential modulation of BK Channel voltage dependent gating by auxiliary γ subunits
The Journal of General Physiology, 2015Co-Authors: Fei Fan, Jiusheng Yan, Ha Rim KwakAbstract:Large conductance Ca2+- and voltage-activated potassium (BK) Channels are comprised of pore-forming α subunits and various regulatory auxiliary subunits. The BK Channel auxiliary γ (BKγ) subunits are a newly identified class of proteins containing an extracellular leucine-rich repeat domain (LRRD), a single transmembrane (TM) segment, and a short cytoplasmic C-terminal tail (C-tail). Although each of the four BKγ proteins shifts the voltage dependence of BK Channel activation in a hyperpolarizing direction, they show markedly different efficacies, mediating shifts over a range of 15–145 mV. Analyses of chimeric BKγ subunits created by swapping individual structural elements, and of BKγ deletion and substitution mutants, revealed that differential modulation of BK gating by the four BKγ subunits depends on a small region consisting of the TM segment and the adjacent intracellular cluster of positively charged amino acids. The γ1 and γ2 TM segments contributed approximately −100 mV, and the γ1 and γ3 C-tails contributed approximately −40 mV, to shifting the voltage dependence of BK Channel activation, whereas the γ3 and γ4 TM segments and the γ2 and γ4 C-tails contributed much less. The large extracellular LRRDs were mainly functionally interchangeable, although the γ1 LRRD was slightly less effective at enhancing (or slightly more effective at attenuating) the shift in BK Channel voltage-dependent gating toward hyperpolarizing potentials than those of the other BKγ subunits. Analysis of mutated BKγ subunits revealed that juxta-membrane clusters of positively charged amino acids determine the functions of the γ1 and γ3 C-tails. Therefore, the modulatory functions of BKγ subunits are coarse- and fine-tuned, respectively, through variations in their TM segments and in the adjacent intracellular positively charged regions. Our results suggest that BK Channel modulation by auxiliary γ subunits depends on intra- and/or juxta-membrane mechanisms.
Michael J. Shipston - One of the best experts on this subject based on the ideXlab platform.
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S-Acylation controls functional coupling of BK Channel pore-forming α-subunits and β1-subunits
Journal of Biological Chemistry, 2019Co-Authors: Peter J. Duncan, Heather Mcclafferty, Danlei Bi, Lijun Tian, Lie Chen, Michael J. ShipstonAbstract:The properties and physiological function of pore-forming α-subunits of large conductance calcium- and voltage-activated potassium (BK) Channels are potently modified by their functional coupling with regulatory subunits in many tissues. However, mechanisms that might control functional coupling are very poorly understood. Here we show that S-acylation, a dynamic post-translational lipid modification of proteins, of the intracellular S0-S1 loop of the BK Channel pore-forming α-subunit controls functional coupling to regulatory β1-subunits. In HEK293 cells, α-subunits that cannot be S-acylated show attenuated cell surface expression, but expression was restored by co-expression with the β1-subunit. However, we also found that nonacylation of the S0-S1 loop reduces functional coupling between α- and β1-subunits by attenuating the β1-subunit-induced left shift in the voltage for half-maximal activation. In mouse vascular smooth muscle cells expressing both α- and β1-subunits, BK Channel α-subunits were endogenously S-acylated. We further noted that S-acylation is significantly reduced in mice with a genetic deletion of the palmitoyl acyltransferase (Zdhhc23) that controls S-acylation of the S0-S1 loop. Genetic deletion of Zdhhc23 or broad-spectrum pharmacological inhibition of S-acylation attenuated endogenous BK Channel currents independently of changes in cell surface expression of the α-subunit. We conclude that functional effects of S-acylation on BK Channels depend on the presence of β1-subunits. In the absence of β1-subunits, S-acylation promotes cell surface expression, whereas in its presence, S-acylation controls functional coupling. S-Acylation thus provides a mechanism that dynamically regulates the functional coupling with β1-subunits, enabling an additional level of conditional, cell-specific control of ion-Channel physiology.
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palmitoylation and membrane association of the stress axis regulated insert strex controls BK Channel regulation by protein kinase c
Journal of Biological Chemistry, 2012Co-Authors: Xiaobo Zhou, Heather Mcclafferty, Michael J. Shipston, Iris Wulfsen, Peter Ruth, Michael Korth, Robert Lukowski, Dobromir Dobrev, Thomas WielandAbstract:Abstract Large-conductance, calcium- and voltage-gated potassium (BK) Channels play an important role in cellular excitability by controlling membrane potential and calcium influx. The stress axis-regulated exon (STREX) at splice site 2 inverts BK Channel regulation by protein kinase A (PKA) from stimulatory to inhibitory. Here we show that palmitoylation of STREX controls BK Channel regulation also by protein kinase C (PKC). In contrast to the 50% decrease of maximal Channel activity by PKC in the insertless (ZERO) splice variant, STREX Channels were completely resistant to PKC. STREX Channel mutants in which Ser700, located between the two regulatory domains of K+ conductance (RCK) immediately downstream of the STREX insert, was replaced by the phosphomimetic amino acid glutamate (S700E) showed a ~50% decrease in maximal Channel activity, whereas the S700A mutant retained its normal activity. BK Channel inhibition by PKC, however, was effectively established when the palmitoylation-mediated membrane-anchor of the STREX insert was removed by either pharmacological inhibition of palmitoyl transferases or site-directed mutagenesis. These findings suggest that STREX confers a conformation on BK Channels where PKC fails to phosphorylate and to inhibit Channel activity. Importantly, PKA which inhibits Channel activity by disassembling the STREX insert from the plasma membrane, allows PKC to further suppress the Channel gating independent from voltage and calcium. Our results present an important example for the cross-talk between ion Channel palmitoylation and phosphorylation in regulation of cellular excitability.
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Palmitoylation Controls BK Channel Regulation By Phosphorylation
Biophysical Journal, 2009Co-Authors: Lijun Tian, Heather Mcclafferty, Fozia Saleem, Iain Rowe, Lie Chen, Owen Jeffries, Adam Molyvdas, Michael J. ShipstonAbstract:Large conductance calcium- and voltage- gated potassium (BK) Channels are important regulators of physiological homeostasis and their function is potently modulated by protein kinase A (PKA) phosphorylation. PKA regulates the Channel through phosphorylation of residues within the intracellular C-terminus of the pore-forming α-subunits. However, how PKA phosphorylation of the α-subunit effects changes in Channel activity are unknown. The STREX variant of BK Channels is inhibited by PKA as a result of phosphorylation of a serine residue within the evolutionary conserved STREX insert. As this inhibition is dependent upon phosphorylation of only a single α-subunit in the Channel tetramer we hypothesised that phosphorylation results in major conformational rearrangements of the C-terminus. Using a combined imaging, biochemical and electrophysiological strategy we have defined the mechanism of PKA-inhibition of BK Channels. We demonstrate that the cytosolic C-terminus of the STREX BK Channel uniquely interacts with the plasma membrane via palmitoylation of evolutionary conserved cysteine residues. PKA-phosphorylation of STREX dissociates the C-terminus from the plasma membrane resulting in Channel inhibition. Abolition of Channel palmitoylation by site-directed mutagenesis or pharmacological inhibition of palmitoyl-transferases prevents PKA-mediated inhibition. Thus PKA inhibition of BK Channels is conditional upon the palmitoylation status of the Channel. Palmitoylation and phosphorylation are both dynamically regulated thus cross-talk between these two major post-translational signalling cascades provides a novel mechanism for conditional regulation of BK Channels. Interplay of these distinct signalling cascades has important implications for the dynamic regulation of BK Channels and the control of physiological homeostasis.
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Characterization of BK Channel splice variants using membrane potential dyes
British Journal of Pharmacology, 2008Co-Authors: Fozia Saleem, Iain Rowe, Michael J. ShipstonAbstract:Background and purpose: Large conductance calcium- and voltage-activated potassium (BK) Channels are encoded by a single gene that displays extensive pre-mRNA splicing. Here we exploited a membrane potential assay to investigate the sensitivity of different BK splice variants to elevations in intracellular free calcium and their inhibition by the BK Channel blocker paxilline. Experimental approach: Murine BK Channel splice variants were expressed in human embryonic kidney 293 cells and their properties analysed in response to ionomycin-induced calcium influx in both fluorescent membrane potential (fluorescent-imaging plate reader) and patch clamp electrophysiological assays. The dose-dependent inhibition of distinct splice variants by the BK Channel-specific blocker paxilline was also investigated. Key results: Ionomycin-induced calcium influx induced a robust hyperpolarization of human embryonic kidney 293 cells expressing distinct BK Channel splice variants: stress regulated exon (STREX), e22 and ZERO. Splice variant expression resulted in membrane hyperpolarization that displayed a rank order of potency in response to calcium influx of STREX > e22 > ZERO. The BK Channel inhibitor paxilline exhibited very similar potency on all three splice variants with IC50s in membrane potential assays of 0.35 ± 0.04, 0.37 ± 0.03 and 0.70 ± 0.02 µmol·L−1 for STREX, ZERO and e22 respectively. Conclusions and implications: BK Channel splice variants can be rapidly discriminated using membrane potential based assays, based on their sensitivity to calcium. BK Channel splice variants are inhibited by the specific blocker paxilline with similar IC50s. Thus, paxilline may be used in functional assays to inhibit BK Channel function, irrespective of the variant expressed.
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Increased large conductance calcium-activated potassium (BK) Channel expression accompanied by STREX variant downregulation in the developing mouse CNS
BMC Developmental Biology, 2006Co-Authors: Stephen H-f Macdonald, Hans-guenther Knaus, Peter Ruth, Michael J. ShipstonAbstract:Background Large conductance calcium- and voltage activated potassium (BK) Channels are important determinants of neuronal excitability through effects on action potential duration, frequency and synaptic efficacy. The pore- forming subunits are encoded by a single gene, KCNMA1 , which undergoes extensive alternative pre mRNA splicing. Different splice variants can confer distinct properties on BK Channels. For example, insertion of the 58 amino acid stress-regulated exon (STREX) insert, that is conserved throughout vertebrate evolution, encodes Channels with distinct calcium sensitivity and regulation by diverse signalling pathways compared to the insertless (ZERO) variant. Thus, expression of distinct splice variants may allow cells to differentially shape their electrical properties during development. However, whether differential splicing of BK Channel variants occurs during development of the mammalian CNS has not been examined. Results Using quantitative real-time polymerase chain reaction (RT-PCR) Taqman™ assays, we demonstrate that total BK Channel transcripts are up regulated throughout the murine CNS during embryonic and postnatal development with regional variation in transcript levels. This upregulation is associated with a decrease in STREX variant mRNA expression and an upregulation in ZERO variant expression. Conclusion As BK Channel splice variants encode Channels with distinct functional properties the switch in splicing from the STREX phenotype to ZERO phenotype during embryonic and postnatal CNS development may provide a mechanism to allow BK Channels to control distinct functions at different times of mammalian brain development.
Peter Ruth - One of the best experts on this subject based on the ideXlab platform.
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amp activated protein kinase in BK Channel regulation and protection against hearing loss following acoustic overstimulation
The FASEB Journal, 2012Co-Authors: Michael Foller, Peter Ruth, Mirko Jaumann, Juliane Dettling, Ambrish Saxena, Tatsiana Pakladok, Carlos Munoz, Mentor Sopjani, Guiscard Seebohm, Lukas RuttigerAbstract:The energy-sensing AMP-activated serine/threonine protein kinase (AMPK) confers cell survival in part by stimulation of cellular energy production and limitation of cellular energy utilization. AMPK-sensitive functions further include activities of epithelial Na+ Channel ENaC and voltage-gated K+ Channel KCNE1/KCNQ1. AMPK is activated by an increased cytosolic Ca2+ concentration. The present study explored whether AMPK regulates the Ca2+-sensitive large conductance and voltage-gated potassium (BK) Channel. cRNA encoding BK Channel was injected into Xenopus oocytes with and without additional injection of wild-type AMPK (AMPKα1+AMPKβ1+AMPKγ1), constitutively active AMPKγR70Q, or inactive AMPKαK45R. BK-Channel activity was determined utilizing the 2-electrode voltage-clamp. Moreover, BK-Channel protein abundance in the cell membrane was determined by confocal immunomicroscopy. As BK Channels are expressed in outer hair cells (OHC) of the inner ear and lack of BK Channels increases noise vulnerability, OHC B...
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palmitoylation and membrane association of the stress axis regulated insert strex controls BK Channel regulation by protein kinase c
Journal of Biological Chemistry, 2012Co-Authors: Xiaobo Zhou, Heather Mcclafferty, Michael J. Shipston, Iris Wulfsen, Peter Ruth, Michael Korth, Robert Lukowski, Dobromir Dobrev, Thomas WielandAbstract:Abstract Large-conductance, calcium- and voltage-gated potassium (BK) Channels play an important role in cellular excitability by controlling membrane potential and calcium influx. The stress axis-regulated exon (STREX) at splice site 2 inverts BK Channel regulation by protein kinase A (PKA) from stimulatory to inhibitory. Here we show that palmitoylation of STREX controls BK Channel regulation also by protein kinase C (PKC). In contrast to the 50% decrease of maximal Channel activity by PKC in the insertless (ZERO) splice variant, STREX Channels were completely resistant to PKC. STREX Channel mutants in which Ser700, located between the two regulatory domains of K+ conductance (RCK) immediately downstream of the STREX insert, was replaced by the phosphomimetic amino acid glutamate (S700E) showed a ~50% decrease in maximal Channel activity, whereas the S700A mutant retained its normal activity. BK Channel inhibition by PKC, however, was effectively established when the palmitoylation-mediated membrane-anchor of the STREX insert was removed by either pharmacological inhibition of palmitoyl transferases or site-directed mutagenesis. These findings suggest that STREX confers a conformation on BK Channels where PKC fails to phosphorylate and to inhibit Channel activity. Importantly, PKA which inhibits Channel activity by disassembling the STREX insert from the plasma membrane, allows PKC to further suppress the Channel gating independent from voltage and calcium. Our results present an important example for the cross-talk between ion Channel palmitoylation and phosphorylation in regulation of cellular excitability.
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osteopenia due to enhanced cathepsin k release by BK Channel ablation in osteoclasts
PLOS ONE, 2011Co-Authors: Ulrike Sausbier, Peter Ruth, Christian Dullin, Jeannine Missbachguentner, Clement Kabagema, Katarina Flockerzie, Gerd Marten Kuscher, Walter Stuehmer, Winfried Neuhuber, Frauke AlvesAbstract:Background The process of bone resorption by osteoclasts is regulated by Cathepsin K, the lysosomal collagenase responsible for the degradation of the organic bone matrix during bone remodeling. Recently, Cathepsin K was regarded as a potential target for therapeutic intervention of osteoporosis. However, mechanisms leading to osteopenia, which is much more common in young female population and often appears to be the clinical pre-stage of idiopathic osteoporosis, still remain to be elucidated, and molecular targets need to be identified. Methodology/Principal Findings We found, that in juvenile bone the large conductance, voltage and Ca2+-activated (BK) K+ Channel, which links membrane depolarization and local increases in cytosolic calcium to hyperpolarizing K+ outward currents, is exclusively expressed in osteoclasts. In juvenile BK-deficient (BK−/−) female mice, plasma Cathepsin K levels were elevated two-fold when compared to wild-type littermates. This increase was linked to an osteopenic phenotype with reduced bone mineral density in long bones and enhanced porosity of trabecular meshwork in BK−/− vertebrae as demonstrated by high-resolution flat-panel volume computed tomography and micro-CT. However, plasma levels of sRANKL, osteoprotegerin, estrogene, Ca2+ and triiodthyronine as well as osteoclastogenesis were not altered in BK−/− females. Conclusion/Significance Our findings suggest that the BK Channel controls resorptive osteoclast activity by regulating Cathepsin K release. Targeted deletion of BK Channel in mice resulted in an osteoclast-autonomous osteopenia, becoming apparent in juvenile females. Thus, the BK−/− mouse-line represents a new model for juvenile osteopenia, and revealed the BK Channel as putative new target for therapeutic controlling of osteoclast activity.
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Dual role of protein kinase C on BK Channel regulation.
Proceedings of the National Academy of Sciences of the United States of America, 2010Co-Authors: Xiaobo Zhou, Iris Wulfsen, Emine Utku, Ulrike Sausbier, Matthias Sausbier, Thomas Wieland, Peter Ruth, Michael KorthAbstract:Large conductance voltage- and Ca2+-activated potassium Channels (BK Channels) are important feedback regulators in excitable cells and are potently regulated by protein kinases. The present study reveals a dual role of protein kinase C (PKC) on BK Channel regulation. Phosphorylation of S695 by PKC, located between the two regulators of K+ conductance (RCK1/2) domains, inhibits BK Channel open-state probability. This PKC-dependent inhibition depends on a preceding phosphorylation of S1151 in the C terminus of the Channel α-subunit. Phosphorylation of only one α-subunit at S1151 and S695 within the tetrameric pore is sufficient to inhibit BK Channel activity. We further detected that protein phosphatase 1 is associated with the Channel, constantly counteracting phosphorylation of S695. PKC phosphorylation at S1151 also influences stimulation of BK Channel activity by protein kinase G (PKG) and protein kinase A (PKA). Though the S1151A mutant Channel is activated by PKA only, the phosphorylation of S1151 by PKC renders the Channel responsive to activation by PKG but prevents activation by PKA. Phosphorylation of S695 by PKC or introducing a phosphomimetic aspartate at this position (S695D) renders BK Channels insensitive to the stimulatory effect of PKG or PKA. Therefore, our findings suggest a very dynamic regulation of the Channel by the local PKC activity. It is shown that this complex regulation is not only effective in recombinant Channels but also in native BK Channels from tracheal smooth muscle.
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aldosterone increases kca1 1 BK Channel mediated colonic k secretion
The Journal of Physiology, 2008Co-Authors: Mads V Sorensen, Ulrike Sausbier, Matthias Sausbier, Peter Ruth, J Matos, Helle A Praetorius, Jens LeipzigerAbstract: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 KCa1.1 (BK) Channels. Here we investigate if aldosterone stimulates colonic K+ secretion via BK Channels. Luminal Ba2+ 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 Ba2+ and IBTX-sensitive short-circuit current in distal mouse colonic mucosa. This effect was absent in BK α-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 α- and β2-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.
Qiang Chai - One of the best experts on this subject based on the ideXlab platform.
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downregulation of BK Channel function and protein expression in coronary arteriolar smooth muscle cells of type 2 diabetic patients
Cardiovascular Research, 2019Co-Authors: Qiang Chai, Xiao Li Wang, Xiaojing Sun, Guoqing Jiao, Jonathan D Furuseth, John M Stulak, Richard C Daly, Kevin L GreasonAbstract:Aims Type 2 diabetes (T2D) is strongly associated with cardiovascular morbidity and mortality in patients. Vascular large conductance Ca2+-activated potassium (BK) Channels, composed of four pore-forming α subunits (BK-α), and four regulatory β1 subunits (BK-β1), are densely expressed in coronary arterial smooth muscle cells (SMCs) and play an important role in regulating vascular tone and myocardial perfusion. However, the role of BK Channels in coronary microvascular dysfunction of human subjects with diabetes is unclear. In this study, we examined BK Channel function and protein expression, and BK Channel-mediated vasodilation in freshly isolated coronary arterioles from T2D patients. Methods and results Atrial tissues were obtained from 16 patients with T2D and 25 matched non-diabetic subjects during cardiopulmonary bypass procedure. Microvessel videomicroscopy and immunoblot analysis were performed in freshly dissected coronary arterioles and inside-out single BK Channel currents was recorded in enzymatically isolated coronary arteriolar SMCs. We found that BK Channel sensitivity to physiological Ca2+ concentration and voltage was downregulated in the coronary arteriolar SMCs of diabetic patients, compared with non-diabetic controls. BK Channel kinetics analysis revealed that there was significant shortening of the mean open time and prolongation of the mean closed time in diabetic patients, resulting in a remarkable reduction of the Channel open probability. Functional studies showed that BK Channel activation by dehydrosoyasaponin-1 was diminished and that BK Channel-mediated vasodilation in response to shear stress was impaired in diabetic coronary arterioles. Immunoblot experiments confirmed that the protein expressions of BK-α and BK-β1 subunits were significantly downregulated, but the ratio of BK-α/BK-β1 was unchanged in the coronary arterioles of T2D patients. Conclusions Our results demonstrated for the first time that BK Channel function and BK Channel-mediated vasodilation were abnormal in the coronary microvasculature of diabetic patients, due to decreased protein expression and altered intrinsic properties of BK Channels.
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role of nrf2 signaling in the regulation of vascular BK Channel β1 subunit expression and BK Channel function in high fat diet induced diabetic mice
Diabetes, 2017Co-Authors: Tong Lu, Yong Li, Qiang Chai, Xiao Li WangAbstract:The large conductance Ca 2+ -activated K + (BK) Channel β1-subunit (BK-β1) is a key modulator of BK Channel electrophysiology and the downregulation of BK-β1 protein expression in vascular smooth muscle cells (SMCs) underlies diabetic vascular dysfunction. In this study, we hypothesized that the nuclear factor erythroid-2–related factor 2 (Nrf2) signaling pathway plays a significant role in the regulation of coronary BK Channel function and vasodilation in high-fat diet (HFD)–induced obese/diabetic mice. We found that the protein expressions of BK-β1 and Nrf2 were markedly downregulated, whereas those of the nuclear factor-κB (NF-κB) and the muscle ring finger protein 1 (MuRF1 [a ubiquitin E3 ligase for BK-β1]) were significantly upregulated in HFD mouse arteries. Adenoviral expression of Nrf2 suppressed the protein expressions of NF-κB and MuRF1 but enhanced BK-β1 mRNA and protein expressions in cultured coronary SMCs. Knockdown of Nrf2 resulted in reciprocal changes of these proteins. Patch-clamp studies showed that coronary BK-β1–mediated Channel activation was diminished in HFD mice. Importantly, the activation of Nrf2 by dimethyl fumarate significantly reduced the body weight and blood glucose levels of HFD mice, enhanced BK-β1 transcription, and attenuated MuRF1-dependent BK-β1 protein degradation, which in turn restored coronary BK Channel function and BK Channel–mediated coronary vasodilation in HFD mice. Hence, Nrf2 is a novel regulator of BK Channel function with therapeutic implications in diabetic vasculopathy.
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Role of Nrf2 Signaling in the Regulation of Vascular BK Channel β1 Subunit Expression and BK Channel Function in High-Fat Diet–Induced Diabetic Mice
Diabetes, 2017Co-Authors: Xiaojing Sun, Qiang Chai, Xiao Li Wang, Chi LeeAbstract:The large conductance Ca2+-activated K+ (BK) Channel β1-subunit (BK-β1) is a key modulator of BK Channel electrophysiology and the downregulation of BK-β1 protein expression in vascular smooth muscle cells (SMCs) underlies diabetic vascular dysfunction. In this study, we hypothesized that the nuclear factor erythroid-2-related factor 2 (Nrf2) signaling pathway plays a significant role in the regulation of coronary BK Channel function and vasodilation in high-fat diet (HFD)-induced obese/diabetic mice. We found that the protein expressions of BK-β1 and Nrf2 were markedly downregulated, whereas those of the nuclear factor-κB (NF-κB) and the muscle ring finger protein 1 (MuRF1 [a ubiquitin E3 ligase for BK-β1]) were significantly upregulated in HFD mouse arteries. Adenoviral expression of Nrf2 suppressed the protein expressions of NF-κB and MuRF1 but enhanced BK-β1 mRNA and protein expressions in cultured coronary SMCs. Knockdown of Nrf2 resulted in reciprocal changes of these proteins. Patch-clamp studies showed that coronary BK-β1-mediated Channel activation was diminished in HFD mice. Importantly, the activation of Nrf2 by dimethyl fumarate significantly reduced the body weight and blood glucose levels of HFD mice, enhanced BK-β1 transcription, and attenuated MuRF1-dependent BK-β1 protein degradation, which in turn restored coronary BK Channel function and BK Channel-mediated coronary vasodilation in HFD mice. Hence, Nrf2 is a novel regulator of BK Channel function with therapeutic implications in diabetic vasculopathy.
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regulation of large conductance ca2 activated k BK Channel β1 subunit expression by muscle ring finger protein 1 in diabetic vessels
Journal of Biological Chemistry, 2014Co-Authors: Huan Wang, Qiang Chai, Xiao Li Wang, Win Kuang Shen, Monte S Willis, Hon Chi LeeAbstract:The large conductance Ca2+-activated K+ (BK) Channel, expressed abundantly in vascular smooth muscle cells (SMCs), is a key determinant of vascular tone. BK Channel activity is tightly regulated by its accessory β1 subunit (BK-β1). However, BK Channel function is impaired in diabetic vessels by increased ubiquitin/proteasome-dependent BK-β1 protein degradation. Muscle RING finger protein 1 (MuRF1), a muscle-specific ubiquitin ligase, is implicated in many cardiac and skeletal muscle diseases. However, the role of MuRF1 in the regulation of vascular BK Channel and coronary function has not been examined. In this study, we hypothesized that MuRF1 participated in BK-β1 proteolysis, leading to the down-regulation of BK Channel activation and impaired coronary function in diabetes. Combining patch clamp and molecular biological approaches, we found that MuRF1 expression was enhanced, accompanied by reduced BK-β1 expression, in high glucose-cultured human coronary SMCs and in diabetic vessels. Knockdown of MuRF1 by siRNA in cultured human SMCs attenuated BK-β1 ubiquitination and increased BK-β1 expression, whereas adenoviral expression of MuRF1 in mouse coronary arteries reduced BK-β1 expression and diminished BK Channel-mediated vasodilation. Physical interaction between the N terminus of BK-β1 and the coiled-coil domain of MuRF1 was demonstrated by pulldown assay. Moreover, MuRF1 expression was regulated by NF-κB. Most importantly, pharmacological inhibition of proteasome and NF-κB activities preserved BK-β1 expression and BK-Channel-mediated coronary vasodilation in diabetic mice. Hence, our results provide the first evidence that the up-regulation of NF-κB-dependent MuRF1 expression is a novel mechanism that leads to BK Channelopathy and vasculopathy in diabetes.
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reactive oxygen species signaling facilitates foxo 3a fbxo dependent vascular BK Channel β1 subunit degradation in diabetic mice
Diabetes, 2012Co-Authors: Qiang Chai, Livius V Duscio, Zvonimir S Katusic, Hon Chi LeeAbstract:Activity of the vascular large conductance Ca(2+)-activated K(+) (BK) Channel is tightly regulated by its accessory β(1) subunit (BK-β(1)). Downregulation of BK-β(1) expression in diabetic vessels is associated with upregulation of the forkhead box O subfamily transcription factor-3a (FOXO-3a)-dependent F-box-only protein (FBXO) expression. However, the upstream signaling regulating this process is unclear. Overproduction of reactive oxygen species (ROS) is a common finding in diabetic vasculopathy. We hypothesized that ROS signaling cascade facilitates the FOXO-3a/FBXO-mediated BK-β(1) degradation and leads to diabetic BK Channel dysfunction. Using cellular biology, patch clamp, and videomicroscopy techniques, we found that reduced BK-β(1) expression in streptozotocin (STZ)-induced diabetic mouse arteries and in human coronary smooth muscle cells (SMCs) cultured with high glucose was attributable to an increase in protein kinase C (PKC)-β and NADPH oxidase expressions and accompanied by attenuation of Akt phosphorylation and augmentation of atrogin-1 expression. Treatment with ruboxistaurin (a PKCβ inhibitor) or with GW501516 (a peroxisome proliferator-activated receptor δ activator) reduced atrogin-1 expression and restored BK Channel-mediated coronary vasodilation in diabetic mice. Our results suggested that oxidative stress inhibited Akt signaling and facilitated the FOXO-3a/FBXO-dependent BK-β(1) degradation in diabetic vessels. Suppression of the FOXO-3a/FBXO pathway prevented vascular BK-β(1) degradation and protected coronary function in diabetes.
Xiaobo Zhou - One of the best experts on this subject based on the ideXlab platform.
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palmitoylation and membrane association of the stress axis regulated insert strex controls BK Channel regulation by protein kinase c
Journal of Biological Chemistry, 2012Co-Authors: Xiaobo Zhou, Heather Mcclafferty, Michael J. Shipston, Iris Wulfsen, Peter Ruth, Michael Korth, Robert Lukowski, Dobromir Dobrev, Thomas WielandAbstract:Abstract Large-conductance, calcium- and voltage-gated potassium (BK) Channels play an important role in cellular excitability by controlling membrane potential and calcium influx. The stress axis-regulated exon (STREX) at splice site 2 inverts BK Channel regulation by protein kinase A (PKA) from stimulatory to inhibitory. Here we show that palmitoylation of STREX controls BK Channel regulation also by protein kinase C (PKC). In contrast to the 50% decrease of maximal Channel activity by PKC in the insertless (ZERO) splice variant, STREX Channels were completely resistant to PKC. STREX Channel mutants in which Ser700, located between the two regulatory domains of K+ conductance (RCK) immediately downstream of the STREX insert, was replaced by the phosphomimetic amino acid glutamate (S700E) showed a ~50% decrease in maximal Channel activity, whereas the S700A mutant retained its normal activity. BK Channel inhibition by PKC, however, was effectively established when the palmitoylation-mediated membrane-anchor of the STREX insert was removed by either pharmacological inhibition of palmitoyl transferases or site-directed mutagenesis. These findings suggest that STREX confers a conformation on BK Channels where PKC fails to phosphorylate and to inhibit Channel activity. Importantly, PKA which inhibits Channel activity by disassembling the STREX insert from the plasma membrane, allows PKC to further suppress the Channel gating independent from voltage and calcium. Our results present an important example for the cross-talk between ion Channel palmitoylation and phosphorylation in regulation of cellular excitability.
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Dual role of protein kinase C on BK Channel regulation.
Proceedings of the National Academy of Sciences of the United States of America, 2010Co-Authors: Xiaobo Zhou, Iris Wulfsen, Emine Utku, Ulrike Sausbier, Matthias Sausbier, Thomas Wieland, Peter Ruth, Michael KorthAbstract:Large conductance voltage- and Ca2+-activated potassium Channels (BK Channels) are important feedback regulators in excitable cells and are potently regulated by protein kinases. The present study reveals a dual role of protein kinase C (PKC) on BK Channel regulation. Phosphorylation of S695 by PKC, located between the two regulators of K+ conductance (RCK1/2) domains, inhibits BK Channel open-state probability. This PKC-dependent inhibition depends on a preceding phosphorylation of S1151 in the C terminus of the Channel α-subunit. Phosphorylation of only one α-subunit at S1151 and S695 within the tetrameric pore is sufficient to inhibit BK Channel activity. We further detected that protein phosphatase 1 is associated with the Channel, constantly counteracting phosphorylation of S695. PKC phosphorylation at S1151 also influences stimulation of BK Channel activity by protein kinase G (PKG) and protein kinase A (PKA). Though the S1151A mutant Channel is activated by PKA only, the phosphorylation of S1151 by PKC renders the Channel responsive to activation by PKG but prevents activation by PKA. Phosphorylation of S695 by PKC or introducing a phosphomimetic aspartate at this position (S695D) renders BK Channels insensitive to the stimulatory effect of PKG or PKA. Therefore, our findings suggest a very dynamic regulation of the Channel by the local PKC activity. It is shown that this complex regulation is not only effective in recombinant Channels but also in native BK Channels from tracheal smooth muscle.
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inducible knockout mutagenesis reveals compensatory mechanisms elicited by constitutive BK Channel deficiency in overactive murine bladder
FEBS Journal, 2009Co-Authors: Franz Sprossmann, Xiaobo Zhou, Ulrike Sausbier, Iancu Bucurenciu, Hong Zhao, Patrick Pankert, Angela Wirth, Johannes Madlung, Andreas Jakob, Tobias LamkemeyerAbstract:The large-conductance, voltage-dependent and Ca2+-dependent K+ (BK) Channel links membrane depolarization and local increases in cytosolic free Ca2+ to hyperpolarizing K+ outward currents, thereby controlling smooth muscle contractility. Constitutive deletion of the BK Channel in mice (BK−/−) leads to an overactive bladder associated with increased intravesical pressure and frequent micturition, which has been revealed to be a result of detrusor muscle hyperexcitability. Interestingly, time-dependent and smooth muscle-specific deletion of the BK Channel (SM-BK−/−) caused a more severe phenotype than displayed by constitutive BK−/− mice, suggesting that compensatory pathways are active in the latter. In detrusor muscle of BK−/− but not SM-BK−/− mice, we found reduced L-type Ca2+ current density and increased expression of cAMP kinase (protein kinase A; PKA), as compared with control mice. Increased expression of PKA in BK−/− mice was accompanied by enhanced β-adrenoceptor/cAMP-mediated suppression of contractions by isoproterenol. This effect was attenuated by about 60–70% in SM-BK−/− mice. However, the Rp isomer of adenosine-3′,5′-cyclic monophosphorothioate, a blocker of PKA, only partially inhibited enhanced cAMP signaling in BK−/− detrusor muscle, suggesting the existence of additional compensatory pathways. To this end, proteome analysis of BK−/− urinary bladder tissue was performed, and revealed additional compensatory regulated proteins. Thus, constitutive and inducible deletion of BK Channel activity unmasks compensatory mechanisms that are relevant for urinary bladder relaxation.
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elevated blood pressure linked to primary hyperaldosteronism and impaired vasodilation in BK Channel deficient mice
Circulation, 2005Co-Authors: Matthias Sausbier, Xiaobo Zhou, Ulrike Sausbier, Claudia Arntz, Iancu Bucurenciu, Hong Zhao, Susanne Feil, Simone Kamm, Kyrill Essin, Claudia A SailerAbstract:Background— Abnormally elevated blood pressure is the most prevalent risk factor for cardiovascular disease. The large-conductance, voltage- and Ca2+-dependent K+ (BK) Channel has been proposed as an important effector in the control of vascular tone by linking membrane depolarization and local increases in cytosolic Ca2+ to hyperpolarizing K+ outward currents. However, the BK Channel may also affect blood pressure by regulating salt and fluid homeostasis, particularly by adjusting the renin-angiotensin-aldosterone system. Methods and Results— Here we report that deletion of the pore-forming BK Channel α subunit leads to a significant blood pressure elevation resulting from hyperaldosteronism accompanied by decreased serum K+ levels as well as increased vascular tone in small arteries. In smooth muscle from small arteries, deletion of the BK Channel leads to a depolarized membrane potential, a complete lack of membrane hyperpolarizing spontaneous K+ outward currents, and an attenuated cGMP vasorelaxation ...