scholarly journals Nanoscale remodeling of ryanodine receptor cluster size underlies cerebral microvascular dysfunction in Duchenne muscular dystrophy

2018 ◽  
Vol 115 (41) ◽  
pp. E9745-E9752 ◽  
Author(s):  
Harry A. T. Pritchard ◽  
Paulo W. Pires ◽  
Evan Yamasaki ◽  
Pratish Thakore ◽  
Scott Earley

Duchenne muscular dystrophy (DMD) results from mutations in the gene encoding dystrophin which lead to impaired function of skeletal and cardiac muscle, but little is known about the effects of the disease on vascular smooth muscle cells (SMCs). Here we used the mdx mouse model to study the effects of mutant dystrophin on the regulation of cerebral artery and arteriole SMC contractility, focusing on an important Ca2+-signaling pathway composed of type 2 ryanodine receptors (RyR2s) on the sarcoplasmic reticulum (SR) and large-conductance Ca2+-activated K+ (BK) channels on the plasma membrane. Nanoscale superresolution image analysis revealed that RyR2 and BKα were organized into discrete clusters, and that the mean size of RyR2 clusters that colocalized with BKα was larger in SMCs from mdx mice (∼62 RyR2 monomers) than in controls (∼40 RyR2 monomers). We further found that the frequency and signal mass of spontaneous, transient Ca2+-release events through SR RyR2s (“Ca2+ sparks”) were greater in SMCs from mdx mice. Patch-clamp electrophysiological recordings indicated a corresponding increase in Ca2+-dependent BK channel activity. Using pressure myography, we found that cerebral pial arteries and parenchymal arterioles from mdx mice failed to develop appreciable spontaneous myogenic tone. Inhibition of RyRs with tetracaine and blocking of BK channels with paxilline restored myogenic tone to control levels, demonstrating that enhanced RyR and BK channel activity is responsible for the diminished pressure-induced constriction of arteries and arterioles from mdx mice. We conclude that increased size of RyR2 protein clusters in SMCs from mdx mice increases Ca2+ spark and BK channel activity, resulting in cerebral microvascular dysfunction.

2014 ◽  
Vol 306 (5) ◽  
pp. C460-C470 ◽  
Author(s):  
Kiril L. Hristov ◽  
Amy C. Smith ◽  
Shankar P. Parajuli ◽  
John Malysz ◽  
Georgi V. Petkov

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.


2001 ◽  
Vol 281 (6) ◽  
pp. C1769-C1775 ◽  
Author(s):  
Guillermo J. Pérez ◽  
Adrian D. Bonev ◽  
Mark T. Nelson

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.


2016 ◽  
Vol 38 (4) ◽  
pp. 1652-1662 ◽  
Author(s):  
Bernat Elvira ◽  
Yogesh Singh ◽  
Jamshed Warsi ◽  
Carlos Munoz ◽  
Florian Lang

Background/Aims: The oxidative stress-responsive kinase 1 (OSR1) and the serine/threonine kinases SPAK (SPS1-related proline/alanine-rich kinase) are under the control of WNK (with-no-K [Lys]) kinases. OSR1 and SPAK participate in diverse functions including cell volume regulation and neuronal excitability. Cell volume and neuronal excitation are further modified by the large conductance Ca2+-activated K+ channels (maxi K+ channel or BK channels). An influence of OSR1 and/or SPAK on BK channel activity has, however, never been shown. The present study thus explored whether OSR1 and/or SPAK modify the activity of BK channels. Methods: cRNA encoding the Ca2+ insensitive BK channel mutant BKM513I+Δ899-903 was injected into Xenopus laevis oocytes without or with additional injection of cRNA encoding wild-type OSR1 or wild-type SPAK, constitutively active T185EOSR1, catalytically inactive D164AOSR1, constitutively active T233ESPAK or catalytically inactive D212ASPAK. K+ channel activity was measured utilizing dual electrode voltage clamp. Results: BK channel activity in BKM513I+Δ899-903 expressing oocytes was significantly decreased by co-expression of OSR1 or SPAK. The effect of wild-type OSR1/SPAK was mimicked by T185EOSR1 and T233ESPAK, but not by D164AOSR1 or D212ASPAK. Conclusions: OSR1 and SPAK suppress BK channels, an effect possibly contributing to cell volume regulation and neuroexcitability.


2018 ◽  
Vol 315 (3) ◽  
pp. F503-F511 ◽  
Author(s):  
Zhizhi Zhuang ◽  
Jia Xiao ◽  
Xinxin Chen ◽  
Xiaohan Hu ◽  
Ruidian Li ◽  
...  

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.


2020 ◽  
Vol 319 (1) ◽  
pp. F52-F62
Author(s):  
Shan Chen ◽  
Xiuyan Feng ◽  
Xinxin Chen ◽  
Zhizhi Zhuang ◽  
Jia Xiao ◽  
...  

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.


Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Tong Lu ◽  
Xiaoli Wang ◽  
Hon-Chi Lee

Angiotensin II (Ang II) type I receptor (ATR 1 ) trafficking into caveolae is essential for Ang II signaling, which is known to be abnormal in diabetic vessels. We have shown that the large conductance Ca 2+ actviated K + (BK) channels are also targeted to caveolae in vascular cells. The potential interaction between Ang II signaling and BK channel function in normal and diabetic vessels is unknown. Using whole-cell patch clamp recordings and molecular biology techniques, we examined the mechanisms through which caveolae targeting facilitates the regulation of BK channels by Ang II signaling. We found that in cultured human coronary arterial smooth muscle cells (CASMC) and in freshly isolated rat CASMC, BK channels, ATR 1 , and Src-family protein tyrosine kinases (Src-PTK) were colocalized and enriched in the low buoyant density, caveolae-rich fractions. 2 μM Ang II inhibited BK channel activity by ∼50% in rat and human CASMC and these effects were completely abolished by 2 μM Losartan (a selective ATR 1 inhibitor), 10 μM PP2 (a selective Src-PTK inhibitor), and by caveolin-1 (cav-1) knockdown using 60 nM siRNA. Similar results were obtained in HEK293 cells coexpressing hSlo, BK-β 1 subunit, ATR 1 , cav-1, and Src-PTK, indicating that inhibition of BK channels by Ang II was mediated through ATR 1 activation of Src-PTK and the integrity of caveolae is critical for Ang II signaling. Culturing human CASMC with high glucose (HG, 22 mM) enhanced Ang II-mediated BK channel inhibition (78.8±16.8% vs. 54.5±15.7% in 5 mM glucose, n=3, p<0.05). Analysis of ATR 1 , Src-PTK, and BK channel distribution by sucrose gradient fractionation and by co-immunoprecipitation with anti-cav-1 antibodies showed that expression of ATR 1 and Src-PTK were up-regulated in human CASMC cultured in HG and in CASMC from streptozotocin-induced diabetic rats. Total BK channel protein in these cells was diminished, but the amount of BK channels co-immunoprecipitated with anti-cav-1 antibody was increased, suggesting increased caveolae targeting of BK channels in diabetes, which leads to enhanced Ang II-mediated BK channel inhibition. These results indicate that Ang II-BK channel interaction is critically dependent upon caveolae targeting under normal conditions and in disease states such as diabetes.


2014 ◽  
Vol 306 (7) ◽  
pp. H981-H988 ◽  
Author(s):  
Gayathri Krishnamoorthy ◽  
Swapnil K. Sonkusare ◽  
Thomas J. Heppner ◽  
Mark T. Nelson

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.


2002 ◽  
Vol 120 (1) ◽  
pp. 15-27 ◽  
Author(s):  
Ronghua ZhuGe ◽  
Kevin E. Fogarty ◽  
Richard A. Tuft ◽  
John V. Walsh

Ca2+ sparks are small, localized cytosolic Ca2+ transients due to Ca2+ release from sarcoplasmic reticulum through ryanodine receptors. In smooth muscle, Ca2+ sparks activate large conductance Ca2+-activated K+ channels (BK channels) in the spark microdomain, thus generating spontaneous transient outward currents (STOCs). The purpose of the present study is to determine experimentally the level of Ca2+ to which the BK channels are exposed during a spark. Using tight seal, whole-cell recording, we have analyzed the voltage-dependence of the STOC conductance (g(STOC)), and compared it to the voltage-dependence of BK channel activation in excised patches in the presence of different [Ca2+]s. The Ca2+ sparks did not change in amplitude over the range of potentials of interest. In contrast, the magnitude of g(STOC) remained roughly constant from 20 to −40 mV and then declined steeply at more negative potentials. From this and the voltage dependence of BK channel activation, we conclude that the BK channels underlying STOCs are exposed to a mean [Ca2+] on the order of 10 μM during a Ca2+ spark. The membrane area over which a concentration ≥10 μM is reached has an estimated radius of 150–300 nm, corresponding to an area which is a fraction of one square micron. Moreover, given the constraints imposed by the estimated channel density and the Ca2+ current during a spark, the BK channels do not appear to be uniformly distributed over the membrane but instead are found at higher density at the spark site.


2010 ◽  
Vol 31 (1) ◽  
pp. 3-16 ◽  
Author(s):  
Masayo Koide ◽  
Matthew A Nystoriak ◽  
Gayathri Krishnamoorthy ◽  
Kevin P O'Connor ◽  
Adrian D Bonev ◽  
...  

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.


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