scholarly journals The extracellular loop of the auxiliary β1-subunit is involved in the regulation of BKCa channel mechanosensitivity

2018 ◽  
Vol 315 (4) ◽  
pp. C485-C493 ◽  
Author(s):  
Fang Xin ◽  
Yuan Cheng ◽  
Jie Ren ◽  
Sitao Zhang ◽  
Ping Liu ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Patch Clamp ◽  
Smooth Muscle Cells ◽  
Single Channel ◽  
Muscle Cells ◽  
Hek293 Cells ◽  
Bkca Channel ◽  
Extracellular Loop ◽  
Α Subunits ◽  
Stretch Sensitivity

The large conductance Ca2+-activated potassium (BKCa) channel is activated by stretch. The stress-regulated exon (STREX) in α-subunits is known to affect the mechanosensitivity of BKCa channels. However, in human colonic smooth muscle cells (HCoSMCs), we found that the α-subunits without STREX (ZERO-BK) and β1-subunits could be detected yet the cells were mechanosensitive. Whether the β1-subunit is involved in the regulation of BKCa mechanosensitivity is unclear. In the present study, ZERO-BK and β1-subunits were individually expressed or coexpressed in HEK293 cells and cell-attached patch-clamp techniques were used to measure BKCa currents defining mechanosensitivity. Single-channel patch-clamp recordings from HEK293 cells cotransfected with ZERO-BK and β1-subunits showed stretch sensitivity, but there was no mechanosensitivity in HEK293 cells transfected only with ZERO-BK. We also showed that expression of the β1-subunit could increase mechanosensitivity of the STREX-type α-subunits (STREX-BK). To identify the domain in β1-subunits responsible for affecting stretch sensitivity, we expressed β1- LoopDel (chimeric β1-subunits without the extracellular loop) or β1- C TermDel (chimeric β1-subunits without COOH terminus) with ZERO-BK in HEK293 cells. The data showed that deletion of the extracellular loop but not the COOH terminus of β1-subunits virtually abolished the mechanosensitivity. These results suggest that the extracellular loop of the β1-subunit is involved in the regulation of BKCa channel mechanosensitivity and that role is independent of STREX.

scholarly journals Properties of single-channel and whole cell Cl− currents in guinea pig detrusor smooth muscle cells

2019 ◽  
Vol 316 (5) ◽  
pp. C698-C710 ◽  
Author(s):  
Viktor Yarotskyy ◽  
John Malysz ◽  
Georgi V. Petkov
Keyword(s):  
Smooth Muscle ◽  
Patch Clamp ◽  
Guinea Pig ◽  
Smooth Muscle Cells ◽  
Single Channel ◽  
Muscle Cells ◽  
Niflumic Acid ◽  
Detrusor Smooth Muscle ◽  
Whole Cell ◽  
Whole Cell Patch Clamp

Multiple types of Cl− channels regulate smooth muscle excitability and contractility in vascular, gastrointestinal, and airway smooth muscle cells. However, little is known about Cl− channels in detrusor smooth muscle (DSM) cells. Here, we used inside-out single channel and whole cell patch-clamp recordings for detailed biophysical and pharmacological characterizations of Cl− channels in freshly isolated guinea pig DSM cells. The recorded single Cl− channels displayed unique gating with multiple subconductive states, a fully opened single-channel conductance of 164 pS, and a reversal potential of −41.5 mV, which is close to the ECl of −65 mV, confirming preferential permeability to Cl−. The Cl− channel demonstrated strong voltage dependence of activation (half-maximum of mean open probability, V0.5, ~−20 mV) and robust prolonged openings at depolarizing voltages. The channel displayed similar gating when exposed intracellularly to solutions containing Ca2+-free or 1 mM Ca2+. In whole cell patch-clamp recordings, macroscopic current demonstrated outward rectification, inhibitions by 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS) and niflumic acid, and insensitivity to chlorotoxin. The outward current was reversibly reduced by 94% replacement of extracellular Cl− with I−, Br−, or methanesulfonate (MsO−), resulting in anionic permeability sequence: Cl−>Br−>I−>MsO−. While intracellular Ca2+ levels (0, 300 nM, and 1 mM) did not affect the amplitude of Cl− current and outward rectification, high Ca2+ slowed voltage-step current activation at depolarizing voltages. In conclusion, our data reveal for the first time the presence of a Ca2+-independent DIDS and niflumic acid-sensitive, voltage-dependent Cl− channel in the plasma membrane of DSM cells. This channel may be a key regulator of DSM excitability.

Activation of BKCa channel is associated with increased apoptosis of cerebrovascular smooth muscle cells in simulated microgravity rats

2010 ◽  
Vol 298 (6) ◽  
pp. C1489-C1500 ◽  
Author(s):  
Man-Jiang Xie ◽  
Yu-Guang Ma ◽  
Fang Gao ◽  
Yun-Gang Bai ◽  
Jiu-Hua Cheng ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Animal Studies ◽  
Simulated Microgravity ◽  
Orthostatic Intolerance ◽  
Cerebral Arteries ◽  
Muscle Cells ◽  
Hek293 Cells ◽  
Bkca Channel ◽  
Sprague Dawley

Cerebral arterial remodeling is one of the critical factors in the occurrence of postspaceflight orthostatic intolerance. We hypothesize that large-conductance calcium-activated K+ (BKCa) channels in vascular smooth muscle cells (VSMCs) may play an important role in regulating cerebrovascular adaptation during microgravity exposure. The aim of this work was to investigate whether activation of BKCa channels is involved in regulation of apoptotic remodeling of cerebral arteries in simulated microgravity rats. In animal studies, Sprague-Dawley rats were subjected to 1-wk hindlimb unweighting to simulate microgravity. Alterations of BKCa channels in cerebral VSMCs were investigated by patch clamp and Western blotting; apoptosis was assessed by electron microscopy and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick-end labeling (TUNEL). To evaluate the correlation of BKCa channel and apoptosis, channel protein and cell nucleus were double-stained. In cell studies, hSloα+β1 channel was coexpressed into human embryonic kidney 293 (HEK293) cells to observe the effects of BKCa channels on apoptosis. In rats, enhanced activities and expression of BKCa channels were found to be correlated with increased apoptosis in cerebral VSMCs after simulated microgravity. In transfected HEK293 cells, activation of cloned BKCa channel induced apoptosis, whereas inhibition of cloned BKCa channel decreased apoptosis. In conclusion, activation of BKCa channels is associated with increased apoptosis in cerebral VSMCs of simulated microgravity rats.

scholarly journals BKCa channel regulates calcium oscillations induced by alpha-2-macroglobulin in human myometrial smooth muscle cells

2016 ◽  
Vol 113 (16) ◽  
pp. E2335-E2344 ◽  
Author(s):  
Monali Wakle-Prabagaran ◽  
Ramón A. Lorca ◽  
Xiaofeng Ma ◽  
Susan J. Stamnes ◽  
Chinwendu Amazu ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Single Channel ◽  
Muscle Cells ◽  
Calcium Oscillations ◽  
Proximity Ligation Assay ◽  
Human Myometrium ◽  
Bkca Channel ◽  
Open Probability ◽  
Oscillatory Pattern

The large-conductance, voltage-gated, calcium (Ca2+)-activated potassium channel (BKCa) plays an important role in regulating Ca2+ signaling and is implicated in the maintenance of uterine quiescence during pregnancy. We used immunopurification and mass spectrometry to identify proteins that interact with BKCa in myometrium samples from term pregnant (≥37 wk gestation) women. From this screen, we identified alpha-2-macroglobulin (α2M). We then used immunoprecipitation followed by immunoblot and the proximity ligation assay to confirm the interaction between BKCa and both α2M and its receptor, low-density lipoprotein receptor-related protein 1 (LRP1), in cultured primary human myometrial smooth muscle cells (hMSMCs). Single-channel electrophysiological recordings in the cell-attached configuration demonstrated that activated α2M (α2M*) increased the open probability of BKCa in an oscillatory pattern in hMSMCs. Furthermore, α2M* caused intracellular levels of Ca2+ to oscillate in oxytocin-primed hMSMCs. The initiation of oscillations required an interaction between α2M* and LRP1. By using Ca2+-free medium and inhibitors of various Ca2+ signaling pathways, we demonstrated that the oscillations required entry of extracellular Ca2+ through store-operated Ca2+ channels. Finally, we found that the specific BKCa blocker paxilline inhibited the oscillations, whereas the channel opener NS11021 increased the rate of these oscillations. These data demonstrate that α2M* and LRP1 modulate the BKCa channel in human myometrium and that BKCa and its immunomodulatory interacting partners regulate Ca2+ dynamics in hMSMCs during pregnancy.

Ketamine blocks Ca2+-activated K+ channels in rabbit cerebral arterial smooth muscle cells

2003 ◽  
Vol 285 (3) ◽  
pp. H1347-H1355 ◽  
Author(s):  
Jin Han ◽  
Nari Kim ◽  
Hyun Joo ◽  
Euiyong Kim
Keyword(s):  
Smooth Muscle ◽  
Patch Clamp ◽  
Smooth Muscle Cells ◽  
Cerebral Arteries ◽  
Muscle Cells ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Arterial Smooth Muscle ◽  
Clamp Technique ◽  
Arterial Smooth Muscle Cells

Although ketamine and Ca2+-activated K+ (KCa) channels have been implicated in the contractile activity regulation of cerebral arteries, no studies have addressed the specific interactions between ketamine and the KCa channels in cerebral arteries. The purpose of this study was to examine the direct effects of ketamine on KCa channel activities using the patch-clamp technique in single-cell preparations of rabbit middle cerebral arterial smooth muscle. We tested the hypothesis that ketamine modulates the KCa channel activity of the cerebral arterial smooth muscle cells of the rabbit. Vascular myocytes were isolated from rabbit middle cerebral arteries using enzymatic dissociation. Single KCa channel activities of smooth muscle cells from rabbit cerebral arteries were recorded using the patch-clamp technique. In the inside-out patches, ketamine in the micromolar range inhibited channel activity with a half-maximal inhibition of the ketamine conentration value of 83.8 ± 12.9 μM. The Hill coefficient was 1.2 ± 0.3. The slope conductance of the current-voltage relationship was 320.1 ± 2.0 pS between 0 and +60 mV in the presence of ketamine and symmetrical 145 mM K+. Ketamine had little effect on either the voltage-dependency or open- and closed-time histograms of KCa channel. The present study clearly demonstrates that ketamine inhibits KCa channel activities in rabbit middle cerebral arterial smooth muscle cells. This inhibition of KCa channels may represent a mechanism for ketamine-induced cerebral vasoconstriction.

scholarly journals Epiandrosterone activates BKCa channel in bovine coronary artery smooth muscle cells

2008 ◽  
Vol 22 (S1) ◽  
Author(s):  
Rikuo Ochi ◽  
Rakhee S Gupte ◽  
Michael S Wolin ◽  
Sachin A Gupte
Keyword(s):  
Smooth Muscle ◽  
Coronary Artery ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
Bkca Channel

Patch clamp techniques to study effects of anesthetics on airway smooth muscle cells

1995 ◽  
Vol 9 (1) ◽  
pp. 111-112
Author(s):  
Michiaki Yamakage ◽  
Thomas L. Croxton ◽  
Carol A. Hirshman
Keyword(s):  
Smooth Muscle ◽  
Patch Clamp ◽  
Smooth Muscle Cells ◽  
Airway Smooth Muscle ◽  
Muscle Cells ◽  
Airway Smooth Muscle Cells

scholarly journals Chronic Prenatal Hypoxia Down-Regulated BK Channel Β1 Subunits in Mesenteric Artery Smooth Muscle Cells of the Offspring

2018 ◽  
Vol 45 (4) ◽  
pp. 1603-1616 ◽  
Author(s):  
Bailin Liu ◽  
Yanping Liu ◽  
Ruixiu Shi ◽  
Xueqin Feng ◽  
Xiang Li ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Single Channel ◽  
Muscle Cells ◽  
Bk Channels ◽  
Bk Channel ◽  
Mesenteric Arteries ◽  
Prenatal Hypoxia ◽  
Pregnant Rats ◽  
Α Subunit

Background/Aims: Chronic hypoxia in utero could impair vascular functions in the offspring, underlying mechanisms are unclear. This study investigated functional alteration in large-conductance Ca2+-activated K+ (BK) channels in offspring mesenteric arteries following prenatal hypoxia. Methods: Pregnant rats were exposed to normoxic control (21% O2, Con) or hypoxic (10.5% O2, Hy) conditions from gestational day 5 to 21, their 7-month-old adult male offspring were tested for blood pressure, vascular BK channel functions and expression using patch clamp and wire myograh technique, western blotting, and qRT-PCR. Results: Prenatal hypoxia increased pressor responses and vasoconstrictions to phenylephrine in the offspring. Whole-cell currents density of BK channels and amplitude of spontaneous transient outward currents (STOCs), not the frequency, were significantly reduced in Hy vascular myocytes. The sensitivity of BK channels to voltage, Ca2+, and tamoxifen were reduced in Hy myocytes, whereas the number of channels per patch and the single-channel conductance were unchanged. Prenatal hypoxia impaired NS1102- and tamoxifen-mediated relaxation in mesenteric arteries precontracted with phenylephrine in the presence of Nω-nitro-L-arginine methyl ester. The mRNA and protein expression of BK channel β1, not the α-subunit, was decreased in Hy mesenteric arteries. Conclusions: Impaired BK channel β1-subunits in vascular smooth muscle cells contributed to vascular dysfunction in the offspring exposed to prenatal hypoxia.

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