Effect And Mechanism Of Orail-BK_Ca Signal Complex Regulating Vascular Smooth Muscle Contraction And Vessel Tension

ObjectiveLarge-conductance calcium-activated K+ channels (BKca), one of the members in voltage-gated potassium channel superfamily, are widely distributed inside the vascular smooth muscle cells (VSMCs) and exert essential regulatory effects on both vasodialation and vasoconstriction. Both depolarization of cell membrane and intracellular calcium increase could trigger the opening of BKCa causing membrane hyperpolarization and subsequently block the opening of voltage-dependent calcium channel (VDCC) to regulate the smooth muscle contraction. Ca2+, a well-recognised intracellular second messenger, is an essential factor for smooth muscle contraction, and store-operated Ca2+ entry (SOCE) is the general approach to regulate the Ca2+ influx. SOCE is mediated by store-operated calcium channel (SOCC), and Orail, the main component of SOCC, is a kind of tetraspanin protein located on the cell membrane. After the depletion of Ca2+ store, STIM1 aggregates and subsequently interactes with Orai1 to lead to Orai1 open, mediating influx of Ca2+. Therefore, we speculate that Orai1 is functionally interacted with BKca. Opening of the SOCC elevates intracellular Ca2+ concentration, which activates the adjacent BKcato prevent the excess contraction of mesenteric VSMCs in responce to contractile agonists. In the present study, our aim is to investigate whether Orail has functional interaction with BKca in mesenteric VSMCs and demonstrate the role of Orail-BKca complex in regulation of VSMCs contraction and development of hypertensive blood vessel pathology.Methods1. Cell culture SPF SD male rats, weighing about 250 g, were used to provide VSMCs. Briefly, rats were killed via asphyxia method and mesenteric artery was dissected under sterile condition. After rubbing off endothelial layer, smooth muscle layer was peeled off and then digested with 0.2% collagenase type IA and 0.9% papain for 1 h. The dispersed VSMCs were cultured in Dulbecco modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS),100 U/mL penicillin and 100μg/mL streptomycin for 5 to 7 days before experiment.2. [Ca2+]i measurementAfter being transfected with Scrambled siRNA or Orail siRNA for 24 hr, primary cultured VSMCs were incubated with 10μmol/L Fluo-8/AM and 0.02% pluronic F-127 (Invitrogen) for 40 min in the dark at 37℃. Ca2+ stores were subsequently depleted by treating cells with 4μmol/L TG for 10 min within 0Ca2+-PSS solution and Ca2+ influx was initiated by applying 1 mmol/L extracellular Ca2+. Fluorescence signal was recorded by Nikon Diaphot inverted microscope. Changes in [Ca2+], were shown as the ratio of fluorescence relative to the intensity before applying extracellular Ca2+(F1/F0).3. Membrane potential measurementPrimary cultured VSMCs were transfected with scrambled siRNA or Orail siRNA for 24 hr and subsequently were treated with 100 nmol/L of (DiBAC4(3)) at 37℃ for 10 min. Cells were then treated with 4μmol/L TG in the dark for 10 min in 0Ca2+-PSS solution. After the depletion of Ca2+ stores, SOCE was initiated by applying 1 mmol/L extracellular Ca2+. Changes in fluorescence were measured by Nikon Diaphot inverted microscope and changes of membrane potential were shown as the ratio of fluorescence relative to the intensity before applying extracellular Ca2+(F1/F0).4. Isometric Tension MeasurementsSegments of the tertiary branches of rat mesenteric artery were incubated in Krebs solution and oxygenated with a gas mixture of 95% O2 and 5% CO2. The segments were mounted in a DMT myograph (model 610M; Danish Myo Technology, Aarhus, Denmark) and were subsequently treated with inhibitor IbTX and Orail siRNA separately. Phe and ET1 were used to detect the response to different contractile agonists. The response was recorded by a DMT myograph (model 610 M).5. Co-immunoprecipitationCo-immunoprcipitation was used to detect the interaction of Orail and BKCa in Mesenteric artery.6. In situ proximity ligation assay (PLA)Interaction of Orail with BKCa was detected by using in situ PLA kit, following the manufacturer’s instructions, and primary VSMCs were isolated from mesenteric arteries and attached on coverslips. Only if the two proteins are in close proximity (<40 nm separation), they can be detected. PLA signals were visualized and recorded through a Leica TCS SP5 confocal microscope.7. Statistical analysesThe experimental data were presented as means±SEM. The significance was analyzed using unpaired t test or two way ANOVA by Sigma Plot 12.5 software. A value of P< 0.05 was considered statistically significant.Results1. SOCE was mediated by Orail and Ca2+ influx via Orail triggered membrane hyperpolarization of VSMCs;2. This Orail-mediated and Ca2+ influx-induced membrane hyperpolarization was inhibited by BKca blocker;3. Knockdown of Orail and preincubation of BKCa blocker markedly enhanced vasocontraction of rat mesenteric arteries in response to contractile agonists;4. Coimmunoprecipitation data revealed that anti-Orail antibody could pull down BKCa and vice versa;5. PLA analysis indicated again that Orail and BKca were physically interacted in VSMCs.ConclusionTaken together, Our findings demonstrated that the Ca2+ influx through Orail, a predominant mediator of SOCE, activates BKCa causing membrane hyperpolarization. Moreover, Orail physically interacts with BKCa to form a signaling complex suppressing agonist-induced membrane depolarization, inhibiting excessive contraction of VSMCs in response to contractile agonists.