Cloning of large-conductance Ca-activated K channel α-subunits in mouse cardiomyocytes

Large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels are widely distributed in cellular membranes of various tissues, but have not previously been found in cardiomyocytes. In this study, we cloned a gene encoding the mouse cardiac BK_Ca channel α-subunit (mCardBKa). Sequence analysis of the cDNA revealed an open reading frame encoding 1154 amino acids. Another cDNA variant, identical in amino acid sequence, was also identified by sequence analysis. The nucleotide sequences of the two mCardBKa cDNAs, type 1 (mCardBKa1) and type 2 (mCardBKa2), differed by three nucleotide insertions and one nucleotide substitution in the N-terminal sequence. The amino acid sequence demonstrated that mCardBKa was a unique BK_Ca channel α-subunit in mouse cardiomyocytes, with amino acids 41-1153 being identical to calcium-activated potassium channel SLO1 and amino acids 1-40 corresponding to BK_Ca channel subfamily M alpha member 1. These findings suggest that a unique BK_Ca channel α-subunit is expressed in mouse cardiomyocytes.     

Large conductance Ca2+-activated K+ channel (BKCa) activating properties of a series of novel N-arylbenzamides: Channel subunit dependent effects

Large conductance calcium activated potassium channels (BK_Ca) are fundamental in the control of cellular excitability. Thus, compounds that activate BK_Ca channels could provide potential therapies in the treatment of pathologies of the cardiovascular and central nervous system. A series of novel N-arylbenzamide compounds, and the reference compound NS1619, were evaluated for BK_Ca channel opener properties in Human Embryonic Kidney (HEK293) cells expressing the human BK_Ca channel α-subunit alone or α + β1-subunit complex.Channel activity was determined using a non-radioactive Rb⁺ efflux assay to construct concentration effect curves for each compound. All N-arylbenzamide compounds and NS1619 evoked significant (p <0.05) concentration related increases in Rb⁺ efflux both in cells expressing α-subunit alone or α + β1-subunits. Co-expression of the β1-subunit modified the Rb⁺ efflux responses, relative to that obtained in cells expressing the α-subunit alone, for most of the N-arylbenzamide compounds, in contrast to NS1619. The EC₄₀ values of NS1619, BKMe1 and BKOEt1 were not significantly affected by the co-expression of the BK_Ca channel α + β1-subunits. In contrast, 5 other N-arylbenzamides (BKPr2, BKPr3, BKPr4, BKH1 and BKVV) showed a significant (p <0.05) 2- to 10-fold increase in EC₄₀ values when tested on the BK_Ca α + β1-subunit expressing cells compared to BK_Ca α-subunit expressing cells. Further, the E_max values for BKPr4, BKVV and BKH1 were lower in the BK_Ca channel α + β1-subunit expressing cells.In conclusion, the N-arylbenzamides studied, like NS1619, were able to activate BK_Ca channels formed of the α-subunit only. The co-expression of the β1-subunit, however, modified the ability of certain compounds to active the channel leading to differentiated pharmacodynamic profiles.     

Coexpression and characterization of the human large-conductance Ca2+-activated K+ channel α + β1 subunits in HEK293 cells

Large-conductance Ca²⁺-activated K⁺ channel is formed by a tetramer of the pore-forming α-subunit and distinct accessory β-subunits (β1–β4) which contribute to BK_Ca channel molecular diversity. Accumulative evidences indicate that not only α-subunit alone but also the α + β subunit complex and/or β-subunit might play an important role in modulating various physiological functions in most mammalian cells. To evaluate the detailed pharmacological and biophysical properties of α + β1 subunit complex or β1-subunit in BK_Ca channel, we established an expression system that reliably coexpress hSloα + β1 subunit complex in HEK293 cells. The coexpression of hSloα + β1 subunit complex was evaluated by western blotting and immunolocalization, and then the single-channel kinetics and pharmacological properties of expressed hSloα + β1 subunit complex were investigated by cell-attached and outside-out patches, respectively. The results in this study showed that the expressed hSloα + β1 subunit complex demonstrated to be fully functional for its typical single-channel traces, Ca²⁺-sensitivity, voltage-dependency, high conductance (151 ± 7 pS), and its pharmacological activation and inhibition.     

Potentiation of large-conductance calcium-activated potassium (BK(Ca)) channels by a specific isoform of protein kinase C

The phosphorylation state of large-conductance calcium-activated potassium (BK_Ca) channels regulates their activity and is dynamically regulated by protein phosphatases and kinases, including protein kinase C (PKC). In this study, we showed that PKC activators up-regulate the activity of the BK_Ca channel alpha (α)-subunit, Slo1, in cell-attached patches of transfected COS7 cells. In an immune complex kinase assay, BK_Ca channels isolated from rat brain were phosphorylated in the presence of PKC activators, without the addition of exogenous PKC, which suggests that PKC and BK_Ca channels functionally interact in vivo. Four different PKC isozymes, including PKCδ, phosphorylated the C-terminus of Slo1 and the addition of purified PKCδ-activated BK_Ca channels in excised patches of transfected HEK293 cells. Our results demonstrate that PKC up-regulates BK_Ca channels and that PKCδ may functionally interact with BK_Ca channel complexes in vivo.     

Enhancement of BKCa channel activity induced by hydrogen peroxide: Involvement of lipid phosphatase activity of PTEN

Large-conductance calcium and voltage-dependent potassium (BK_Ca) channel is an important determinant of vascular tone. It is activated by hydrogen peroxide (H₂O₂) which occurs in various physiological and pathological processes. However, the regulation mechanism is not fully understood. In the present study, the mSlo in the presence or absence of hβ1 were cotransfected with the PTEN_wt, PTEN_C124S, PTEN_G129E in HEK 293 cells. Typical BK_Ca channel currents could be recorded in cell-attached configurations. We found that PTEN_wt reduced the H₂O₂-induced BK_Ca channel activation during the initial 10 min treatment. In contrast, coexpression with catalytically inactive PTEN_C124S/PTEN_G129E mutants that lack lipid phosphatase activity produced no regulation on the H₂O₂-induced BK_Ca channel activation. These results demonstrated that PTEN regulated the H₂O₂-induced BK_Ca channel activation through phosphatidylinositol 3-phosphatse. However, the inhibitory effect of PTEN on the H₂O₂-induced BK_Ca channel activation was attenuated when cells were treated with H₂O₂ at concentrations higher than 100 μM or at 100 μM for long-term treatment. In addition, the p-AKT expression level in PTEN_wt overexpressing cells was lower than that in control cells, and the increase of cytoplasmic free calcium concentration ([Ca²⁺]_i) induced by H₂O₂ was also inhibited. These findings may elucidate a new mechanism for H₂O₂-induced BK_Ca channel activation and provide some evidences for the role of PTEN on vasodilation induced by H₂O₂.     

N-terminal Isoforms of the Large-conductance Ca2+-activated K+ Channel Are Differentially Modulated by the Auxiliary β1-Subunit

The large-conductance Ca²⁺-activated K⁺ (BK_Ca) channel is essential for maintaining the membrane in a hyperpolarized state, thereby regulating neuronal excitability, smooth muscle contraction, and secretion. The BK_Ca α-subunit has three predicted initiation codons that generate proteins with N-terminal ends starting with the amino acid sequences MANG, MSSN, or MDAL. Because the N-terminal region and first transmembrane domain of the α-subunit are required for modulation by auxiliary β1-subunits, we examined whether β1 differentially modulates the N-terminal BK_Ca α-subunit isoforms. In the absence of β1, all isoforms had similar single-channel conductances and voltage-dependent activation. However, whereas β1 did not modulate the voltage-activation curve of MSSN, β1 induced a significant leftward shift of the voltage activation curves of both the MDAL and MANG isoforms. These shifts, of which the MDAL was larger, occurred at both 10 μm and 100 μm Ca²⁺. The β1-subunit increased the open dwell times of all three isoforms and decreased the closed dwell times of MANG and MDAL but increased the closed dwell times of MSSN. The distinct modulation of voltage activation by the β1-subunit may be due to the differential effect of β1 on burst duration and interburst intervals observed among these isoforms. Additionally, we observed that the related β2-subunit induced comparable leftward shifts in the voltage-activation curves of all three isoforms, indicating that the differential modulation of these isoforms was specific to β1. These findings suggest that the relative expression of the N-terminal isoforms can fine-tune BK_Ca channel activity in cells, highlighting a novel mechanism of BK_Ca channel regulation.     

Functional and molecular consequences of ionizing irradiation on large conductance Ca2+-activated K+ channels in rat aortic smooth muscle cells

AimsThe goal of this study was to evaluate the influence of γ-irradiation on Ca²⁺-activated K⁺ channel (BK_Ca) function and expression in rat thoracic aorta.Main methodsAortic cells or tissues were studied by the measurement of force versus [Ca²⁺]_i, patch-clamp technique, and RT-PCR.Key findingsStimulation of smooth muscle cells with depolarizing voltage steps showed expression of outward K⁺ currents. Paxilline, an inhibitor of BK_Ca channels, decreased outward K⁺ current density. Outward currents in smooth muscle cells obtained from irradiated animals 9 and 30 days following radiation exposure demonstrated a significant decrease in K⁺ current density. Paxilline decreased K⁺ current in cells obtained 9 days, but was without effect 30 days after irradiation suggesting the absence of BK_Ca channels. Aortic tissue from irradiated animals showed progressively enhanced contractile responses to phenylephrine in the post-irradiation period of 9 and 30 days. The concomitant Ca²⁺ transients were significantly smaller, as compared to tissues from control animals, 9 days following irradiation but were increased above control levels 30 days following irradiation. Irradiation produced a decrease in BK_Ca α- and β₁-subunit mRNA levels in aortic smooth muscle cells suggesting that the vasorelaxant effect of these channels may be diminished.SignificanceThese results suggest that the enhanced contractility of vascular tissue from animals exposed to radiation may result from an increase in myofilament Ca²⁺ sensitivity in the early post-irradiation period and a decrease in BK_Ca channel expression in the late post-irradiation period.     

Biphasic effects of H2O2 on BKCa channels

Abstract

The inhibitory or activating effect of H₂O₂ on large conductance calcium and voltage-dependent potassium (BK_Ca) channels has been reported. However, the mechanism by which this occurs is unclear. In this paper, BK_Ca channels encoded by mouse Slo were expressed in HEK 293 cells and BK_Ca channel activity was measured by electrophysiology. The results showed that H₂O₂ inhibited BK_Ca channel activity in inside-out patches but enhanced BK_Ca channel activity in cell-attached patches. The inhibition by H₂O₂ in inside-out patches may be due to oxidative modification of cysteine residues in BK_Ca channels or other membrane proteins that regulate BK_Ca channel function. PI3K/AKT signaling modulates the H₂O₂-induced BK_Ca channel activation in cell-attached patches. BK_Ca channels and PI3K signaling pathway were involved in H₂O₂-induced vasodilation and H₂O₂-induced vasodilation by PI3K pathway was mainly due to modulation of BK_Ca channel activity.     

多不饱和脂肪酸对钾通道的调控作用及机理

多不饱和脂肪酸具有包括离子通道在内的众多作用靶点,通过作用于这些靶点,可以有效保护免疫系统、神经系统和心血管系统的功能,在一定程度上保护人体健康。电压门控钾离子通道家族K_V7通道和大电导钙离子激活的钾离子通道(BK_Ca)广泛表达于机体的各类组织中,具有重要的生理或病理功能。本综述围绕K_V7和BK_Ca通道,根据对已有报道的汇总,多不饱和脂肪酸可以增大K_V7和BK_Ca通道的电流幅值,其中对K_V7通道电流的影响主要是改变其电压依赖特性和最大电导值,而对BK_Ca通道电流的影响主要是改变其孔道区域关闭态的构象。此外,多不饱和脂肪酸对K_V7和BK_Ca通道功能的调节也会受到共表达的辅助亚基影响,但相关机制有待进一步阐明。深入理解多不饱和脂肪酸对K_V7和BK_Ca通道调节作用效果和分子机制,有助于全面理解K_V7和BK     

Ginsenoside Rg<Subscript>3</Subscript> enhances large conductance Ca<Superscript>2+</Superscript>-activated potassium channel currents: A role of Tyr360 residue

Ginsenosides, active ingredients of Panax ginseng, are known to exhibit neuroprotective effects. Large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels are key modulators of cellular excitability of neurons and vascular smooth muscle cells. In the present study, we examined the effects of ginsenosides on rat brain BK_Ca (rSlo) channel activity heterologously expressed in Xenopus oocytes to elucidate the molecular mechanisms how ginsenoside regulates the BK_Ca channel activity. Ginsenoside Rg₃ (Rg₃) enhanced outward BK_Ca channel currents. The Rg₃-enhancement of outward BK_Ca channel currents was concentration-dependent, voltage-dependent, and reversible. The EC₅₀ was 15.1 ± 3.1 μM. Rg₃ actions were not desensitized by repeated treatment. Tetraetylammonium (TEA), a K⁺ channel blocker, inhibited BK_Ca channel currents. We examined whether extracellular TEA treatment could alter the Rg₃ action and vice versa. TEA caused a rightward shift of the Rg₃ concentration-response curve (i.e., much higher concentration of Rg₃ is required for the activation of BK_Ca channel compared to the absence of TEA), while Rg₃ caused a rightward shift of the TEA concentration-response curve in wild-type channels. Mutation of the extracellular TEA binding site Y360 to Y360I caused a rightward shift of the TEA concentration-response curve and almost abolished both the Rg₃ action and Rg₃-induced rightward shift of TEA concentration-response curve. These results indicate that Tyr360 residue of BK_Ca channel plays an important role in the Rg₃-enhancement of BK_Ca channel currents.     

Activation of cloned BK<Subscript>Ca</Subscript> channels in nitric oxide-induced apoptosis of HEK293 cells

The large conductance Ca²⁺-activated K⁺ (BK_Ca) channels are highly expressed in vascular smooth muscle cells (VSMCs) and play an essential role in the regulation of various physiological functions. Besides its electrophysiological function in vascular relaxation, BK_Ca has also been reported to be implicated in nitric oxide (NO)-induced apoptosis of VSMCs. However, the molecular mechanism is not clear and has not been determined on cloned channels. The present study was designed to clarify whether activation of cloned BK_Ca channel was involved in NO-induced apoptosis in human embryonic kidney 293 (HEK293) cell. The cDNA encoding the α-subunit of BK_Ca channel, hSloα, was transiently transfected into HEK293 cells. The apoptotic death in HEK-hSloα cells was detected using immunocytochemistry, analysis of fragmented DNA by agarose gel electrophoresis, MTT test, and flow cytometry assays. Whole-cell and single-channel characteristics of HEK-hSloα cells exhibited functional features similar to native BK_Ca channel in VSMCs. Exposuring of HEK- hSloα cells to S-nitroso-N-acetyl-penicillamine increased the hSloα channel activities of whole-cell and single-channel, and then increased percentage of cells undergoing apoptosis. However, blocking hSloα channels with 1 mM tetraethylammonia or 100 nM iberiotoxin significantly decreased the NO-induced apoptosis, whereas 30 μM NS1619, the specific agonist of BK_Ca, independently increased hSloα currents and induced apoptosis. These results indicated that activation of cloned BK_Ca channel was involved in NO-induced apoptosis of HEK293 cells.     

膜脂物理属性及化学成分对BKCa钾离子通道的调节作用

大电导钙激活电压门控型钾离子通道(BKCa通道)广泛分布于各种组织中,主要由胞内钙离子浓度增加和细胞膜去极化而激活.此外多种膜脂,如脂肪酸、胆固醇、鞘糖脂等可修饰该通道的功能.该通道参与细胞内信号转导、细胞的兴奋及代谢调节等多种生理过程,其功能异常牵涉到特发性癫痫、高血压等疾病的发生.因而对BKCa通道功能的调节作用的研究具有重要的生理学及病理学意义.文章将主要介绍膜脂对BKCa通道功能的调节作用.     

Palmitoylation of STREX domain confers cerebroside sensitivity to the BKCa channel

Our previous study reported that cerebrosides from traditional Chinese medicine Baifuzi directly interact with the STREX domain of BK_Ca channels, which in turn results in the therapeutic effect of Baifuzi on ischemic stroke. However, it is not known how cerebrosides in the plasma membrane could interact with the STREX domain that is in the cytoplasmic side. Using patch-clamp technique, effects of different cerebrosides on the BK_Ca channel were studied by measuring single channel currents in CHO cells expressing wild type or mutated BK_Ca channels. Palmitoylation of the STREX domain was removed either by site-directed mutagenesis or pharmacological inhibition. Removal of palmitoylation sites at C646 and C647 by mutating the residues to Ala abolished the ability of cerebrosides to activate the BK_Ca channel. In contrast, the mutation neither changed the single channel conductance nor voltage sensitivity of the channel. Both palmitoylation inhibitors tunicamycin and palmitic acid analog 2-bromopalmitate attenuated the activation of the BK_Ca channel by cerebrosides. Furthermore, confocal images on STREX-EGFP fragments demonstrated that STREX fragments no longer associated with the plasma membrane when the palmitoylation was removed or blocked. These findings suggest that palmitoylation of the STREX domain is necessary for cerebrosides to activate the BK_Ca channel and provide insight into the mechanism of how Baifuzi could exert therapeutic effect on ischemic stroke.     

Plasticity and emerging role of BKCa channels in nociceptive control in neuropathic pain

Large‐conductance Ca²⁺‐activated K⁺ (BK_Ca, MaxiK) channels are important for the regulation of neuronal excitability. Peripheral nerve injury causes plasticity of primary afferent neurons and spinal dorsal horn neurons, leading to central sensitization and neuropathic pain. However, little is known about changes in the BK_Ca channels in the dorsal root ganglion (DRG) and spinal dorsal horn and their role in the control of nociception in neuropathic pain. Here we show that L5 and L6 spinal nerve ligation in rats resulted in a substantial reduction in both the mRNA and protein levels of BK_Ca channels in the DRG but not in the spinal cord. Nerve injury primarily reduced the BK_Ca channel immunoreactivity in small‐ and medium‐sized DRG neurons. Furthermore, although the BK_Ca channel immunoreactivity was decreased in the lateral dorsal horn, there was an increase in the BK_Ca channel immunoreactivity present on dorsal horn neurons near the dorsal root entry zone. Blocking the BK_Ca channel with iberiotoxin at the spinal level significantly reduced the mechanical nociceptive withdrawal threshold in control and nerve‐injured rats. Intrathecal injection of the BK_Ca channel opener [1,3‐dihydro‐1‐[2‐hydroxy‐5‐(trifluoromethyl)phenyl]‐5‐(trifluoromethyl)‐2H‐benzimidazol‐2‐one] dose dependently reversed allodynia and hyperalgesia in nerve‐ligated rats but it had no significant effect on nociception in control rats. Our study provides novel information that nerve injury suppresses BK_Ca channel expression in the DRG and induces a redistribution of BK_Ca channels in the spinal dorsal horn. BK_Ca channels are increasingly involved in the control of sensory input in neuropathic pain and may represent a new target for neuropathic pain treatment.     

IP3 decreases coronary artery tone via activating the BKCa channel of coronary artery smooth muscle cells in pigs

Large conductance Ca²⁺-activated K⁺ channel (BK_Ca) is a potential target for coronary artery-relaxing medication, but its functional regulation is largely unknown. Here, we report that inositol trisphosphate (IP3) activated BK_Ca channels in isolated porcine coronary artery smooth muscle cells and by which decreased the coronary artery tone. Both endogenous and exogenous IP3 increased the spontaneous transient outward K⁺ currents (STOC, a component pattern of BK_Ca currents) in perforated and regular whole-cell recordings, which was dependent on the activity of IP3 receptors. IP3 also increased the macroscopic currents (MC, another component pattern of BK_Ca currents) via an IP3 receptor- and sarcoplasmic Ca²⁺ mobilization-independent pathway. In inside-out patch recordings, direct application of IP3 to the cytosolic side increased the open probability of single BK_Ca channel in an IP3 receptor-independent manner. We conclude that IP3 is an activator of BK_Ca channels in porcine coronary smooth muscle cells and exerts a coronary artery-relaxing effect. The activation of BK_Ca channels by IP3 involves the enhancement of STOCs via IP3 receptors and stimulation of MC by increasing the Ca²⁺ sensitivity of the channels.     

BK Ca Channels Activating at Resting Potential without Calcium in LNCaP Prostate Cancer Cells

Large-conductance Ca²⁺-dependent K⁺ (BK_Ca) channels are activated by intracellular Ca²⁺ and membrane depolarization in an allosteric manner. We investigated the pharmacological and biophysical characteristics of a BK_Ca-type K⁺ channel in androgen-dependent LNCaP (lymph node carcinoma of the prostate) cells with novel functional properties, here termed BK_L. K⁺ selectivity, high conductance, activation by Mg²⁺ or NS1619, and inhibition by paxilline and penitrem A largely resembled the properties of recombinant BK_Ca channels. However, unlike conventional BK_Ca channels, BK_L channels activated in the absence of free cytosolic Ca²⁺ at physiological membrane potentials; the half-maximal activation voltage was shifted by about −100 mV compared with BK_Ca channels. Half-maximal Ca²⁺-dependent activation was observed at 0.4 μM for BK_L (at −20 mV) and at 4.1 μM for BK_Ca channels (at +50 mV). Heterologous expression of hSlo1 in LNCaP cells increased the BK_L conductance. Expression of hSlo-β1 in LNCaP cells shifted voltage-dependent activation to values between that of BK_L and BK_Ca channels and reduced the slope of the P_open (open probability)-voltage curve. We propose that LNCaP cells harbor a so far unknown type of BK_Ca subunit, which is responsible for the BK_L phenotype in a dominant manner. BK_L-like channels are also expressed in the human breast cancer cell line T47D. In addition, functional expression of BK_L in LNCaP cells is regulated by serum-derived factors, however not by androgens.     

Baifuzi reduces transient ischemic brain damage through an interaction with the STREX domain of BKCa channels

Stroke is a long-term disability and one of the leading causes of death. However, no successful therapeutic intervention is available for the majority of stroke patients. In this study, we explored a traditional Chinese medicine Baifuzi (Typhonium giganteum Engl.). We show, at first, that the ethanol extract of Baifuzi exerts neuroprotective effects against brain damage induced by transient global or focal cerebral ischemia in rats and mice. Second, the extract activated large-conductance Ca²⁺-activated K⁺ channel (BK_Ca) channels, and BK_Ca channel blockade suppressed the neuroprotection of the extract, suggesting that the BK_Ca is the molecular target of Baifuzi. Third, Baifuzi cerebroside (Baifuzi-CB), purified from its ethanol extract, activated BK_Ca channels in a manner similar to that of the extract. Fourth, the stress axis hormone-regulated exon (STREX) domain of the BK_Ca channel directly interacted with Baifuzi-CB, and its deletion suppressed channel activation by Baifuzi-CB. These results indicate that Baifuzi-CB activated the BK_Ca channel through its direct interaction with the STREX domain of the channel and suggests that Baifuzi-CB merits exploration as a potential therapeutic agent for treating brain ischemia.     

Caveolin-1 Facilitates the Direct Coupling between Large Conductance Ca2+-activated K+ (BKCa) and Cav1.2 Ca2+ Channels and Their Clustering to Regulate Membrane Excitability in Vascular Myocytes

L-type voltage-dependent Ca²⁺ channels (LVDCC) and large conductance Ca²⁺-activated K⁺ channels (BK_Ca) are the major factors defining membrane excitability in vascular smooth muscle cells (VSMCs). The Ca²⁺ release from sarcoplasmic reticulum through ryanodine receptor significantly contributes to BK_Ca activation in VSMCs. In this study direct coupling between LVDCC (Cav1.2) and BK_Ca and the role of caveoline-1 on their interaction in mouse mesenteric artery SMCs were examined. The direct activation of BK_Ca by Ca²⁺ influx through coupling LVDCC was demonstrated by patch clamp recordings in freshly isolated VSMCs. Using total internal reflection fluorescence microscopy, it was found that a large part of yellow fluorescent protein-tagged BK_Ca co-localized with the cyan fluorescent protein-tagged Cav1.2 expressed in the plasma membrane of primary cultured mouse VSMCs and that the two molecules often exhibited FRET. It is notable that each BKα subunit of a tetramer in BK_Ca can directly interact with Cav1.2 and promotes Cav1.2 cluster in the molecular complex. Furthermore, caveolin-1 deficiency in knock-out (KO) mice significantly reduced not only the direct coupling between BK_Ca and Cav1.2 but also the functional coupling between BK_Ca and ryanodine receptor in VSMCs. The measurement of single cell shortening by 40 mm K⁺ revealed enhanced contractility in VSMCs from KO mice than wild type. Taken together, caveolin-1 facilitates the accumulation/clustering of BK_Ca-LVDCC complex in caveolae, which effectively regulates spatiotemporal Ca²⁺ dynamics including the negative feedback, to control the arterial excitability and contractility.