Rationale T-type (CaV3.1/CaV3.2) Ca2+ channels are expressed in rat cerebral arterial smooth muscle. Although present, their functional significance remains uncertain with findings pointing to a variety of roles. Objective This study tested whether CaV3.2 channels mediate a negative feedback response by triggering Ca2+ sparks, discrete events that initiate arterial hyperpolarization by activating large-conductance Ca2+-activated K+ channels. Methods and Results Micromolar Ni2+, an agent that selectively blocks CaV3.2 but not CaV1.2/CaV3.1, was first shown to depolarize/constrict pressurized rat cerebral arteries; no effect was observed in CaV3.2−/− arteries. Structural analysis using 3-dimensional tomography, immunolabeling, and a proximity ligation assay next revealed the existence of microdomains in cerebral arterial smooth muscle which comprised sarcoplasmic reticulum and caveolae. Within these discrete structures, CaV3.2 and ryanodine receptor resided in close apposition to one another. Computational modeling revealed that Ca2+ influx through CaV3.2 could repetitively activate ryanodine receptor, inducing discrete Ca2+-induced Ca2+ release events in a voltage-dependent manner. In keeping with theoretical observations, rapid Ca2+ imaging and perforated patch clamp electrophysiology demonstrated that Ni2+ suppressed Ca2+ sparks and consequently spontaneous transient outward K+ currents, large-conductance Ca2+-activated K+ channel mediated events. Additional functional work on pressurized arteries noted that paxilline, a large-conductance Ca2+-activated K+ channel inhibitor, elicited arterial constriction equivalent, and not additive, to Ni2+. Key experiments on human cerebral arteries indicate that CaV3.2 is present and drives a comparable response to moderate constriction. Conclusions These findings indicate for the first time that CaV3.2 channels localize to discrete microdomains and drive ryanodine receptor–mediated Ca2+ sparks, enabling large-conductance Ca2+-activated K+ channel activation, hyperpolarization, and attenuation of cerebral arterial constriction.

The intracellular mechanism underlying the Ca(2+)-induced enhancement of the L-type Ca2+ current (ICa) was examined in adult rabbit cardiac ventricular myocytes by using patch-clamp methodology. Internal Ca2+ was elevated by flash photolysis of the Ca2+ chelator Nitr 5, and intracellular Ca2+ levels were simultaneously monitored by Fluo 3 fluorescence. Flash photolysis of Nitr 5 produced a rapid (< 1-second) elevation of internal Ca2+, which led to enhancement (39% to 51% above control) of the peak inward Ca2+ current after a delay of 20 to 120 seconds. Internal dialysis of myocytes with synthetic inhibitory peptides derived from the pseudosubstrate (peptide 273-302) and calmodulin binding (peptide 291-317) regions within the regulatory domain of multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) blocked enhancement of ICa produced by elevation of internal Ca2+ but not that produced by beta-adrenergic stimulation. These inhibitory peptides also had no effect on the elevation of internal Ca2+ produced by flash photolysis of Nitr 5. A pseudosubstrate inhibitory peptide derived from protein kinase C had no significant effect on Ca(2+)-dependent enhancement of ICa. We conclude that CaM kinase mediates the Ca(2+)-induced enhancement of ICa in mammalian cardiac myocytes by a mechanism likely involving direct phosphorylation of the L-type Ca2+ channel complex or an associated regulatory protein.

tissue level diversity is predicted. Importantly, heterogeneity in BK Ca channel activity may contribute to tissue-specific differences in regulation of myogenic vasoconstriction, allowing local hemodynamics to be matched to metabolic requirements. Knowledge of such variability will be important to exploiting the BK Ca channel as a therapeutic target and understanding systemic effects of its pharmacological manipulation.

Arteriolar myogenic vasoconstriction occurs when increased stretch or membrane tension leads to smooth muscle cell depolarization and opening of voltage-gated Ca2+ channels. To prevent positive feedback and excessive pressure-induced vasoconstriction, studies in cerebral artery smooth muscle have suggested that activation of large conductance, Ca 2+ -activated K + channels (BK Ca ) provides an opposing hyperpolarizing influence reducing Ca 2+ channel activity. We have hypothesized that this mechanism may not equally apply to all vascular beds. To establish the existence of such heterogeneity in vascular reactivity, studies were performed on rat vascular smooth muscle (VSM) cells from cremaster muscle arterioles and cerebral arteries. Whole cell K + currents were determined at pipette [Ca 2+ ] of 100 nm or 5 μm in the presence and absence of the BK Ca inhibitor, iberiotoxin (IBTX; 0.1 μm). Similar outward current densities were observed for the two cell preparations at the lower pipette Ca 2+ levels. At 5 μm Ca 2+ , cremaster VSM showed a significantly (P < 0.05) lower current density compared to cerebral VSM (34.5 ± 1.9 vs 45.5 ± 1.7 pA pF −1 at +70 mV). Studies with IBTX suggested that the differences in K + conductance at 5 μm intracellular [Ca 2+ ] were largely due to activity of BK Ca . 17β-Oestradiol (1 μm), reported to potentiate BK Ca current via the channel's β-subunit, caused a greater effect on whole cell K + currents in cerebral vessel smooth muscle cells (SMCs) compared to those of cremaster muscle. In contrast, the α-subunit-selective BK Ca opener, NS-1619 (20 μm), exerted a similar effect in both preparations. Spontaneously transient outward currents (STOCs) were more apparent (frequency and amplitude) and occurred at more negative membrane potentials in cerebral compared to cremaster SMCs. Also consistent with decreased STOC activity in cremaster SMCs was an absence of detectable Ca 2+ sparks (0 of 76 cells) compared to that in cerebral SMCs (76 of 105 cells). Quantitative PCR showed decreased mRNA expression for the β1 subunit and a decrease in the β 1: α ratio in cremaster arterioles compared to cerebral vessels. Similarly, cremaster arterioles showed a decrease in total BK Ca protein and the β 1: α-subunit ratio. The data support vascular heterogeneity with respect to the activity of BK Ca in terms of both β-subunit regulation and interaction with SR-mediated Ca 2+ signalling.

L-type, voltage-gated Ca2؉ channels (Ca L ) play critical roles in brain and muscle cell excitability. Here we show that currents through heterologously expressed neuronal and smooth muscle Ca L channel isoforms are acutely potentiated following ␣5␤1 integrin activation. Only the ␣ 1C pore-forming channel subunit is critical for this process. Truncation and site-directed mutagenesis strategies reveal that regulation of Cav1.2 by ␣5␤1 integrin requires phosphorylation of ␣ 1C C-terminal residues Ser 1901 and Tyr 2122 . These sites are known to be phosphorylated by protein kinase A (PKA) and c-Src, respectively, and are conserved between rat neuronal (Cav1.2c) and smooth muscle (Cav1.2b) isoforms. Kinase assays are consistent with phosphorylation of these two residues by PKA and c-Src. Following ␣5␤1 integrin activation, native Ca L channels in rat arteriolar smooth muscle exhibit potentiation that is completely blocked by combined PKA and Src inhibition. Our results demonstrate that integrin-ECM interactions are a common mechanism for the acute regulation of Ca L channels in brain and muscle. These findings are consistent with the growing recognition of the importance of integrin-channel interactions in cellular responses to injury and the acute control of synaptic and blood vessel function.Voltage-gated calcium channels play critical roles in the regulation of calcium entry across the plasma membranes of excitable cells. L-type calcium channels (Ca L ), 5 which are highly expressed in brain and muscle, are heteromeric transmembrane proteins composed of a poreforming ␣ 1C (Cav1.2) subunit along with accessory ␤, ␣ 2 , ␦, and sometimes ␥ subunits (1, 2). The ␣ 1C subunit contains four highly conserved repeat regions with 24 membrane-spanning domains, in addition to a variable length N terminus and relatively long, intracellular C terminus. The three ␣ 1C isoforms (neuronal, Cav1.2c; smooth muscle, Cav1.2b; cardiac, Cav1.2a) exhibit significant sequence differences in their N and C termini but all are regulated by intracellular kinases in ways that uniquely determine calcium entry and cell excitability.The regulation of Ca L channels by serine-threonine kinases has been extensively investigated. PKG phosphorylates a conserved serine reside in the cytoplasmic I-II linker (3) of all three ␣ 1C isoforms, leading to inhibition of current. PKC phosphorylates N-terminal threonine residues in cardiac and smooth muscle isoforms (4 -6) leading in most cases to potentiation of current. PKA phosphorylates all three ␣ 1C isoforms at a conserved C-terminal serine (Ser 1901 in Cav1.2c; Ser 1928 in Cav1.2a), thereby mediating ␤-adrenergic potentiation of the calcium current in cardiac myocytes and neurons (7-9). PKA also regulates ␣ 1C in smooth muscle, but the functional consequences on calcium current are complicated by crossover activation of PKG, which is expressed at high levels in that tissue (10).We recently demonstrated that Ca L currents in vascular smooth muscle (VSM) are acutely regulated by the integrin class of cel...

Large conductance, calcium-sensitive K ؉ channels (BK Ca channels) contribute to the control of membrane potential in a variety of tissues, including smooth muscle, where they act as the target effector for intracellular "calcium sparks" and the endothelium-derived vasodilator nitric oxide. Various signal transduction pathways, including protein phosphorylation can regulate the activity of BK Ca channels, along with many other membrane ion channels. In our study, we have examined the regulation of BK Ca channels by the cellular Src gene product (cSrc), a soluble tyrosine kinase that has been implicated in the regulation of both voltage-and ligand-gated ion channels. Using a heterologous expression system, we observed that co-expression of murine BK Ca channel and the human cSrc tyrosine kinase in HEK 293 cells led to a calcium-sensitive enhancement of BK Ca channel activity in excised membrane patches. In contrast, co-expression with a catalytically inactive cSrc mutant produced no change in BK Ca channel activity, demonstrating the requirement for a functional cSrc molecule. Furthermore, we observed that BK Ca channels underwent direct tyrosine phosphorylation in cells co-transfected with BK Ca channels and active cSrc but not in cells co-transfected with the kinase inactive form of the enzyme. A single Tyr to Phe substitution in the C-terminal half of the channel largely prevented this observed phosphorylation. Given that cSrc may become activated by receptor tyrosine kinases or G-protein-coupled receptors, these findings suggest that cSrc-dependent tyrosine phosphorylation of BK Ca channels in situ may represent a novel regulatory mechanism for altering membrane potential and calcium entry.In the large family of voltage-gated K ϩ channels, large conductance, calcium-sensitive potassium (maxi-K or BK Ca ) 1 channels represent a unique class whose gating depends primarily on membrane voltage but which can be shifted in the negative direction by intracellular free calcium. A direct physiologic consequence of this behavior is that BK Ca channels act as "coincidence detectors" and regulate, in a feedback fashion, cellular processes stimulated by close temporal changes in membrane potential and intracellular calcium. That BK Ca channels indeed play such a role is evidenced by the fact that blocking these channels increases the degree of myogenic tone observed in arterial smooth muscle (1-3) and enhances the presynaptic calcium-dependent release of neurotransmitter at neuromuscular junctions (4, 5).Given their potential to influence cellular processes, it is not surprising that BK Ca channels are also targets of cellular signaling pathways, including phosphorylation and dephosphorylation reactions (6 -11), heterotrimeric GTP-binding proteins (12, 13), and the endothelium-derived vasodilator nitric oxide (14). To date, however, many of the molecular aspects of these regulatory events remain poorly understood.Of these various cellular pathways, protein phosphorylation remains as one of the most common forms of intrace...

Recent data have led us to hypothesize that selective activation of endothelial small- and/or intermediate-conductance, calcium-activated potassium channels (SK(Ca) and IK(Ca) channels, respectively) by the opener compounds NS309 and DCEBIO would augment stimulated nitric oxide (NO) synthesis and vasodilation in resistance arteries. Experimentally, ATP-evoked changes in membrane potential, cytosolic Ca(2+), and NO synthesis were recorded by patch clamp and microfluorimetry in single human endothelial cells. Agonist-evoked inhibition of myogenic tone in isolated, pressurized arterioles from rat cremaster skeletal muscle was analyzed by video microscopy. NS309 and DCEBIO enhanced ATP-evoked membrane hyperpolarization and cytosolic Ca(2+) transients, along with acute NO synthesis in isolated endothelial cells. The acetylcholine-mediated inhibition of myogenic tone (IC(50)=237 nM) was left-shifted in the presence of NS309 and DCEBIO (10, 100, and 1000 nM) to IC(50) values of 101, 78, and 43 nM; endothelial denudation inhibited this drug effect. L-NAME attenuated the acetylcholine-induced inhibition of myogenic tone but did not interfere with NS309 and DCEBIO-evoked vasodilation. Collectively, our data demonstrate that drug-induced enhancement of endothelial SK(Ca) and IK(Ca) channel activities represents a novel cellular mechanism to increase vasodilation of small-resistance arterioles, thereby highlighting these channels as potential therapeutic targets in cardiovascular disease states associated with compromised NO signaling.

The contribution of small-conductance (SK(Ca)) and intermediate-conductance Ca(2+)-activated K(+) (IK(Ca)) channels to the generation of nitric oxide (NO) by Ca(2+)-mobilizing stimuli was investigated in human umbilical vein endothelial cells (HUVECs) by combining single-cell microfluorimetry with perforated patch-clamp recordings to monitor agonist-evoked NO synthesis, cytosolic Ca(2+) transients, and membrane hyperpolarization in real time. ATP or histamine evoked reproducible elevations in NO synthesis and cytosolic Ca(2+), as judged by 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM) and fluo-3 fluorescence, respectively, that were tightly associated with membrane hyperpolarizations. Whereas evoked NO synthesis was unaffected by either tetraethylammonium (10 mmol/l) or BaCl(2) (50 micromol/l) + ouabain (100 micromol/l), depleting intracellular Ca(2+) stores by thapsigargin or removing external Ca(2+) inhibited NO production, as did exposure to high (80 mmol/l) external KCl. Importantly, apamin and charybdotoxin (ChTx)/ triarylmethane (TRAM)-34, selective blockers SK(Ca) and IK(Ca) channels, respectively, abolished both stimulated NO synthesis and membrane hyperpolarization and decreased evoked Ca(2+) transients. Apamin and TRAM-34 also inhibited an agonist-induced outwardly rectifying current characteristic of SK(Ca) and IK(Ca) channels. Under voltage-clamp control, we further observed that the magnitude of agonist-induced NO production varied directly with the degree of membrane hyperpolarization. Mechanistically, our data indicate that SK(Ca) and IK(Ca) channel-mediated hyperpolarization represents a critical early event in agonist-evoked NO production by regulating the influx of Ca(2+) responsible for endothelial NO synthase activation. Moreover, it appears that the primary role of agonist-induced release of intracellular Ca(2+) stores is to trigger the opening of both K(Ca) channels along with Ca(2+) entry channels at the plasma membrane. Finally, the observed inhibition of stimulated NO synthesis by apamin and ChTx/TRAM-34 demonstrates that SK(Ca) and IK(Ca) channels are essential for NO-mediated vasorelaxation.