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Ca2+sparklets are local elevations in intracellular Ca2+produced by the opening of a single or a cluster of L-type Ca2+channels. In arterial myocytes, Ca2+sparklets regulate local and global intracellular Ca2+. At present, the molecular identity of the L-type Ca2+channels underlying Ca2+sparklets in these cells is undetermined. Here, we tested the hypotheses that voltage-gated calcium channel-α 1.3 subunit (Cav1.3) can produce Ca2+sparklets and that Cav1.2 and/or Cav1.3 channels are responsible for Ca2+sparklets in mouse arterial myocytes. First, we investigated the functional properties of single Cav1.3 channels in tsA201 cells. With 110 mM Ba2+as the charge carrier, Cav1.3 channels had a conductance of 20 pS. This value is similar to that of Cav1.2 and native L-type Ca2+channels. As previously shown for Cav1.2 channels, Cav1.3 channels can operate in two gating modes characterized by short and long open times. Expressed Cav1.3 channels also produced Ca2+sparklets. Cav1.3 sparklets had properties similar to those produced by Cav1.2 and native L-type channels, including quantal amplitude, dihydropyridine sensitivity, bimodal gating, and dual-event duration times. However, the voltage dependencies of conductance and steady-state inactivation of the Ca2+current ( ICa) in arterial myocytes were similar to those recorded from cells expressing Cav1.2 but not Cav1.3 channels. Furthermore, nifedipine (10 μM) eliminated Ca2+sparklets in wild-type myocytes but not in myocytes expressing dihydropyridine-insensitive Cav1.2 channels. Accordingly, Cav1.3 transcript and protein were not detected in isolated arterial myocytes. We conclude that although Cav1.3 channels can produce Ca2+sparklets, Cav1.2 channels underlie ICa, Ca2+sparklets, and hence dihydropyridine-sensitive Ca2+influx in mouse arterial myocytes.

Background: Arteriovenous (AV) malformations (AVMs) are characterized by abnormal AV shunts that displace intervening capillaries. Brain AVMs (BAVMs) may cause life-threatening strokes and have limited treatment options. Mechanisms underlying AVM pathogenesis remain poorly understood, hindering therapeutic development. Rationale: We reported that endothelial expression of constitutively active Notch4 (Notch4*) in mice initiates BAVMs de novo through enlargement of microvessels without an increase in endothelial cell number or proliferation. Thus, we hypothesized that Notch4* initiates BAVMs by disrupting normal vasodilation and vascular tone. Nitric oxide (NO) regulates vasodilation and vascular tone, and Notch activation induces NO synthesis. Thus, we further hypothesized that Notch4* disrupts NO signaling, thereby permitting vessel enlargement and AV shunting. Results: Cerebral arteries isolated from Notch4* mutant mice were less constricted and exhibited decreased arterial tone than that of controls. Administering the NO synthase (NOS) inhibitor NG-nitro-L-arginine (L-NNA, 30 mg/kg, 1x/day) attenuated Notch4*-induced AV shunt initiation: AV connection diameter in L-NNA-treated Notch4* mutants was decreased compared to that of saline-treated Notch4* mutants (7.0±1.1 vs. 15.1±2.3 μm) at postnatal day 12, when vessel enlargement and AV shunting first become apparent. Although L-NNA treatment attenuated AV shunt initiation, it did not prevent AV shunt formation or its progression; LNNA-treated Notch4* mutants still exhibited BAVM-associated pathologies and mortality. In moribund L-NNA-treated Notch4* mutants, AV connection diameter was decreased compared to that of saline-treated Notch4* mutants (18.9±6.5 vs. 30.6±8.2 μm), but remained ~4 fold that of genetic controls (~5 μm). A reduction in diameter in L-NNA-treated Notch4* mutants was accompanied by an increase in median survival time (30 vs. 25 days in saline-treated Notch4* mutants). Conclusions: Our results suggest that arterial dysfunction may contribute to Notch4*-mediated BAVM formation, and that inhibiting NOS attenuates BAVM formation. These data support a role for NO pathway in Notch4*-mediated BAVM formation.

Enhanced arterial tone is a leading cause of vascular complications during diabetes. Voltage-gated K(+) (KV) channels are key regulators of vascular smooth muscle cells (VSMCs) contractility and arterial tone. Whether impaired KV channel function contributes to enhance arterial tone during diabetes is unclear. Here, we demonstrate a reduction in KV-mediated currents (IKv) in VSMCs from a high fat diet (HFD) mouse model of type 2 diabetes. In particular, IKv sensitive to stromatoxin (ScTx), a potent KV2 blocker, were selectively reduced in diabetic VSMCs. This was associated with decreased KV2-mediated regulation of arterial tone and suppression of the KV2.1 subunit mRNA and protein in VSMCs/arteries isolated from HFD mice. We identified protein kinase A anchoring protein 150 (AKAP150), via targeting of the phosphatase calcineurin (CaN), and the transcription factor nuclear factor of activated T-cells c3 (NFATc3) as required determinants of KV2.1 suppression during diabetes. Interestingly, substantial reduction in transcript levels for KV2.1 preceded down-regulation of large conductance Ca(2+)-activated K(+) (BKCa) channel β1 subunits, which are ultimately suppressed in chronic hyperglycemia to a similar extent. Together, our study supports the concept that transcriptional suppression of KV2.1 by activation of the AKAP150-CaN/NFATc3 signaling axis contributes to enhanced arterial tone during diabetes.

In the heart, reliable activation of Ca2+ release from the sarcoplasmic reticulum during the plateau of the ventricular action potential requires synchronous opening of multiple CaV1.2 channels. Yet the mechanisms that coordinate this simultaneous opening during every heartbeat are unclear. Here, we demonstrate that CaV1.2 channels form clusters that undergo dynamic, reciprocal, allosteric interactions. This ‘functional coupling’ facilitates Ca2+ influx by increasing activation of adjoined channels and occurs through C-terminal-to-C-terminal interactions. These interactions are initiated by binding of incoming Ca2+ to calmodulin (CaM) and proceed through Ca2+/CaM binding to the CaV1.2 pre-IQ domain. Coupling fades as [Ca2+]i decreases, but persists longer than the current that evoked it, providing evidence for ‘molecular memory’. Our findings suggest a model for CaV1.2 channel gating and Ca2+-influx amplification that unifies diverse observations about Ca2+ signaling in the heart, and challenges the long-held view that voltage-gated channels open and close independently. DOI: http://dx.doi.org/10.7554/eLife.05608.001, eLife digest To pump blood around the body, the muscle cells within the heart must contract and relax together with a regular rhythm. A contraction begins when proteins called CaV1.2 channels embedded in the cell membranes of heart cells open to allow calcium ions to enter the cells. The calcium ions that enter through these CaV1.2 channels trigger the release of calcium ions from storage compartments within the cells, which leads to the heart contracting. However, to trigger this release of calcium ions, many CaV1.2 channels have to open at the same time and we do not yet know how this is co-ordinated. Dixon et al. studied CaV1.2 channels in heart muscle cells from mice. The experiments show that these proteins are arranged in clusters of eight, on average, in the cell membrane. When calcium ions enter the cell they bind to a protein called calmodulin, which in turn binds to a CaV1.2 channel. This allows the CaV1.2 channels within a cluster to interact with each other. The physical association between CaV1.2 channels within clusters enables them to work cooperatively; they open at the same time to allow more calcium ions to enter and then close together to allow the cell to relax. Dixon et al. found that even when levels of calcium ions in the cells declined, the CaV1.2 channels within clusters remained open for a little while longer before they closed. This suggests that the interactions between the CaV1.2 channels act as a type of ‘molecular memory’ that may alter how the cells respond to future activity. These results challenge the previously held view that the CaV1.2 channels open and close independently of one another. Future studies will seek to understand the molecular details of how these channels cluster together, and how this clustering affects changes in heart rate and heart abnormalities like long QT syndrome. DOI: http://dx.doi.org/10.7554/eLife.05608.002

A recent tryptophan scanning of the alpha-TM3 domain of the Torpedo californica AChR demonstrated that this domain can modulate ion-channel gating [Guzman, G., Santiago, J., Ricardo, A., Martí-Arbona, R., Rojas, L., Lasalde-Dominicci, J. (2003) Biochemistry 42, 12243-12250]. Here we extend the study of the alpha-TM3 domain to the muscle-type AChR by examining functional consequences of single tryptophan substitutions at five conserved positions (alphaM282, alphaF284, alphaV285, alphaA287, and alphaI290) homologous to the alpha-TM3 positions that were recently characterized in the Torpedo AChR. Similarly to the Torpedo AChR, mutations alphaM282W and alphaV285W, which are presumed to face the interior of the protein, did not exhibit functional channel activity. Nevertheless, significant expression levels of these mutants were observed at the oocyte surface. In contrast to the Torpedo AChR, in the muscle-type AChR, tryptophan substitution at positions F284, A287, and I290 produces a significant increase in normalized macroscopic response. Single-channel recordings at low ACh concentration revealed that the increase in AChR sensitivity for the F284W, A287W, and I290W is due to an increase in the mean open duration. These results suggest that tryptophan substitution directly affects channel gating, primarily the channel closing rate. Our results suggest that residues facing the interior of the protein (i.e., alphaM282 and alphaV285) may similarly affect channel gating in Torpedo and muscle-type AChR. However, equivalent mutations (i.e., F284W and I290W) presumably facing the lipid environment display a very different functional response between these two AChR species.

We investigated the basis for a novel form of the slow-channel congenital myasthenic syndrome presenting in infancy in a single individual as progressive weakness and impaired neuromuscular transmission without overt degeneration of the motor endplate. Prolonged low-amplitude synaptic currents in biopsied anconeus muscle at 9 years of age suggested a kinetic disorder of the muscle acetylcholine receptor. Ultrastructural studies at 16 months, at 9 years, and at 15 years of age showed none of the typical degenerative changes of the endplate associated with the slow-channel congenital myasthenic syndrome, and acetylcholine receptor numbers were not significantly reduced. We identified a novel C-to-T substitution in exon 8 of the delta-subunit that results in a serine to phenylalanine mutation in the region encoding the second transmembrane domain that lines the ion channel. Using Xenopus oocyte in vitro expression studies we confirmed that the deltaS268F mutation, as with other slow-channel congenital myasthenic syndrome mutations, causes delayed closure of acetylcholine receptor ion channels. In addition, unlike other mutations in slow-channel congenital myasthenic syndrome, this mutation also causes delayed opening of the channel, a finding that readily explains the marked congenital weakness in the absence of endplate degeneration. Finally, we used serial morphometric analysis of electron micrographs to explore the basis for the progressive weakness and decline of amplitude of endplate currents over a period of 14 years. We demonstrated a progressive widening and accumulation of debris in the synaptic cleft, resulting in loss of efficacy of released neurotransmitter and reduced safety factor. These studies demonstrate the role of previously unrecognized mechanisms of impairment of synaptic transmission caused by a novel mutation and show the importance of serial in vitro studies to elucidate novel disease mechanisms.

Angiotensin signaling promotes interactions between AKAP150, PKC, and TRPV4 channels to form signaling domains that control Ca2+ influx into arterial myocytes., Transient receptor potential vanilloid 4 (TRPV4) channels are Ca2+-permeable, nonselective cation channels expressed in multiple tissues, including smooth muscle. Although TRPV4 channels play a key role in regulating vascular tone, the mechanisms controlling Ca2+ influx through these channels in arterial myocytes are poorly understood. Here, we tested the hypothesis that in arterial myocytes the anchoring protein AKAP150 and protein kinase C (PKC) play a critical role in the regulation of TRPV4 channels during angiotensin II (AngII) signaling. Super-resolution imaging revealed that TRPV4 channels are gathered into puncta of variable sizes along the sarcolemma of arterial myocytes. Recordings of Ca2+ entry via single TRPV4 channels (“TRPV4 sparklets”) suggested that basal TRPV4 sparklet activity was low. However, Ca2+ entry during elementary TRPV4 sparklets was ∼100-fold greater than that during L-type CaV1.2 channel sparklets. Application of the TRPV4 channel agonist GSK1016790A or the vasoconstrictor AngII increased the activity of TRPV4 sparklets in specific regions of the cells. PKC and AKAP150 were required for AngII-induced increases in TRPV4 sparklet activity. AKAP150 and TRPV4 channel interactions were dynamic; activation of AngII signaling increased the proximity of AKAP150 and TRPV4 puncta in arterial myocytes. Furthermore, local stimulation of diacylglycerol and PKC signaling by laser activation of a light-sensitive Gq-coupled receptor (opto-α1AR) resulted in TRPV4-mediated Ca2+ influx. We propose that AKAP150, PKC, and TRPV4 channels form dynamic subcellular signaling domains that control Ca2+ influx into arterial myocytes.

Movement of Ca2+ from intracellular stores into the cytosol is an essential component of excitation–contraction coupling in muscle. In cardiac and smooth muscle, Ca2+ is released from the sarcoplasmic reticulum (SR) in response to Ca2+ influx by a process of Ca2+-induced Ca2+ release (CICR). First described by Lederer and coworkers in cardiac myocytes (Cheng et al., 1993), highly localized cytosolic Ca2+ transients, called Ca2+ sparks, originating from ryanodine-sensitive Ca2+-release channels (ryanodine receptors [RyRs]) in the SR membrane, are thought to be the elementary intracellular Ca2+ release events in excitation–contraction coupling, not only in cardiac muscle but also in skeletal (Klein et al., 1996) and smooth muscle (Nelson et al., 1995). RyRs are formed by a family of intracellular Ca2+ channel subunits (RyR1–3), which span the SR membrane of all muscle cells. RyR1 is limited primarily to skeletal muscle, and cardiac cells express chiefly RyR2 (Fill and Copello, 2002). Although RyR2 and RyR3 may be functional there is sufficient evidence to suggest that RyR2 predominates with respect to Ca2+ sparks in smooth muscle (Ji et al., 2004). Smooth muscle Ca2+ sparks, which result from a concerted opening of clusters of RyRs (Blatter et al., 1997; Mejia-Alvarez et al., 1999), generate microdomains of elevated (>10 μM) Ca2+ without changing global intracellular Ca2+ concentrations (Nelson et al., 1995; Soeller and Cannell, 1997; Perez et al., 2001). Because the surface membrane and peripheral SR of smooth muscle cells often are closely apposed (≈10 nm) and form periodic “surface couplings” (Devine et al., 1972), Ca2+-sensitive plasmalemmal proteins, such as ion channels, are exposed to intense pulses of elevated Ca2+. Consistent with this scenario, Ca2+ sparks in smooth muscle are important regulators of membrane potential and hence smooth muscle excitability. In these cells, the Ca2+ signal produced by a spark is tightly coupled to large-conductance Ca2+-activated K+ (BK) channels. Stimulation of BK channel activity by Ca2+ sparks produces spontaneous transient outward currents (STOCs) resulting in membrane hyperpolarization, which causes relaxation (Nelson et al., 1995). In contrast, Ca2+ spark activation of Ca2+-activated Cl− channels in portal vein produces analogous inward currents (STICs) and membrane depolarization (Wang et al., 1992). Thus, the functional role(s) of Ca2+ sparks depend on the molecular identity and relative location of nearby Ca2+-sensitive proteins. The close apposition between plasma and SR membranes has functional consequences on CICR as well. In ventricular myocytes, brief openings of voltage-gated, dihydropyridine-sensitive L-type Ca2+ channels allows a small amount of Ca2+ to enter the small cytosolic volume separating this junctional space. This local increase in [Ca2+]i activates nearby RyRs (to cause the Ca2+ sparks) by the mechanism of CICR. Thus, in ventricular myocytes, the function of sarcolemmal L-type Ca2+ channels and SR RyRs is tightly coupled. Ca2+ influx during the action potential results in a coordinated, synchronous activation of multiple Ca2+ sparks that culminates in a global [Ca2+]i transient that activates the contractile apparatus. Such a relationship is essential for efficient, spatially uniform excitation–contraction coupling in the heart. Because of the physical proximity of plasma and SR membranes in smooth muscle, one would expect the same relationship to hold also in smooth muscle. Experimental evidence suggests otherwise. In contrast to cardiac myocytes, the functional coupling between L-type Ca2+ channels and RyRs is not obvious, and minimal at best. As proposed by Kotlikoff and coworkers, CICR in smooth muscle, when it does occur, results from a “loose coupling” between L-type Ca2+ channels and RyRs (Collier et al., 2000). The basis for loose coupling is unclear at present. Perhaps related to the loose coupling between L-type Ca2+ channels and RyRs is the observation that the spatial distribution of Ca2+ sparks in smooth muscle, spontaneous and evoked, is not stochastic but rather is limited to discrete subcellular sites called “frequent discharge sites” (Gordienko et al., 1998). These sites of Ca2+ spark activity generally correspond to a region of perinuclear SR just underneath the plasma membrane (Pucovsky and Bolton, 2006). Interestingly, RyRs are broadly expressed throughout smooth muscle cells (Devine et al., 1972; Gordienko et al., 1998). These previous studies raise two important questions. Are RyRs outside of frequent discharge sites functional? And, if so, how are they activated? Addressing these two questions is critical to understanding the function and mechanisms underlying spatially heterogeneous SR Ca2+ release activity in smooth muscle. In this issue of the Journal of General Physiology, Ji et al. (p. 225) describe an elegant series of experiments designed to examine the mechanisms of Ca2+ release from the SR of urinary bladder smooth muscle. Ji et al. (2006) used two-photon and confocal microscopy to image [Ca2+]i and locally uncage Ca2+ via flash photolysis in urinary bladder smooth muscle, respectively. Using these approaches, the authors make three novel observations regarding the mechanisms of SR Ca2+ release in these cells. First, they found that local photorelease of Ca2+ evoked SR Ca2+ release in the form of Ca2+ sparks and Ca2+ waves throughout the cell. Second, loss of FK506-binding protein 12.6 expression lowers the threshold and increases the rate of activation of RyRs by local increases in Ca2+ in smooth muscle. Third, SR Ca2+ release in response to local photorelease of Ca2+ required activation of RyRs and IP3 receptors. These findings provide the first direct and compelling demonstration that smooth muscle expresses functional RyRs throughout the cell and that they can be activated by CICR. Related to that, the findings by Ji et al. (2006) suggest that RyRs outside frequent discharge sites could, if the appropriate signal for activation is provided, produce Ca2+ sparks and, together with nearby IP3 receptors, be involved in the propagation of Ca2+ waves in smooth muscle. The latter observation provides strong support for the view that IP3 receptors can be activated by CICR and that there may be bidirectional communication between RyRs and IP3 receptors. The findings from Ji et al. (2006) imply that during membrane depolarization, activation of Ca2+ influx throughout the cell may provide a Ca2+ signal to trigger SR Ca2+ by the mechanism of CICR. As the authors convincingly demonstrate, given the appropriate signal, Ca2+ release from the SR can be evoked throughout the cell. Because BK channels are broadly distributed throughout the sarcolemma of smooth muscle cells (Amberg and Santana, 2003), these findings indicate that RyR–BK channel communication is not necessarily limited to the frequent discharge sites but could occur in multiple locations within the cell. However, as with any good study, the work by Ji et al. (2006) not only answers several important questions, it also raises a new set of issues. For example, why are spontaneous Ca2+ sparks limited to specific locations within a cell (i.e., frequent discharge sites) even though Ca2+ sparks can be evoked throughout the cell? Recent studies, together with the study by Ji et al. (2006), may hint a potential answer to this difficult, yet important, question. In smooth muscle, Ca2+ influx, like Ca2+ sparks, seems to occur through specialized regions of the cell in which small clusters of Ca2+ channels operate in high open probability mode that create sites of nearly continual Ca2+ influx (“persistent Ca2+ sparklets”) (Navedo et al., 2005). Persistent Ca2+ sparklets could increase local [Ca2+]i to a similar extent as Ca2+ sparks or the artificial Ca2+ sparks evoked by Ji et al. (2006) using flash photolysis. Although multiple aspects of Ca2+ sparklet activity need to be elucidated (e.g., molecular identity of the channels involved, mechanisms of activation), maybe persistent Ca2+ sparklets allow for spatially restricted modulation of SR Ca2+ release by providing a localized source of Ca2+ that could increase, presumably via loose coupling mechanisms (Collier et al., 2000), Ca2+ spark activity. Future studies should examine the relationship between Ca2+ sparklet and frequent Ca2+ spark sites in smooth muscle. To conclude, the work described here by Ji et al. (2006) highlights the exquisite, localized control of SR Ca2+ release in smooth muscle and the role of local Ca2+ signals in activating SR Ca2+ release. Future studies will undoubtedly expand on these findings and reveal the mechanisms underlying recurrent, spontaneous Ca2+ spark activity in smooth muscle.

The activity of persistent Ca2+sparklets, which are characterized by longer and more frequent channel open events than low-activity sparklets, contributes substantially to steady-state Ca2+entry under physiological conditions. Here, we addressed two questions related to the regulation of Ca2+sparklets by PKC-α and c-Src, both of which increase whole cell Cav1.2 current: 1) Does c-Src activation enhance persistent Ca2+sparklet activity? 2) Does PKC-α activate c-Src to produce persistent Ca2+sparklets? With the use of total internal reflection fluorescence microscopy, Ca2+sparklets were recorded from voltage-clamped tsA-201 cells coexpressing wild-type (WT) or mutant Cav1.2c (the neuronal isoform of Cav1.2) constructs ± active or inactive PKC-α/c-Src. Cells expressing Cav1.2c exhibited both low-activity and persistent Ca2+sparklets. Persistent Ca2+sparklet activity was significantly reduced by acute application of the c-Src inhibitor PP2 or coexpression of kinase-dead c-Src. Cav1.2c constructs mutated at one of two COOH-terminal residues (Y2122F and Y2139F) were used to test the effect of blocking putative phosphorylation sites for c-Src. Expression of Y2122F but not Y2139F Cav1.2c abrogated the potentiating effect of c-Src on Ca2+sparklet activity. We could not detect a significant change in persistent Ca2+sparklet activity or density in cells coexpressing Cav1.2c + PKC-α, regardless of whether WT or Y2122F Cav1.2c was used, or after PP2 application, suggesting that PKC-α does not act upstream of c-Src to produce persistent Ca2+sparklets. However, our results indicate that persistent Ca2+sparklet activity is promoted by the action of c-Src on residue Y2122of the Cav1.2c COOH terminus.

Smooth muscle cells are ultimately responsible for determining vascular luminal diameter and blood flow. Dynamic changes in intracellular calcium are a critical mechanism regulating vascular smooth muscle contractility. Processes influencing intracellular calcium are therefore important regulators of vascular function with physiological and pathophysiological consequences. In this review we discuss the major dynamic calcium signals identified and characterized in vascular smooth muscle cells. These signals vary with respect to their mechanisms of generation, temporal properties, and spatial distributions. The calcium signals discussed include calcium waves, junctional calcium transients, calcium sparks, calcium puffs, and L-type calcium channel sparklets. For each calcium signal we address underlying mechanisms, general properties, physiological importance, and regulation.

Author(s): Shen, Y; Pfluger, T; Ferreira, F; Liang, J; Navedo, M; Zeng, Q; Reid, B; Zhao, M

Ca 2+ influx via L-type Ca v 1.2 channels is essential for multiple physiological processes, including gene expression, excitability, and contraction. Amplification of the Ca 2+ signals produced by the opening of these channels is a hallmark of many intracellular signaling cascades, including excitation-contraction coupling in heart. Using optogenetic approaches, we discovered that Ca v 1.2 channels form clusters of varied sizes in ventricular myocytes. Physical interaction between these channels via their C-tails renders them capable of coordinating their gating, thereby amplifying Ca 2+ influx and excitation-contraction coupling. Light-induced fusion of WT Ca v 1.2 channels with Ca v 1.2 channels carrying a gain-of-function mutation that causes arrhythmias and autism in humans with Timothy syndrome (Ca v 1.2-TS) increased Ca 2+ currents, diastolic and systolic Ca 2+ levels, contractility and the frequency of arrhythmogenic Ca 2+ fluctuations in ventricular myocytes. Our data indicate that these changes in Ca 2+ signaling resulted from Ca v 1.2-TS increasing the activity of adjoining WT Ca v 1.2 channels. Collectively, these data support the concept that oligomerization of Ca v 1.2 channels via their C termini can result in the amplification of Ca 2+ influx into excitable cells.

Within neurons, Ca2+-dependent inactivation (CDI) of voltage-gated L-type Ca2+ channels shapes cytoplasmic Ca2+ signals. CDI is initiated by Ca2+ binding to channel-associated calmodulin and subsequent Ca2+/calmodulin activation of the Ca2+-dependent phosphatase, calcineurin (CaN), which is targeted to L channels by the A-kinase-anchoring protein AKAP79/150. Here, we report that CDI of neuronal L channels was abolished by inhibition of PKA activity or PKA anchoring to AKAP79/150 and that CDI was also suppressed by stimulation of PKA activity. Although CDI was reduced by positive or negative manipulation of PKA, interference with PKA anchoring or activity lowered Ca2+ current density whereas stimulation of PKA activity elevated it. In contrast, inhibition of CaN reduced CDI but had no effect on current density. These results suggest a model wherein PKA-dependent phosphorylation enhances neuronal L current, thereby priming channels to undergo CDI, and Ca2+/calmodulin-activated CaN actuates CDI by reversing PKA-mediated enhancement of channel activity.

Introduction Voltage-gated L-type Ca2+ channels are expressed in the surface membrane of neurons and muscle cells ([Catterall, 2000][1]). There, they regulate multiple processes including excitability, contraction, gene expression, and memory storage. L-type Ca2+ channels are heteromeric

Ca2+ sparklets are a fluorescence signal associated with Ca2+ entry through L-type calcium (Cav1.2) channels, which are primary Ca2+ entry pathway in many excitable cells. Ca2+ sparklets are quantal in nature (q = 34 nM Δ[Ca2+]i) and their activity is categorized as low (0 0.2), where n = no. of quantal levels and Ps = active sparklet probability. Both types of Ca2+ sparklets are present in vascular smooth muscle cells (VSMCs) but only low activity Ca2+ sparklets are present in heterologously expressed Cav1.2 channels unless PKCα is activated. The latter effect presumably requires Cav1.2 phosphorylation, perhaps at the canonical PKA phosphorylation site, S1901, yet the exact phosphorylation site remains unclear. Using TIRF microscopy in conjunction with whole cell voltage clamp, we detected persistent Ca2+ sparklet activity in HEK 293 cells co-expressing S1901A Cav1.2c and PKCα, indicating that PKCα does not phosphorylate S1901. Also, persistent Ca2+ sparklets were detectable in cells expressing WT Cav1.2c in the absence of PKCα if c-Src was co-expressed. Furthermore, Ca2+ sparklet activity was reduced in cells expressing WT Cav1.2c and kinase dead c-Src (7/9 cells). To test if phosphorylation of a previously identified Cav1.2c tyrosine phosphorylation site (Y2122) by c-Src mediated persistent Ca2+ sparklet activity, we co-expressed Y2122F Cav1.2c and c-Src in HEK cells. Persistent Ca2+ sparklet activity was present under these conditions; however, Ca2+ sparklet activity was reduced in cells co-expressing Y2139FCav1.2c and c-Src. These data suggest that c-Src may phosphorylate Cav1.2c at Y2139 under basal conditions to produce persistent Ca2+ sparklet activity. Future experiments on HEK cells expressing Y2139FCav1.2c and PKCα will allow us to determine if persistent Ca2+ sparklet activity in VSMCs is evoked by a common mechanism involving PKCα and c-Src.

Ca+ sparklets are subcellular Ca2+ signals produced by the opening of L-type Ca2+ channels (LTCCs). In cerebral arterial myocytes, Ca2+ sparklet activity varies regionally, resulting in low and high activity, “persistent” Ca2+ sparklet sites. Although increased Ca2+ influx via LTCCs in arterial myocytes has been implicated in the chain of events contributing to vascular dysfunction during acute hyperglycemia and diabetes, the mechanisms underlying these pathological changes remain unclear. Here, we tested the hypothesis that increased Ca2+ sparklet activity contributes to higher Ca2+ influx in cerebral artery smooth muscle during acute hyperglycemia and in an animal model of non-insulin-dependent, type 2 diabetes: the dB/dB mouse. Consistent with this hypothesis, acute elevation of extracellular glucose from 10 to 20 mM increased the density of low activity and persistent Ca2+ sparklet sites as well as the amplitude of LTCC currents in wild-type cerebral arterial myocytes. Furthermore, Ca2+ sparklet activity and LTCC currents were higher in dB/dB than in control myocytes. We found that activation of PKA contributed to higher Ca2+ sparklet activity during hyperglycemia and diabetes. In addition, we found that the interaction between PKA and the scaffolding protein A-kinase anchoring protein was critical for the activation of persistent Ca2+ sparklets by PKA in cerebral arterial myocytes after hyperglycemia. Accordingly, PKA inhibition equalized Ca2+ sparklet activity between dB/dB and wild-type cells. These findings suggest that hyperglycemia increases Ca2+ influx by increasing Ca2+ sparklet activity via a PKA-dependent pathway in cerebral arterial myocytes and contributes to vascular dysfunction during diabetes.

In this article, we review the biophysical basis and functional implications of a novel Ca(2+) signal (called "Ca(2+) sparklets") produced by Ca(2+) influx via L-type Ca(2+) channels (LTCCs) in smooth muscle. Ca(2+) sparklet activity is bimodal. In low activity mode, Ca(2+) sparklets are produced by random, brief openings of solitary LTCCs. In contrast, small clusters of LTCCs can function in a high activity mode that creates sites of continual Ca(2+) influx called "persistent Ca(2+) sparklets". Low activity and persistent Ca(2+) sparklets contribute to Ca(2+) influx in arterial, colonic, and venous smooth muscle. Targeting of PKCalpha by the scaffolding protein AKAP150 to specific sarcolemmal domains is required for the activation of persistent Ca(2+) sparklets. Calcineurin, which is also associated with AKAP150, opposes the actions of PKCalpha on Ca(2+) sparklets. At hyperpolarized potentials, Ca(2+) sparklet activity is low and hence does not contribute to global [Ca(2+)](i). Membrane depolarization increases low and persistent Ca(2+) sparklet activity, thereby increasing local and global [Ca(2+)](i). Ca(2+) sparklet activity is increased in arterial myocytes during hypertension, thus increasing Ca(2+) influx and activating the transcription factor NFATc3. We discuss a model for subcellular variations in Ca(2+) sparklet activity and their role in the regulation of excitation-contraction coupling and excitation-transcription coupling in smooth muscle.

Many excitable cells express L-type Ca 2+ channels (LTCCs), which participate in physiological and pathophysiological processes ranging from memory, secretion, and contraction to epilepsy, heart failure, and hypertension. Clusters of LTCCs can operate in a PKCα-dependent, high open probability mode that generates sites of sustained Ca 2+ influx called “persistent Ca 2+ sparklets.” Although increased LTCC activity is necessary for the development of vascular dysfunction during hypertension, the mechanisms leading to increased LTCC function are unclear. Here, we tested the hypothesis that increased PKCα and persistent Ca 2+ sparklet activity contributes to arterial dysfunction during hypertension. We found that PKCα and persistent Ca 2+ sparklet activity is indeed increased in arterial myocytes during hypertension. Furthermore, in human arterial myocytes, PKCα-dependent persistent Ca 2+ sparklets activated the prohypertensive calcineurin/NFATc3 signaling cascade. These events culminated in three hallmark signs of hypertension-associated vascular dysfunction: increased Ca 2+ entry, elevated arterial [Ca 2+ ] i , and enhanced myogenic tone. Consistent with these observations, we show that PKCα ablation is protective against the development of angiotensin II-induced hypertension. These data support a model in which persistent Ca 2+ sparklets, PKCα, and calcineurin form a subcellular signaling triad controlling NFATc3-dependent gene expression, arterial function, and blood pressure. Because of the ubiquity of these proteins, this model may represent a general signaling pathway controlling gene expression and cellular function.

The role of position L8', located in transmembrane domain 1 of the neuronal nicotinic alpha3 subunit, was characterized by using two-electrode voltage clamp in Xenopus oocytes. Four amino acids (Ala, Ser, Phe, and Tyr) were inserted at this conserved position, and the mutant subunit was coexpressed with either wild-type beta2 or beta4 subunits. These substitutions led to significant alterations in the pharmacodynamic parameters of cholinergic agents, resulting in loss of function. Ala and Ser substitutions resulted in losses in agonist (ACh, nicotine, and DMPP) potency and intrinsic activity at both alpha3beta2 and alpha3beta4 receptors. Similarly, significant changes in antagonist potency were produced by the Ala and Ser substitutions. Phe and Tyr mutations did not alter the receptor's EC(50) for ACh or nicotine but reduced the EC(50) for DMPP at both receptors. The Phe mutation also reduced the intrinsic activity of all agonists tested at both receptors. The Tyr mutation, though, led to a decrease in intrinsic activity for all agonists at the alpha3beta2 receptor, yet resulted in no changes for DMPP, a decrease for nicotine, and an increase for ACh at the alpha3beta4 receptor. The most dramatic changes in the receptor's functional properties were produced by substitutions that introduced the largest changes in amino acid volume. Additional replacements (Gly, Thr, and Val) suggested an inverse correlation between amino acid volume at position alpha3L8' and EC(50) for alpha3beta4 nAChRs; however, alpha3beta2 nAChRs displayed a nonlinear correlation. These data demonstrate that structural alterations at position alpha3L8' could propagate to the agonist-binding site.

Hypertension is a perplexing multiorgan disease involving renal primary pathology and enhanced angiotensin II vascular reactivity. Here, we report that a novel form of a local Ca 2+ signaling in arterial smooth muscle is linked to the development of angiotensin II–induced hypertension. Long openings and reopenings of L-type Ca 2+ channels in arterial myocytes produce stuttering persistent Ca 2+ sparklets that increase Ca 2+ influx and vascular tone. These stuttering persistent Ca 2+ sparklets arise from the molecular interactions between the L-type Ca 2+ channel and protein kinase Cα at only a few subsarcolemmal regions in resistance arteries. We have identified AKAP150 as the key protein, which targets protein kinase Cα to the L-type Ca 2+ channels and thereby enables its regulatory function. Accordingly, AKAP150 knockout mice (AKAP150 −/− ) were found to lack persistent Ca 2+ sparklets and have lower arterial wall intracellular calcium ([Ca 2+ ] i ) and decreased myogenic tone. Furthermore, AKAP150 −/− mice were hypotensive and did not develop angiotensin II–induced hypertension. We conclude that local control of L-type Ca 2+ channel function is regulated by AKAP150-targeted protein kinase Cα signaling, which controls stuttering persistent Ca 2+ influx, vascular tone, and blood pressure under physiological conditions and underlies angiotensin II–dependent hypertension.

In arterial smooth muscle, protein kinase Calpha (PKCalpha) coerces discrete clusters of L-type Ca2+ channels to operate in a high open probability mode, resulting in subcellular domains of nearly continual Ca2+ influx called 'persistent Ca2+ sparklets'. Our previous work suggested that steady-state Ca2+ entry into arterial myocytes, and thus global [Ca2+]i, is regulated by Ca2+ influx through clusters of L-type Ca2+ channels operating in this persistently active mode in addition to openings of solitary channels functioning in a low-activity mode. Here, we provide the first direct evidence supporting this 'Ca2+ sparklet' model of Ca2+ influx at a physiological membrane potential and external Ca2+ concentration. In support of this model, we found that persistent Ca2+ sparklets produced local and global elevations in [Ca2+]i. Membrane depolarization increased Ca2+ influx via low-activity and high-activity persistent Ca2+ sparklets. Our data indicate that Ca2+ entering arterial smooth muscle through persistent Ca2+ sparklets accounts for approximately 50% of the total dihydropyridine-sensitive (i.e. L-type Ca2+ channel) Ca2+ influx at a physiologically relevant membrane potential (-40 mV) and external Ca2+ concentration (2 mm). Consistent with this, inhibition of basal PKCalpha-dependent persistent Ca2+ sparklets decreased [Ca2+]i by about 50% in isolated arterial myocytes and intact pressurized arteries. Taken together, these data support the conclusion that in arterial smooth muscle steady-state Ca2+ entry and global [Ca2+]i are regulated by low-activity and PKCalpha-dependent high-activity persistent Ca(2+) sparklets.

The second transmembrane domain (TMD2) of the Cys-loop family of ligand-gated ion channels forms the channel pore. The functional role of the amino acid residues contributing to the channel pore in neuronal nicotinic a3 receptors is not well understood. We characterized the contribution of TMD2 position V7 0 to channel gating in neuronal nicotinic a3 receptors. Site-directed mutagenesis was used to substitute position a3 (V7 0 ) with four different amino acids (A, F, S, or Y) and coexpressed each mutant subunit with wild-type (WT) b 2o r b4 subunits in Xenopus oocytes. Whole-cell voltage clamp experiments show that substitution for an alanine, serine, or phenylalanine decreased by 2.3–6.2fold the ACh-EC50 for a3b2 and a3b4 receptor subtypes. Interestingly, mutation V7 0 Y did not produce a significant change in ACh-EC50 when coexpressed with the b2 subunit but showed a significant approximately two-fold increase with b4. Similar responses were obtained with nicotine as the agonist. The antagonist sensitivity of the mutant channels was assessed by using dihydro-b-erythroidine (DHbE) and methyllycaconitine (MLA). The apparent potency of DHbE as an antagonist increased by *3.7- and 11-fold for the a3b2 V7 0 S and V7 0 F mutants, respectively, whereas no evident changes in antagonist potency were observed for the V7 0 A and V7 0 Y mutants. The V7 0 S and V7 0 F mutations increase MLA antagonist potency for the a3b4 receptor by *6.2- and *9.3-fold, respectively. The V7 0 A mutation selectively increases the MLA antagonist potency for the a3b4 receptor by *18.7-fold. These results indicate that position V7 0 contributes to channel gating kinetics and pharmacology of the neuronal nicotinic a3 receptors. V V C 2006 Wiley-Liss, Inc. Neurotransmitter gated channels are key components of the communication process between neurons, i.e., synaptic transmission. Upon agonist binding, the receptor undergoes a series of conformational changes leading to opening of the ion channel pore, flux of specific ions into the cell, and inactivation (desensitization) of the channel after prolonged exposure to the agonist. Nicotinic acetylcholine receptors (nAChRs) are members of a superfamily of neurotransmitter-gated ion

In arterial smooth muscle, single or small clusters of Ca(2+) channels operate in a high probability mode, creating sites of nearly continual Ca(2+) influx (called "persistent Ca(2+) sparklet" sites). Persistent Ca(2+) sparklet activity varies regionally within any given cell. At present, the molecular identity of the Ca(2+) channels underlying Ca(2+) sparklets and the mechanisms that give rise to their spatial heterogeneity remain unclear. Here, we used total internal reflection fluorescence (TIRF) microscopy to directly investigate these issues. We found that tsA-201 cells expressing L-type Cavalpha1.2 channels recapitulated the general features of Ca(2+) sparklets in cerebral arterial myocytes, including amplitude of quantal event, voltage dependencies, gating modalities, and pharmacology. Furthermore, PKCalpha activity was required for basal persistent Ca(2+) sparklet activity in arterial myocytes and tsA-201 cells. In arterial myocytes, inhibition of protein phosphatase 2A (PP2A) and 2B (PP2B; calcineurin) increased Ca(2+) influx by evoking new persistent Ca(2+) sparklet sites and by increasing the activity of previously active sites. The actions of PP2A and PP2B inhibition on Ca(2+) sparklets required PKC activity, indicating that these phosphatases opposed PKC-mediated phosphorylation. Together, these data unequivocally demonstrate that persistent Ca(2+) sparklet activity is a fundamental property of L-type Ca(2+) channels when associated with PKC. Our findings support a novel model in which the gating modality of L-type Ca(2+) channels vary regionally within a cell depending on the relative activities of nearby PKCalpha, PP2A, and PP2B.

We traced the cause of a slow-channel syndrome (SCS) in a patient with progressive muscle weakness, repetitive compound muscle action potential and prolonged low amplitude synaptic currents to a V --> F substitution in the M1 domain of the beta subunit (betaV229F) of the muscle acetylcholine receptor (AChR). In vitro expression studies in Xenopus oocytes indicated that the novel mutation betaV229F expressed normal amounts of AChRs and decreased the ACh EC50 by 10-fold compared to wild type. Kinetic analysis indicated that the mutation displayed prolonged mean open duration and repeated openings during activation. Prolonged openings caused by the betaV229F mutation were due to a reduction in the channel closing rate and an increase in the effective channel opening rate. Repeated openings of the channel during activation were caused by a significant reduction in the agonist dissociation constant. In addition, the betaV229F mutation produced an increase in calcium permeability. The kinetic and permeation studies presented in this work are sufficient to explain the consequences of the betaV229F mutation on the miniature endplate currents and thus are direct evidence that the betaV229F mutation is responsible for compromising the safety margin of neuromuscular transmission in the patient.

Ca 2+ influx through L-type Ca 2+ channels (LTCCs) influences numerous physiological processes ranging from contraction in muscle and memory in neurons to gene expression in many cell types. However, the spatiotemporal organization of functional LTCCs has been nearly impossible to investigate because of methodological limitations. Here, we examined LTCC function with high temporal and spatial resolution using evanescent field fluorescence microscopy. Surprisingly, we found that LTCCs operated in functionally organized clusters, not necessarily as individual proteins. Furthermore, LTCC function in these clusters does not appear to be controlled by simple stochastic gating but instead by a PKC-dependent switch mechanism. This work suggests that resting intracellular free calcium concentration in arterial myocytes is predominantly controlled by this process in combination with rare voltage-dependent openings of individual LTCCs. We propose that Ca 2+ influx via persistent LTCCs may be an important mechanism regulating steady-state local and global Ca 2+ signals.

Voltage-gated K+ (Kv) channels control the excitability of arterial smooth muscle. However, the molecular mechanisms regulating Kv channel function in smooth muscle remain unclear. We examined the hypothesis that the vasoactive peptide angiotensin II (Ang II) regulates arterial smooth muscle Kv channel function via calcineurin-dependent activation of the transcription factor NFAT. We found that sustained administration of Ang II decreased Kv currents (IKv) by reducing the expression of Kv2.1 K+ channel subunits. This effect of Ang II was independent of pressure but required Ca2+ influx through L-type Ca2+ channels. Consistent with our hypothesis, we found that calcineurin and NFATc3 are obligatory components of the signaling cascade mediating reduced IKv by Ang II. We conclude that sustained Ang II exposure increases smooth muscle Ca2+, which leads to activation of calcineurin and NFATc3, culminating in decreased Kv2.1 expression and reduced IKv function. These results support the novel concept that NFATc3 controls the excitability of arterial smooth muscle by regulating Kv2.1 expression.

We improved the statistical sieve algorithm developed by Izu et al. (Biophys. J. 92(12):4458-65) and simplified the user interface. In this new version, the normalized image (Norm) and background fluorescence is automatically calculated by the program. After reading the stack of images for a given cell, a template is created by averaging all the images. This template will constitute the norm against which each individual frame will be normalized. The cell border is detected from this template, and fluorescence outside this area is considered the background that is subtracted from all images. Then each frame is normalized by the Norm image. The next step highlights events as potential sparks, if fluorescence is ≥ 3 standard deviations from the Norm value. A final statistical test in the time domain is applied at each potential event. If the amplitude of the event is statistically different from the mean value of the previous 10 frames, the event is considered a spark. A table with the amplitude, FWHMx, FHHMy, Tau, Frame and location (x,y) is generated. Additionally the user can inspect each of the sparks in a 3D plot or its fitting profiles in x,y and t axis. A user friendly interface implemented in LabVIEW with intuitive menus guide the user through the whole process. We tested the functionality of the program by analyzing Ca2+ sparks from vascular smooth muscle. SparkLAB2 presents the advantages of being a statistically based spark detector with minimum intervention of the user thus reducing the possibility of human bias.

Ca2+ sparklets are local elevations in intracellular Ca2+ produced by the opening of a single or a small cluster of sarcolemmal L-type Cav1.2 Ca2+ channels. At present, however, the spatial organization and mechanisms of modulation of Ca2+ sparklets in cardiac myocytes is unknown. Here, we tested the hypothesis that Ca2+ sparklets activity varies within the sarcolemma of neonatal cardiac myocytes and that chemically-induced translocation of PKCα increases the Ca2+ sparklet activity in these cells. Consistent with this hypothesis we found that application of the PKC activator phorbol 12,13-dibutyrate (PDBu; 500 nM) increased Ca2+ sparklet activity in neonatal cardiac myocytes. Analysis of the spatial distribution Ca2+ sparklet activity was not random (i.e. did not have a Poisson distribution). Rather, as in reported in smooth muscle cells, Ca2+ sparklet activity was higher at specific regions of the cell. Translocation of PKCα to the sarcolemma of neonatal cardiac myocytes resulted in an increase in Ca2+ sparklet activity in specific regions of the cell. Data will be presented on the relationship between Ca2+ sparklet activity and Ca2+ release from the sarcoplasmic reticulum via ryanodine receptors (i.e. Ca2+ sparks) during excitation-contraction coupling. Our data suggest that Ca2+ sparklet (i.e. L-type Ca2+ channels) activity varies within the sarcolemma of neonatal cardiac myocytes and that they are modulated by PKCα, potentially regulating SR Ca2+ release during EC coupling in heart.

Calcium influx through L-type calcium channels (LTCCs) influences numerous physiological processes in excitable cells ranging from contraction, memory and gene expression. Clusters of LTCCs can operate in a PKCalpha-dependent, high open probability mode that generates sites of sustained calcium influx called “persistent calcium sparklets”. In vascular smooth muscle, persistent calcium sparklets contribute to local and global calcium. Calcium sparklets activity varies regionally within smooth muscle cells. At present however, the mechanisms underlying heterogeneous sparklet activity are incompletely understood. Here, we use TIRF microscopy and whole-cell patch clamp electrophysiology to investigate the role calmodulin in the modulation of calcium sparklet activity. We found that inhibition of calmodulin increases calcium sparklet activity in wild type (WT) smooth muscle cells. Inhibition of calmodulin in PKCalpha KO cells, which are devoid of persistent calcium sparklets, increased calcium influx by evoking new persistent calcium sparklet sites and by increasing the activity of previously low activity sites in these cells. On the basis of these finding, we hypothesize that calmodulin plays a critical role in determining the activity of calcium sparklet sites in arterial smooth muscle. This work was supported by the National Institutes of Health and the American Heart Association.

Activity-dependent CREB phosphorylation and gene expression are critical for long-term neuronal plasticity. Local signaling at CaV1 channels triggers these events, but how information is relayed onward to the nucleus remains unclear. Here, we report a mechanism that mediates long-distance communication within cells: a shuttle that transports Ca(2+)/calmodulin from the surface membrane to the nucleus. We show that the shuttle protein is γCaMKII, its phosphorylation at Thr287 by βCaMKII protects the Ca(2+)/CaM signal, and CaN triggers its nuclear translocation. Both βCaMKII and CaN act in close proximity to CaV1 channels, supporting their dominance, whereas γCaMKII operates as a carrier, not as a kinase. Upon arrival within the nucleus, Ca(2+)/CaM activates CaMKK and its substrate CaMKIV, the CREB kinase. This mechanism resolves long-standing puzzles about CaM/CaMK-dependent signaling to the nucleus. The significance of the mechanism is emphasized by dysregulation of CaV1, γCaMKII, βCaMKII, and CaN in multiple neuropsychiatric disorders.

In smooth muscle, activation of the Ca2+-sensitive phosphatase calcineurin dephosphorylates the transcription factor nuclear factor of activated T cells c3 (NFATc3). Upon de-phosphorylation, NFATc3 translocates into the nucleus of arterial smooth muscle cells where it modulates the expression of multiple genes including Kv2.1 and the α and β1 subunits of large conductance K+ (BK) channels. Recent work by our group, suggested that L-type Ca2+ channels, the A-kinase anchoring protein 150 (AKAP150), calcineurin, and PKCα form a signaling triad that controls Ca2+ influx into these cells. Ca2+ influx via L-type Ca2+channels is necessary for NFATc3 activation in smooth muscle cells during angiotensin II signaling. In this study, we tested the hypothesis that AKAP150 is required for the down regulation of Kv2.1 and BK channel α and β1 subunits by calcineurin/NFATc3 signaling during chronic angiotensin II signaling.To test this hypothesis, we used wild type and AKAP150 null (AKAP150-/-) arteries. Experiments involved measurement of calcineurin activity in wild type (WT) and AKAP150 null (AKAP150-/-) myocytes. We also used TIRF and confocal microscopy to image local Ca2+ signals by Ca2+ influx via L-type Ca2+ channels and nuclear NFATc3 translocation in WT and AKAP150-/- before and after application of angiotensin II. Finally, we examined the effects of chronic activation of angiotensin II and NFATc3 signaling on the expression of Kv2.1 as well as the α and β1 subunits of the BK channels in WT and AKAP150-/- arteries. We found that sustained activation of angiotensin II signaling down regulated these genes in arterial smooth muscle from WT but not AKAP150-/- arteries. These results suggest a model, in which AKAP150, calcineurin, and L-type Ca2+ channels form a signaling unit that regulates Ca2+ influx and gene expression in smooth muscle.

In arterial smooth muscle, L-type Ca2+ channels play a critical role in multiple physiological processes including excitability, contraction, and gene expression. Recent work suggests that TRPV4 channels are also important contributors to Ca2+ influx in these cells. We used optical approaches to record Ca2+ sparklets produced by Ca2+ influx via TRPV4 and L-type Ca2+ channels in arterial myocytes under physiological conditions. We found that TRPV4 and L-type Ca2+ sparklet activity varies throughout the sarcolemmal of arterial myocytes. Our data suggest that these regional variations in sparklet activity arise from interactions between channels, the scaffolding protein AKAP150, and associated proteins at only a few sub-sarcolemmal regions in arterial smooth muscle cells. We will present data obtained using biochemical and optogenetic approaches to investigate the mechanisms leading to subcellular variations in TRPV4 and L-type Ca2+ sparklet activity as well as the functional consequences of these local Ca2+ signals in arterial smooth muscle. These findings will form the basis for a new model for the local control and amplification Ca2+ influx via TRPV4 and voltage-gated L-type Ca2+ channels in resistance artery smooth muscle.