TRP channels: a provocative rationalization for local Ca2+ control in arterial tone development.

Arterial networks are controlled by the consolidated output of stimuli that set "how much" (magnitude) and "where" (distribution) blood flow is delivered. While notable changes in magnitude are tied to network wide responses, altered distribution often arises from focal changes in tone, whose mechanistic foundation remains unclear. We propose herein a framework of focal vasomotor contractility being controlled by pharmacomechanical coupling and the generation of Ca2+ waves via the sarcoplasmic reticulum. We argue the latter is sustained by receptor operated, transient receptor potential (TRP) channels through direct extracellular Ca2+ influx or indirect Na+ influx, reversing the Na+/Ca2+ exchanger. We view this focal regulatory mechanism as complementary, but not redundant with, electromechanical coupling in the precision tuning of blood flow delivery.

Capillary oxygen regulates demand-supply coupling by triggering connexin40-mediated conduction: Rethinking the metabolic hypothesis.

Coupling red blood cell (RBC) supply to O2 demand is an intricate process requiring O2 sensing, generation of a stimulus, and signal transduction that alters upstream arteriolar tone. Although actively debated, this process has been theorized to be induced by hypoxia and to involve activation of endothelial inwardly rectifying K+ channels (KIR) 2.1 by elevated extracellular K+ to trigger conducted hyperpolarization via connexin40 (Cx40) gap junctions to upstream resistors. This concept was tested in resting healthy skeletal muscle of Cx40-/- and endothelial KIR2.1-/- mice using state-of-the-art live animal imaging where the local tissue O2 environment was manipulated using a custom gas chamber. Second-by-second capillary RBC flow responses were recorded as O2 was altered. A stepwise drop in PO2 at the muscle surface increased RBC supply in capillaries of control animals while elevated O2 elicited the opposite response; capillaries were confirmed to express Cx40. The RBC flow responses were rapid and tightly coupled to O2; computer simulations did not support hypoxia as a driving factor. In contrast, RBC flow responses were significantly diminished in Cx40-/- mice. Endothelial KIR2.1-/- mice, on the other hand, reacted normally to O2 changes, even when the O2 challenge was targeted to a smaller area of tissue with fewer capillaries. Conclusively, microvascular O2 responses depend on coordinated electrical signaling via Cx40 gap junctions, and endothelial KIR2.1 channels do not initiate the event. These findings reconceptualize the paradigm of blood flow regulation in skeletal muscle and how O2 triggers this process in capillaries independent of extracellular K+.

CaV3.1 channels facilitate calcium wave generation and myogenic tone development in mouse mesenteric arteries.

The arterial myogenic response to intraluminal pressure elicits constriction to maintain tissue perfusion. Smooth muscle [Ca2+] is a key determinant of constriction, tied to L-type (CaV1.2) Ca2+ channels. While important, other Ca2+ channels, particularly T-type could contribute to pressure regulation within defined voltage ranges. This study examined the role of one T-type Ca2+ channel (CaV3.1) using C57BL/6 wild type and CaV3.1-/- mice. Patch-clamp electrophysiology, pressure myography, blood pressure and Ca2+ imaging defined the CaV3.1-/- phenotype relative to C57BL/6. CaV3.1-/- mice had absent CaV3.1 expression and whole-cell current, coinciding with lower blood pressure and reduced mesenteric artery myogenic tone, particularly at lower pressures (20-60 mmHg) where membrane potential is hyperpolarized. This reduction coincided with diminished Ca2+ wave generation, asynchronous events of Ca2+ release from the sarcoplasmic reticulum, insensitive to L-type Ca2+ channel blockade (Nifedipine, 0.3 µM). Proximity ligation assay (PLA) confirmed IP3R1/CaV3.1 close physical association. IP3R blockade (2-APB, 50 µM or xestospongin C, 3 µM) in nifedipine-treated C57BL/6 arteries rendered a CaV3.1-/- contractile phenotype. Findings indicate that Ca2+ influx through CaV3.1 contributes to myogenic tone at hyperpolarized voltages through Ca2+-induced Ca2+ release tied to the sarcoplasmic reticulum. This study helps establish CaV3.1 as a potential therapeutic target to control blood pressure.

Functional tuning of Vascular L-type Ca2+ channels.

Vascular smooth muscle contraction is intimately tied to membrane potential and the rise in intracellular Ca2+ enabled by the opening of L-type Ca2+ channels. While voltage is often viewed as the single critical factor gating these channels, research is starting to reveal a more intricate scenario whereby their function is markedly tuned. This emerging concept will be the focus of this three-part review, the first part articulating the mechanistic foundation of contractile development in vascular smooth muscle. Part two will extend this foundational knowledge, introducing readers to functional coupling and how neighboring L-type Ca2+ channels work cooperatively through signaling protein complexes, to facilitate their open probability. The final aspect of this review will discuss the impact of L-type Ca2+ channel trafficking, a process tied to cytoskeleton dynamics. Cumulatively, this brief manuscript provides new insight into how voltage, along with channel cooperativity and number, work in concert to tune Ca2+ responses and smooth muscle contraction.

Genetic ablation of smooth muscle KIR2.1 is inconsequential to the function of mouse cerebral arteries.

Cerebral blood flow is a finely tuned process dependent on coordinated changes in arterial tone. These changes are strongly tied to smooth muscle membrane potential and inwardly rectifying K+ (KIR) channels are thought to be a key determinant. To elucidate the role of KIR2.1 in cerebral arterial tone development, this study examined the electrical and functional properties of cells, vessels and living tissue from tamoxifen-induced smooth muscle cell (SMC)-specific KIR2.1 knockout mice. Patch-clamp electrophysiology revealed a robust Ba2+-sensitive inwardly rectifying K+ current in cerebral arterial myocytes irrespective of KIR2.1 knockout. Immunolabeling clarified that KIR2.1 expression was low in SMCs while KIR2.2 labeling was remarkably abundant at the membrane. In alignment with these observations, pressure myography revealed that the myogenic response and K+-induced dilation were intact in cerebral arteries post knockout. At the whole organ level, this translated to a maintenance of brain perfusion in SMC KIR2.1-/- mice, as assessed with arterial spin-labeling MRI. We confirmed these findings in superior epigastric arteries and implicated KIR2.2 as more functionally relevant in SMCs. Together, these results suggest that subunits other than KIR2.1 play a significant role in setting native current in SMCs and driving arterial tone.

Inward Rectifier Potassium Channels: Membrane Lipid-Dependent Mechanosensitive Gates in Brain Vascular Cells.

Cerebral arteries contain two primary and interacting cell types, smooth muscle (SMCs) and endothelial cells (ECs), which are each capable of sensing particular hemodynamic forces to set basal tone and brain perfusion. These biomechanical stimuli help confer tone within arterial networks upon which local neurovascular stimuli function. Tone development is intimately tied to arterial membrane potential (V M ) and changes in intracellular [Ca2+] driven by voltage-gated Ca2+ channels (VGCCs). Arterial V M is in turn set by the dynamic interplay among ion channel species, the strongly inward rectifying K+ (Kir) channel being of special interest. Kir2 channels possess a unique biophysical signature in that they strongly rectify, display negative slope conductance, respond to elevated extracellular K+ and are blocked by micromolar Ba2+. While functional Kir2 channels are expressed in both smooth muscle and endothelium, they lack classic regulatory control, thus are often viewed as a simple background conductance. Recent literature has provided new insight, with two membrane lipids, phosphatidylinositol 4,5-bisphosphate (PIP2) and cholesterol, noted to (1) stabilize Kir2 channels in a preferred open or closed state, respectively, and (2) confer, in association with the cytoskeleton, caveolin-1 (Cav1) and syntrophin, hemodynamic sensitivity. It is these aspects of vascular Kir2 channels that will be the primary focus of this review.

Defining a role of NADPH oxidase in myogenic tone development.

OBJECTIVE: The myogenic response sets the foundation for blood flow control. Recent findings suggest a role for G protein-coupled receptors (GPCR) and signaling pathways tied to the generation of reactive oxygen species (ROS). In this regard, this study ascertained the impact of NADPH oxidase (Nox) on myogenic tone in rat cerebral resistance arteries. METHODS: The study employed real-time qPCR (RT-qPCR), pressure myography, and immunohistochemistry. RESULTS: Gq blockade abolished myogenic tone in rat cerebral arteries, linking GPCR to mechanosensation. Subsequent work revealed that general (TEMPOL) and mitochondrial specific (MitoTEMPO) ROS scavengers had little impact on myogenic tone, whereas apocynin, a broad spectrum Nox inhibitor, initiated transient dilation. RT-qPCR revealed Nox1 and Nox2 mRNA expression in smooth muscle cells. Pressure myography defined Nox1 rather than Nox2 is facilitating myogenic tone. We rationalized that Nox1-generated ROS was initiating this response by impairing the ability of the CaV 3.2 channel to elicit negative feedback via BKCa . This hypothesis was confirmed in functional experiments. The proximity ligation assay further revealed that Nox1 and CaV 3.2 colocalize within 40 nm of one another. CONCLUSIONS: Our data highlight that vascular pressurization augments Nox1 activity and ensuing ROS production facilitates myogenic tone by limiting Ca2+ influx via CaV 3.2.

Myogenic Vasoconstriction Requires Canonical Gq/11 Signaling of the Angiotensin II Type 1 Receptor.

Background Blood pressure and tissue perfusion are controlled in part by the level of intrinsic (myogenic) arterial tone. However, many of the molecular determinants of this response are unknown. We previously found that mice with targeted disruption of the gene encoding the angiotensin II type 1a receptor (AT1AR) (Agtr1a), the major murine angiotensin II type 1 receptor (AT1R) isoform, showed reduced myogenic tone; however, uncontrolled genetic events (in this case, gene ablation) can lead to phenotypes that are difficult or impossible to interpret. Methods and Results We tested the mechanosensitive function of AT1R using tamoxifen-inducible smooth muscle-specific AT1aR knockout (smooth muscle-Agtr1a-/-) mice and studied downstream signaling cascades mediated by Gq/11 and/or ß-arrestins. FR900359, Sar1Ile4Ile8-angiotensin II (SII), TRV120027 and TRV120055 were used as selective Gq/11 inhibitor and biased agonists to activate noncanonical ß-arrestin and canonical Gq/11 signaling of the AT1R, respectively. Myogenic and Ang II-induced constrictions were diminished in the perfused renal vasculature, mesenteric and cerebral arteries of smooth muscle-Agtr1a-/- mice. Similar effects were observed in arteries of global mutant Agtr1a-/- but not Agtr1b-/- mice. FR900359 decreased myogenic tone and angiotensin II-induced constrictions whereas selective biased targeting of AT1R-ß-arrestin signaling pathways had no effects. Conclusions This study demonstrates that myogenic arterial constriction requires Gq/11-dependent signaling pathways of mechanoactivated AT1R but not G protein-independent, noncanonical pathways in smooth muscle cells.

Gestational long-term hypoxia induces metabolomic reprogramming and phenotypic transformations in fetal sheep pulmonary arteries.

Gestational long-term hypoxia increases the risk of myriad diseases in infants including persistent pulmonary hypertension. Similar to humans, fetal lamb lung development is susceptible to long-term intrauterine hypoxia, with structural and functional changes associated with the development of pulmonary hypertension including pulmonary arterial medial wall thickening and dysregulation of arterial reactivity, which culminates in decreased right ventricular output. To further explore the mechanisms associated with hypoxia-induced aberrations in the fetal sheep lung, we examined the premise that metabolomic changes and functional phenotypic transformations occur due to intrauterine, long-term hypoxia. To address this, we performed electron microscopy, Western immunoblotting, calcium imaging, and metabolomic analyses on pulmonary arteries isolated from near-term fetal lambs that had been exposed to low- or high-altitude (3,801 m) hypoxia for the latter 110+ days of gestation. Our results demonstrate that the sarcoplasmic reticulum was swollen with high luminal width and distances to the plasma membrane in the hypoxic group. Hypoxic animals were presented with higher endoplasmic reticulum stress and suppressed calcium storage. Metabolically, hypoxia was associated with lower levels of multiple omega-3 polyunsaturated fatty acids and derived lipid mediators (e.g., eicosapentaenoic acid, docosahexaenoic acid, α-linolenic acid, 5-hydroxyeicosapentaenoic acid (5-HEPE), 12-HEPE, 15-HEPE, prostaglandin E3, and 19(20)-epoxy docosapentaenoic acid) and higher levels of some omega-6 metabolites (P < 0.02) including 15-keto prostaglandin E2 and linoleoylglycerol. Collectively, the results reveal broad evidence for long-term hypoxia-induced metabolic reprogramming and phenotypic transformations in the pulmonary arteries of fetal sheep, conditions that likely contribute to the development of persistent pulmonary hypertension.

Conceptualizing conduction as a pliant electrical response: impact of gap junctions and ion channels.

Vasomotor responses conduct among resistance arteries to coordinate blood flow delivery pursuant to energetic demand. Conduction is set by the electrical and mechanical properties of vascular cells, the former tied to how gap junctions and ion channels distribute and dissipate charge, respectively. These membrane proteins are subject to modulation; thus, conduction could be viewed as "pliant" to the current regulatory state. This study used in silico approaches to conceptualize electrical pliancy and to illustrate how gap junctional and ion channel properties distinctly impact conduction along a single skeletal muscle artery or a branching cerebrovascular network. Initial simulations revealed how vascular cells encoded with electrotonic properties best reproduced spreading behavior; the endothelium's importance as a charge source and a longitudinal conduit was readily observed. Alterations in gap junctional conductance produced unique electrical fingerprints: 1) decreased endothelial coupling impaired longitudinal but enhanced radial spread, and 2) reduced myoendothelial coupling limited radial but enhanced longitudinal spread. Subsequent simulations illustrated how tuning ion channel activity, e.g., inward rectifying- and voltage-gated K+ channels, modified charge dissipation, resting membrane potential, and the spread of the electrical phenomenon. Restricting ion channel tuning to a network subregion then revealed how electrical spread could be locally shaped in accordance with the aggregate changes in membrane resistance. In summary, our analysis frames and reimagines electrical conduction as a pliable process, with subtle regulatory changes to membrane proteins shaping network spread and tissue perfusion.NEW & NOTEWORTHY Conducted vasomotor responses depend on initiation and spread of electrical phenomena along arterial walls and their translation into contractile responses. Using computational approaches, we show how subtle but widespread regulation of gap junctions and ion channels can modulate the range and amplitude of electrical spread. Ion channels are regulated by endocrine and mechanical signals and may differ regionally in networks. Subregional electrical changes are not spatially confined but may affect electrical conduction in neighboring regions.

Conceptualizing Conduction as a Pliant Vasomotor response: Impact of Ca2+ fluxes and Ca2+ Sensitization.

Coordinating blood flow to active tissue requires vasomotor responses to conduct among resistance arteries. Vasomotor spread is governed by the electrical and mechanical properties of vessels; the latter being linked to the sigmoid relations between membrane potential (VM), [Ca2+], and smooth muscle contractility. Proteins guiding electrical-to-tone translation are subject to regulation; thus, vasomotor conduction could be viewed as "pliant" to the current regulatory state. Using simple in silico approaches, we explored vasomotor pliancy and how the regulation of contractility impacts conduction along a skeletal muscle artery and a branching cerebrovascular network. Initial simulations revealed how limited electromechanical linearity affects the translation of electrical spread into arterial tone. Subtle changes to the VM-[Ca2+] or [Ca2+]-diameter relationship, akin to regulatory alterations in Ca2+ influx and Ca2+ sensitivity, modified the distance and amplitude of the conducted vasomotor response. Simultaneous changes to both relationships, consistent with agonist stimulation, augmented conduction although the effect varied with stimulus strength and polarity (depolarization vs hyperpolarization). Final simulations using our cerebrovascular network revealed how localized changes to the VM-[Ca2+] or [Ca2+]-diameter relationships could regionally shape conduction without interfering with the electrical spread. We conclude that regulatory changes to key effector proteins (e.g. L-type Ca2+ channels, myosin light chain phosphatase), integral to voltage translation, not only impact conducted vasomotor tone but likely blood flow delivery to active tissues.

KIR channels in the microvasculature: Regulatory properties and the lipid-hemodynamic environment.

Basal tone and perfusion control is set in cerebral arteries by the sensing of pressure and flow, key hemodynamic stimuli. These forces establish a contractile foundation within arterial networks upon which local neurovascular stimuli operate. This fundamental process is intimately tied to arterial VM and the rise in cytosolic [Ca2+] by the graded opening of voltage-operated Ca2+ channels. Arterial VM is in turn controlled by a dynamic interaction among several resident ion channels, KIR being one of particular significance. As the name suggests, KIR displays strong inward rectification, retains a small outward component, potentiated by extracellular K+ and blocked by micromolar Ba2+. Cerebrovascular KIR is unique from other K+ currents as it is present in both smooth muscle and endothelium yet lacking in classical regulatory modulation. Such observations have fostered the view that KIR is nothing more than a background conductance, activated by extracellular K+ and which passively facilitates dilation. Recent work in cell model systems has; however, identified two membrane lipids, phosphatidylinositol 4,5-bisphosphate (PIP2) and cholesterol, that interact with KIR2.x, to stabilize the channel in the preferred open or silent state, respectively. Translating this unique form of regulation, recent studies have demonstrated that specific lipid-protein interactions enable unique KIR populations to sense distinct hemodynamic stimuli and set basal tone. This review summarizes the current knowledge of vascular KIR channels and how the lipid and hemodynamic impact their activity.

A stepwise approach to resolving small ionic currents in vascular tissue.

Arterial membrane potential (Vm) is set by an active interplay among ion channels whose principal function is to set contractility through the gating of voltage-operated Ca2+ channels. To garner an understanding of this electrical parameter, the activity of each channel must be established under near-physiological conditions, a significant challenge given their small magnitude. The inward rectifying K+ (KIR) channel is illustrative of the problem, as its outward "physiological" component is almost undetectable. This study describes a stepwise approach to dissect small ionic currents at physiological Vm using endothelial and smooth muscle cells freshly isolated from rat cerebral arteries. We highlight three critical steps, beginning with the voltage clamping of vascular cells bathed in physiological solutions while maintaining a giga-ohm seal. KIR channels are then inhibited (micromolar Ba2+) so that a difference current can be created, once Ba2+ traces are corrected for the changing seal resistance and subtle instrument drift, pulling the reversal potential rightward. The latter is a new procedure and entails the alignment of whole cell current traces at a voltage where KIR is silent and other channels exhibit limited activity. We subsequently introduced corrected and uncorrected currents into computer models of the arterial wall to show how these subtle adjustments markedly impact the importance of KIR in Vm and arterial tone regulation. We argue that this refined approach can be used on an array of vascular ion channels to build a complete picture of how they dynamically interact to set arterial tone in key organs like the brain.NEW & NOTEWORTHY This work describes a stepwise approach to resolve small ionic currents involved in controlling Vm in resistance arteries. Using this new methodology, we particularly resolved the outward component of the KIR current in native vascular cells, voltage clamped in near-physiological conditions. This novel approach can be applied to any other vascular currents and used to better interpret how vascular ion channels cooperate to control arterial tone.

Intercellular Conduction Optimizes Arterial Network Function and Conserves Blood Flow Homeostasis During Cerebrovascular Challenges.

OBJECTIVE: Cerebral arterial networks match blood flow delivery with neural activity. Neurovascular response begins with a stimulus and a focal change in vessel diameter, which by themselves is inconsequential to blood flow magnitude, until they spread and alter the contractile status of neighboring arterial segments. We sought to define the mechanisms underlying integrated vascular behavior and considered the role of intercellular electrical signaling in this phenomenon. Approach and Results: Electron microscopic and histochemical analysis revealed the structural coupling of cerebrovascular cells and the expression of gap junctional subunits at the cell interfaces, enabling intercellular signaling among vascular cells. Indeed, robust vasomotor conduction was detected in human and mice cerebral arteries after focal vessel stimulation: a response attributed to endothelial gap junctional communication, as its genetic alteration attenuated this behavior. Conducted responses were observed to ascend from the penetrating arterioles, influencing the contractile status of cortical surface vessels, in a simulated model of cerebral arterial network. Ascending responses recognized in vivo after whisker stimulation were significantly attenuated in mice with altered endothelial gap junctional signaling confirming that gap junctional communication drives integrated vessel responses. The diminishment in vascular communication also impaired the critical ability of the cerebral vasculature to maintain blood flow homeostasis and hence tissue viability after stroke. CONCLUSIONS: Our findings highlight the integral role of intercellular electrical signaling in transcribing focal stimuli into coordinated changes in cerebrovascular contractile activity and expose, a hitherto unknown mechanism for flow regulation after stroke.

Membrane Lipid-KIR2.x Channel Interactions Enable Hemodynamic Sensing in Cerebral Arteries.

Objective- Inward rectifying K+ (KIR) channels are present in cerebral arterial smooth muscle and endothelial cells, a tandem arrangement suggestive of a dynamic yet undiscovered role for this channel. This study defined whether distinct pools of cerebral arterial KIR channels were uniquely modulated by membrane lipids and hemodynamic stimuli. Approach and Results- A Ba2+-sensitive KIR current was isolated in smooth muscle and endothelial cells of rat cerebral arteries; molecular analyses subsequently confirmed KIR2.1/KIR2.2 mRNA and protein expression in both cells. Patch-clamp electrophysiology next demonstrated that each population of KIR channels was sensitive to key membrane lipids and hemodynamic stimuli. In this regard, endothelial KIR was sensitive to phosphatidylinositol 4,5-bisphosphate content, with depletion impairing the ability of laminar shear stress to activate this channel pool. In contrast, smooth muscle KIR was sensitive to membrane cholesterol content, with sequestration blocking the ability of pressure to inhibit channel activity. The idea that membrane lipids help confer shear stress and pressure sensitivity of KIR channels was confirmed in intact arteries using myography. Virtual models integrating structural/electrical observations reconceptualized KIR as a dynamic regulator of membrane potential working in concert with other currents to set basal tone across a range of shear stresses and intravascular pressures. Conclusions- The data show for the first time that specific membrane lipid-KIR interactions enable unique channel populations to sense hemodynamic stimuli and drive vasomotor responses to set basal perfusion in the cerebral circulation.

An assessment of KIR channel function in human cerebral arteries.

In the rodent cerebral circulation, inward rectifying K+ (KIR) channels set resting tone and the distance over which electrical phenomena spread along the arterial wall. The present study sought to translate these observations into human cerebral arteries obtained from resected brain tissue. Computational modeling and a conduction assay first defined the impact of KIR channels on electrical communication; patch-clamp electrophysiology, quantitative PCR, and immunohistochemistry then characterized KIR2.x channel expression/activity. In keeping with rodent observations, computer modeling highlighted that KIR blockade should constrict cerebral arteries and attenuate electrical communication if functionally expressed. Surprisingly, Ba2+ (a KIR channel inhibitor) had no effect on human cerebral arterial tone or intercellular conduction. In alignment with these observations, immunohistochemistry and patch-clamp electrophysiology revealed minimal KIR channel expression/activity in both smooth muscle and endothelial cells. This absence may be reflective of chronic stress as dysphormic neurons, leukocyte infiltrate, and glial fibrillary acidic protein expression was notable in the epileptic cortex. In closing, KIR2.x channel expression is limited in human cerebral arteries from patients with epilepsy and thus has little impact on resting tone or the spread of vasomotor responses. NEW & NOTEWORTHY KIR2.x channels are expressed in rodent cerebral arterial smooth muscle and endothelial cells. As they are critical to setting membrane potential and the distance signals conduct, we sought to translate this work into humans. Surprisingly, KIR2.x channel activity/expression was limited in human cerebral arteries, a paucity tied to chronic brain stress in the epileptic cortex. Without substantive expression, KIR2.x channels were unable to govern arterial tone or conduction.

Caveolae Link CaV3.2 Channels to BKCa-Mediated Feedback in Vascular Smooth Muscle.

Objective- This study examined whether caveolae position CaV3.2 (T-type Ca2+ channel encoded by the α-3.2 subunit) sufficiently close to RyR (ryanodine receptors) for extracellular Ca2+ influx to trigger Ca2+ sparks and large-conductance Ca2+-activated K+ channel feedback. Approach and Results- Using smooth muscle cells from mouse mesenteric arteries, the proximity ligation assay confirmed that CaV3.2 reside within 40 nm of caveolin 1, a key caveolae protein. Methyl-ß-cyclodextrin, a cholesterol depleting agent that disrupts caveolae, suppressed CaV3.2 activity along with large-conductance Ca2+-activated K+-mediated spontaneous transient outward currents in cells from C57BL/6 but not CaV3.2-/- mice. Genetic deletion of caveolin 1, a perturbation that prevents caveolae formation, also impaired spontaneous transient outward current production but did so without impairing Ca2+ channel activity, including CaV3.2. These observations indicate a mistargeting of CaV3.2 in caveolin 1-/- mice, a view supported by a loss of Ni2+-sensitive Ca2+ spark generation and colocalization signal (CaV3.2-RyR) from the proximity ligation assay. Vasomotor and membrane potential measurements confirmed that cellular disruption of the CaV3.2-RyR axis functionally impaired the ability of large-conductance Ca2+-activated K+ to set tone in pressurized caveolin 1-/- arteries. Conclusions- Caveolae play a critical role in protein targeting and preserving the close structural relationship between CaV3.2 and RyR needed to drive negative feedback control in resistance arteries.