Osama F Harraz1, Rasha R Abd El-Rahman1, Kamran Bigdely-Shamloo1, Sean M Wilson1, Suzanne E Brett1, Monica Romero1, Albert L Gonzales1, Scott Earley1, Edward J Vigmond1, Anders Nygren1, Bijoy K Menon1, Rania E Mufti1, Tim Watson1, Yves Starreveld1, Tobias Furstenhaupt1, Philip R Muellerleile1, David T Kurjiaka1, Barry D Kyle1, Andrew P Braun1, Donald G Welsh2. 1. From the Department of Physiology and Pharmacology, Hotchkiss Brain and Libin Cardiovascular Institutes (O.F.H., R.R.A.E.-R., K.B.-S., S.E.B., R.E.M., B.D.K., A.P.B., D.G.W.), Department of Electrical and Computer Engineering (K.B.-S., E.J.V., A.N.), Department of Clinical Neurosciences (B.K.M., T.W., Y.S.), and Microscopy Imaging Facility (T.F.), University of Calgary, Calgary, Alberta, Canada; Department of Pharmacology and Toxicology, Alexandria University, Alexandria, Egypt (O.F.H.); Division of Pharmacology, Loma Linda University, CA (S.M.W., M.R.); Department of Biomedical Sciences, Colorado State University, Fort Collins (A.L.G.); Department of Pharmacology, University of Nevada, Reno (S.E.); LIRYC Institute and Lab IMB, University of Bordeaux, Bordeaux, France (E.J.V.); and Department of Biomedical Sciences, Grand Valley State University, Allendale, MI (P.R.M., D.T.K.). 2. From the Department of Physiology and Pharmacology, Hotchkiss Brain and Libin Cardiovascular Institutes (O.F.H., R.R.A.E.-R., K.B.-S., S.E.B., R.E.M., B.D.K., A.P.B., D.G.W.), Department of Electrical and Computer Engineering (K.B.-S., E.J.V., A.N.), Department of Clinical Neurosciences (B.K.M., T.W., Y.S.), and Microscopy Imaging Facility (T.F.), University of Calgary, Calgary, Alberta, Canada; Department of Pharmacology and Toxicology, Alexandria University, Alexandria, Egypt (O.F.H.); Division of Pharmacology, Loma Linda University, CA (S.M.W., M.R.); Department of Biomedical Sciences, Colorado State University, Fort Collins (A.L.G.); Department of Pharmacology, University of Nevada, Reno (S.E.); LIRYC Institute and Lab IMB, University of Bordeaux, Bordeaux, France (E.J.V.); and Department of Biomedical Sciences, Grand Valley State University, Allendale, MI (P.R.M., D.T.K.). dwelsh@ucalgary.
Abstract
RATIONALE: T-type (CaV3.1/CaV3.2) Ca(2+) 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 Ca(2+) sparks, discrete events that initiate arterial hyperpolarization by activating large-conductance Ca(2+)-activated K(+) channels. METHODS AND RESULTS: Micromolar Ni(2+), 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 Ca(2+) influx through CaV3.2 could repetitively activate ryanodine receptor, inducing discrete Ca(2+)-induced Ca(2+) release events in a voltage-dependent manner. In keeping with theoretical observations, rapid Ca(2+) imaging and perforated patch clamp electrophysiology demonstrated that Ni(2+) suppressed Ca(2+) sparks and consequently spontaneous transient outward K(+) currents, large-conductance Ca(2+)-activated K(+) channel mediated events. Additional functional work on pressurized arteries noted that paxilline, a large-conductance Ca(2+)-activated K(+) channel inhibitor, elicited arterial constriction equivalent, and not additive, to Ni(2+). 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 Ca(2+) sparks, enabling large-conductance Ca(2+)-activated K(+) channel activation, hyperpolarization, and attenuation of cerebral arterial constriction.
RATIONALE: T-type (CaV3.1/CaV3.2) Ca(2+) 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 Ca(2+) sparks, discrete events that initiate arterial hyperpolarization by activating large-conductance Ca(2+)-activated K(+) channels. METHODS AND RESULTS: Micromolar Ni(2+), 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 Ca(2+) influx through CaV3.2 could repetitively activate ryanodine receptor, inducing discrete Ca(2+)-induced Ca(2+) release events in a voltage-dependent manner. In keeping with theoretical observations, rapid Ca(2+) imaging and perforated patch clamp electrophysiology demonstrated that Ni(2+) suppressed Ca(2+) sparks and consequently spontaneous transient outward K(+) currents, large-conductance Ca(2+)-activated K(+) channel mediated events. Additional functional work on pressurized arteries noted that paxilline, a large-conductance Ca(2+)-activated K(+) channel inhibitor, elicited arterial constriction equivalent, and not additive, to Ni(2+). 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 Ca(2+) sparks, enabling large-conductance Ca(2+)-activated K(+) channel activation, hyperpolarization, and attenuation of cerebral arterial constriction.
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