CaV3.2 Channels and the Induction of Negative Feedback in Cerebral Arteries

Rationale: T-type (Ca(V)3.1/Ca(V)3.2) 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 Ca(V)3.2 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 Ca(V)3.2 but not Ca(V)1.2/Ca(V)3.1, was first shown to depolarize/constrict pressurized rat cerebral arteries; no effect was observed in Ca(V)3.2(-/-) 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, Ca(V)3.2 and ryanodine receptor resided in close apposition to one another. Computational modeling revealed that Ca2+ influx through Ca(V)3.2 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 Ca(V)3.2 is present and drives a comparable response to moderate constriction. Conclusions: These findings indicate for the first time that Ca(V)3.2 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.