Ca V 3.2 Channels and the Induction of Negative Feedback in Cerebral Arteries

Abstract

Rationale: T-type (Ca V 3.1/Ca V 3.2) 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 Ca V 3.2 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 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 Ca 2+ influx through Ca V 3.2 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 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 Ca 2+ sparks, enabling large-conductance Ca 2+ -activated K + channel activation, hyperpolarization, and attenuation of cerebral arterial constriction.