Ca_v3.2 channels and the induction of negative feedback in cerebral arteries

Rationale: T-type (Ca_V3.1/Ca_V3.2) 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_V3.2 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_V3.2 but not Ca_V1.2/Ca_V3.1, was first shown to depolarize/constrict pressurized rat cerebral arteries; no effect was observed in Ca_V3.2^-/- 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_V3.2 and ryanodine receptor resided in close apposition to one another. Computational modeling revealed that Ca²⁺ influx through Ca_V3.2 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_V3.2 is present and drives a comparable response to moderate constriction. Conclusions: These findings indicate for the first time that Ca_V3.2 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.