Rationale T-type (CaV3.1/CaV3.2) 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 CaV3.2 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 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 Ca2+ influx through CaV3.2 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 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 Ca2+ sparks, enabling large-conductance Ca2+-activated K+ channel activation, hyperpolarization, and attenuation of cerebral arterial constriction.

channel inhibition reduced tone at 20 and 80 mmHg, with the greatest effect at high pressure when the vessel is depolarized. In comparison, the effect of T-type Ca 2ϩ channel blockade on myogenic tone was more limited, with their greatest effect at low pressure where vessels are hyperpolarized. Blood flow modeling revealed that the vasomotor responses induced by T-type Ca 2ϩ blockade could alter arterial flow by ϳ20 -50%. Overall, our findings indicate that L-and T-type Ca 2ϩ channels are expressed in cerebral arterial smooth muscle and can be electrically isolated from one another. Both conductances contribute to myogenic tone, although their overall contribution is unequal. influx from the extracellular space (9). Voltage-gated Ca 2ϩ channels are the principal conductances that regulate extracellular Ca 2ϩ influx. These membrane channels are hetero-oligomeric complexes that comprise a pore-forming ␣ 1 -subunit and accessory proteins that influence gating characteristics and protein trafficking (24). The ␣ 1 -subunit is composed of four domains, each of which contain six transmembrane segments, a S4 voltage sensor, and a P loop that confers ion selectivity (21, 50). Molecular studies have identified three classes of ␣ 1 -subunits (Ca v 1-3), and within each category there are several subtypes. Ca v 1/Ca v 2 subunits display electrical properties characteristic of high voltage-activated Ca 2ϩ channels (i.e., L-, P/Q-, N-, and R types) (5). In contrast, Ca v 3 subunits encode for Ca 2ϩ channels activated by lower voltages (i.e., T type) (20,34 channels was more limited and best observed at lower pressures in hyperpolarized vessels. Although the contribution of the channels to tone development is limited, computational