Ca(V)3.2 channels and the induction of negative feedback in cerebral arteries.

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.

Identification of L- and T-type Ca2+ channels in rat cerebral arteries: role in myogenic tone development.

L-type Ca(2+) channels are broadly expressed in arterial smooth muscle cells, and their voltage-dependent properties are important in tone development. Recent studies have noted that these Ca(2+) channels are not singularly expressed in vascular tissue and that other subtypes are likely present. In this study, we ascertained which voltage-gated Ca(2+) channels are expressed in rat cerebral arterial smooth muscle and determined their contribution to the myogenic response. mRNA analysis revealed that the α(1)-subunit of L-type (Ca(v)1.2) and T-type (Ca(v)3.1 and Ca(v)3.2) Ca(2+) channels are present in isolated smooth muscle cells. Western blot analysis subsequently confirmed protein expression in whole arteries. With the use of patch clamp electrophysiology, nifedipine-sensitive and -insensitive Ba(2+) currents were isolated and each were shown to retain electrical characteristics consistent with L- and T-type Ca(2+) channels. The nifedipine-insensitive Ba(2+) current was blocked by mibefradil, kurtoxin, and efonidpine, T-type Ca(2+) channel inhibitors. Pressure myography revealed that L-type Ca(2+) 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(2+) 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(2+) blockade could alter arterial flow by ∼20-50%. Overall, our findings indicate that L- and T-type Ca(2+) 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.