Summary
Recent investigations have identified that T-type Ca2+ channels (Ca V 3.x) are expressed in rat cerebral arterial smooth muscle. In the study reported here, we isolated the T-type conductance, differentiated the current into the Ca V 3.1/Ca V 3.2 subtypes and determined whether they are subject to protein kinase regulation. Using patch clamp electrophysiology, whole-cell Ba 2+ current was monitored and initially subdivided into nifedipine-sensitive and -insensitive components. The latter conductance was abolished by T-type Ca 2+ channel blockers and was faster with leftward shifted activation/inactivation properties, reminiscent of a T-type channel. Approximately 60% of this T-type conductance was blocked by 50 mM Ni 2+ , a concentration that selectively interferes with Ca V 3.2 channels. Subsequent work revealed that the whole-cell T-type conductance was subject to protein kinase A (PKA) modulation. Specifically, positive PKA modulators (db-cAMP, forskolin, isoproterenol) suppressed T-type currents and evoked a hyperpolarized shift in steady-state inactivation. Blocking PKA (with KT5720) masked this suppression without altering the basal T-type conductance. A similar effect was observed with stHt31, a peptide inhibitor of A-kinase anchoring proteins. A final set of experiments revealed that PKA-induced suppression targeted the Ca V 3.2 subtype. In summary, this study revealed that a T-type Ca 2+ channel conductance can be isolated in arterial smooth muscle, and differentiated into Ca V 3.1 and Ca V 3.2 components. It also showed that vasodilatory signaling cascades inhibit this conductance by targeting Ca V 3.2. Such targeting would impact Ca 2+ dynamics and consequent tone regulation in the cerebral circulation.