Protein kinase a regulation of T-type Ca2+ channels in rat cerebral arterial smooth muscle

Abstract

Recent investigations have reported that T-type Ca2+ channels (CaV3.x) are expressed in rat cerebral arterial smooth muscle. In this study, we isolate the T-type conductance, differentiate the current into the CaV3.1/CaV3.2 subtypes and determine whether they are subject to protein kinase regulation. Using patch clamp electrophysiology, whole-cell Ba2+ current was monitored and initially subdivided into nifedipine-sensitive and -insensitive components. The latter conductance was abolished by T-type Ca2+ channel blockers and displayed faster and leftward shifted activation/inactivation properties, reminiscent of T-type channel. Approximately 60% of this T-type conductance was blocked by 50 µM Ni2+, a concentration that selectively interferes with CaV3.2 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 (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 CaV3.2 subtype. In closing, this study revealed that a T-type Ca2+ channel conductance can be isolated in arterial smooth muscle, and differentiated into a CaV3.1 and CaV3.2 component. It also showed that vasodilatory signaling cascades inhibit this conductance by targeting CaV3.2. Such targeting will impact Ca2+ dynamics and consequent tone regulation in the cerebral circulation.