Electrophysiological and molecular characteristics of voltage-dependent calcium (Ca2+) channels were studied using whole-cell patch clamp, polymerase chain reaction and Western blotting in smooth muscle cells freshly isolated from dog basilar artery. Inward currents evoked by depolarizing steps from a holding potential of -50 or -90 mV in 10 m m barium consisted of low- (LVA) and high-voltage activated (HVA) components. LVA current comprised more than half of total current in 24 (12%) of 203 cells and less than 10% of total current in 52 (26%) cells. The remaining cells (127 cells, 62%) had LVA currents between one tenth and one half of total current. LVA current was rapidly inactivating, slowly deactivating, inhibited by high doses of nimodipine and mibefradil (> 0.3 μm), not affected by ω-agatoxin GVIA (γ100 nm), ω-conotoxin IVA (1 μm) or SNX-482 (200 nm) and probably carried by T-type Ca2+ channels based on the presence of messenger ribonucleic acid (mRNA) and protein for Cav3.1 and Cav3.3 α 1 subunits of these channels. LVA currents exhibited window current with a maximum of 13% of the LVA current at -37.4 mV. HVA current was slowly inactivating and rapidly deactivating. It was inhibited by nimodipine (IC50 = 0.018 μm), mibefradil (IC50 = 0.39 μm) and ω-conotoxin IV (1 μm). Smooth muscle cells also contained mRNA and protein for L- (Cav1.2 and Cav1.3), N- (Cav2.2) and T-type (Cav3.1 and Cav3.3) α1 Ca2+ channel subunits. Confocal microscopy showed Cav1.2 and Cav1.3 (L-type), Cav2.2 (N-type) and Cav3.1 and Cav3.3 (T-type) protein in smooth muscle cells. Relaxation of intact arteries under isometric tension in vitro to nimodipine (1 μM) and mibefradil (1 μM) but not to ω-agatoxin GVIA (100 nm), ω-conotoxin IVA (1 μM) or SNX-482 (1 μM) confirmed the functional significance of L- and T-type voltage-dependent Ca2+ channel subtypes but not N-type. These results show that dog basilar artery smooth muscle cells express functional voltage-dependent Ca2+ channels of multiple types.
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