Voltage-dependent calcium channels of dog basilar artery

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 mM 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 mu M), not affected by omega-agatoxin GVIA (gamma 100 nM), omega-conotoxin IVA (1 mu M) 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 Ca-v3.1 and Ca-v3.3 alpha(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 mu M), mibefradil (IC50 = 0.39 mu M) and omega-conotoxin IV (1 mu M). Smooth muscle cells also contained mRNA and protein for L- (Ca-v1.2 and Ca-v1.3), N- (Ca-v2.2) and T-type (Ca-v3.1 and Ca-v3.3) alpha(1) Ca2+ channel subunits. Confocal microscopy showed Ca-v1.2 and Ca-v1.3 (L-type), Ca-v2.2 (N-type) and Ca-v3.1 and Ca-v3.3 (T-type) protein in smooth muscle cells. Relaxation of intact arteries under isometric tension in vitro to nimodipine (1 mu M) and mibefradil (1 mu M) but not to omega-agatoxin GVIA (100 nM), omega-conotoxin IVA (1 mu M) or SNX-482 (1 mu M) 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.