Abstract
Electrophysiological and molecular characteristics of voltage-dependent calcium (Ca(2+)) 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 microM), not affected by omega-agatoxin GVIA (gamma100 nM), omega-conotoxin IVA (1 microM) or SNX-482 (200 nM) and probably carried by T-type Ca(2+) 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 (IC(50) = 0.018 microM), mibefradil (IC(50) = 0.39 microM) and omega-conotoxin IV (1 microM). 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) Ca(2+) 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 microM) and mibefradil (1 microM) but not to omega-agatoxin GVIA (100 nM), omega-conotoxin IVA (1 microM) or SNX-482 (1 microM) confirmed the functional significance of L- and T-type voltage-dependent Ca(2+) channel subtypes but not N-type. These results show that dog basilar artery smooth muscle cells express functional voltage-dependent Ca(2+) channels of multiple types.