Calcium channel blockers inhibit the (Ca 2+ + Mg 2+)-ATPase activity and the 125I-calmodulin binding in brain membranes

Abstract

Ca 2+ channel blockers belongingn to three distinct chemical groups (dihydropyridines, phenylalkylamines and diphenylalkylamines) differentially inhibit the (Ca 2+ + Mg 2+)-ATPase activity of synaptic plasma membranes (Santos et al., J. Neurochem. 52, S49D, 1989). We now report that (−)-desmethoxyverapamil and flunarizine are the most potent inhibitors of the Ca 2+-activated ATPase activity of synaptic plasma membranes, decreasing the V max by 41% and 37%, respectively, with no significant effects on the K m for Ca 2+ (162.7 ± 14.9 nM free [Ca 2+]), while nitrendipine did not affect these parameters. Trifluoperazine was the most potent inhibitor of the Ca 2+-activated ATPase of synaptic plasma membranes with an IC 50 of 8–10 μM. To clarify whether the inhibitory effects of Ca 2+ channel blockers and of trifluoroperazine on the (Ca 2+ + Mg 2+)-ATPase occur through the inhibition of the interaction of calmodulin with the enzyme, we studied their effects on the binding of 125-calmodulin to the membrane proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by electrotransfer to nitrocellulose and autoradiography. The autoradiograms revealed Ca 2+-dependent CaM binding proteins of about 140, 70 and 55 kDa. Trifluoperazine (30–40 μM) inhibited by 50–60% the binding of 125I-calmodulin to the 140 kDa band, which probably includes the (Ca 2+ + Mg 2+)-ATPase protein. Flunarizine and (−)-desmethoxyverapamil (100 μM) inhibited the 125I-calmodulin binding to the 140 kDa peptides by 100 and 90%, respectively, and they inhibited by 55 and 40%, respectively, the binding of 125I-calmodulin to the peptides in the 70−55 kDa range, whereas nitrendipine did not show any effect. The results suggest that the inhibitory effects of (−)-desmethoxyverapamil and flunarizine, as well as trifluoperazine, on the (Ca 2+ + Mg 2+)-ATPase activity of synaptic plasma membranes are mediated by inhibition of the calmodulin interaction with the enzyme.