Calcium channel blockers inhibit the (Ca2+ + Mg2+)-ATPase activity and the 125I-calmodulin binding in brain membranes.
Ca2+ channel blockers belonging to three distinct chemical groups (dihydropyridines, phenylalkylamines and diphenylalkylamines) differentially inhibit the (Ca2+ + Mg2+)-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 Vmax by 41% and 37%, respectively, with no significant effects on the Km for Ca2+ (162.7 +/- 14.9 nM free [Ca2+]), 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 IC50 of 8-10 microM. To clarify whether the inhibitory effects of Ca2+ channel blockers and of trifluoperazine on the (Ca2+ + Mg2+)-ATPase occur through the inhibition of the interaction of calmodulin with the enzyme, we studied their effects on the binding of 125I-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 microM) inhibited by 50-60% the binding of 125I-calmodulin to the 140 kDa band, which probably includes the (Ca2+ + Mg2+)-ATPase protein. Flunarizine and (-)-desmethoxyverapamil (100 microM) 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 (Ca2+ + Mg2+)-ATPase activity of synaptic plasma membranes are mediated by inhibition of the calmodulin interaction with the enzyme.
Santos
D L
DL
Department of Biology, University of Trás-os-Montes and Alto Douro, Vila Real, Portugal.
Lopes
M C
MC
Carvalho
C M
CM
D000818:Animals; D001345:Autoradiography; D001665:Binding Sites / Q000187:drug effects; D001921:Brain / Q000187:drug effects* / Q000378:metabolism; D017301:Ca(2+) Mg(2+)-ATPase / Q000037:antagonists & inhibitors*; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D002147:Calmodulin / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005444:Flunarizine / Q000494:pharmacology; D007457:Iodine Radioisotopes; D009568:Nitrendipine / Q000494:pharmacology; D012756:Sheep; D013237:Stereoisomerism; D013570:Synaptic Membranes / Q000187:drug effects* / Q000378:metabolism; D014268:Trifluoperazine / Q000494:pharmacology; D014700:Verapamil / Q000031:analogs & derivatives / Q000494:pharmacology
D002121:Calcium Channel Blockers; D002147:Calmodulin; D007457:Iodine Radioisotopes; D014268:Trifluoperazine; C043184:4-desmethoxyverapamil; D009568:Nitrendipine; D014700:Verapamil; D017301:Ca(2+) Mg(2+)-ATPase; D005444:Flunarizine