Calmodulin Interactions with Erythrocyte Membranes

Two new vascular smooth muscle relaxants, bepridil and cetiedil, were found to possess specific CaM-inhibitory properties which resembled those of trifluoperazine. Trifluoperazine, bepridil, and cetiedil inhibited Ca2+-dependent '251-CaM binding to erythrocyte membranes and CaM activation of membrane Ca2+-ATPase with IC50 values of -12, 17, and -40 MAM, respectively. This does not appear to be the result of a nonspecific hydrophobic interaction since inhibition was not observed with micromolar concentrations of many other hydrophobic agents. The predominant inhibition of binding and Ca2+-ATPase activation was competitive with respect to CaM. Bepridil and cetiedil bind directly to CaM since these drugs displaced [3H]trifluoperazine from sites on CaM. Inhibition of Ca2+-ATPase and binding by the drugs was not due to interference with the catalytic activity of this enzyme since: (a) neither inhibition of CaM-independent basal Ca2+-ATPase activity nor inhibition of proteolyticallyactivated Ca2+-ATPase activities were produced by these agents, and (b) no drug-induced inhibition of CaM binding was detected when membranes were preincubated with these agents but washed prior to addition of I25I-CaM. Thus, bepridil and cetiedil competitively inhibit Ca2+_ Dr. Agre is the recipient of a Clinical Investigator Award from the National Heart, Lung, and Blood Institute. Dr. Bennett is the recipient of a Research Career Development Award from the National Heart, Lung, and Blood Institute. Dr. Virshup is a member of the Pediatric House Staff at Johns Hopkins Hospital. Address reprint requests to Dr. Bennett, Department of Cell Biology and Anatomy, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205. Received for publication 17 August 1983 and in revised form 3 May 1984. dependent interactions of CaM with erythrocyte membranes, most likely by a direct interaction between these drugs and CaM. The principal clinical actions of these drugs may be explained by their interactions with CaM or CaM-related proteins leading to reduced activation of Ca2"-regulated enzymes in certain other tissues, such as myosin light chain kinase in vascular smooth muscle.