Molecular Pharmacology, Vol 19, 264-269, Copyright © 1981 by the American Society for Pharmacology and Experimental Therapeutics

Photoaffinity Labeling of Calmodulin by Phenothiazine Antipsychotics

¹ Department of Pharmacology, Medical College of Pennsylvania, Philadelphia, Pennsylvania 19129

Previous studies have shown that the binding of radiolabeled phenothiazine antipsychotics to calmodulin can be reversed by removing calcium or by dialyzing against an excess of nonradiolabeled phenothiazine. The present studies show that the binding of [³H]-chlorpromazine or [³H]trifiuoperazine to calmodulin can be made irreversible by irradiating the samples with UV light. Like the reversible binding of these agents to calmodulin, the irreversible binding was enhanced by calcium. This calcium-dependent binding was saturable, with approximately one binding site per molecule of calmodulin; one-half maximal binding occurred at a chlorpromazine concentration of approximately 10 µM and at a trifluoperazine concentration of 5 µM. The irreversible binding of chlorpromazine to calmodulin probably involves the formation of a covalent bond, since extensive dialysis and treatment with denaturing agents failed to displace the bound chlorpromazine from calmodulin. Chlorpromazine displayed little or no irreversible calcium-dependent binding to bovine serum albumin, trypsin, histone, or hemoglobin. Nonphenothiazine antipsychotics, such as penfluridol and haloperidol, and a number of other centrally active drugs, such as diazepam, apomorphine, and dopamine, showed little or no irreversible binding to calmodulin. However, the nonphenothiazine antipsychotics did block the irreversible binding of chlorpromazine and trifluoperazine to calmodulin. Psychotropic drugs with little antipsychotic activity did not prevent the irreversible binding of the phenothiazines. UV-irradiated samples of calmodulin and chlorpromazine or trifluoperazine failed to activate phosphodiesterase even after extensive dialysis, indicating that the irreversible binding of the phenothiazines to calmodulin results in an irreversible inhibition of the biological activity of calmodulin. These results suggest that the irreversible binding of antipsychotics to calmodulin may account for some of the long-term actions of these drugs and may provide a means with which to study the location and turnover of this calcium-binding protein.

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ACKNOWLEDGMENTS We thank Ms. Becky Simon for her excellent technical assistance and Ms. Josie Weycis for her help with the manuscript.