Interaction of quaternary phenothiazine salts with calmodulin

To clarify the role of electrostatic forces in the binding of phenothiazines to calmodulin, the quaternary methiodide salts of several 10-alkylaminophenothiazines were prepared and examined for anticalmodulin activity. In addition, the methiodide salt of [3H]chlorpromazine was prepared and its binding to calmodulin was studied. Both the hydrochloride and methiodide salts of a variety of 10-alkylaminophenothiazines inhibited the calmodulin-induced activation of phosphodiesterase at concentrations that had no effect on the basal activity of the enzyme. The IC50 values for the inhibition of calmodulin by the methiodide salts were similar to those of the corresponding hydrochloride salts, with the compounds displaying the order of potency: trifluopromazine greater than chlorpromazine greater than promazine greater than promethazine greater than chlorpromazine sulfoxide. The acidic compound 3-(2-chloro-10-phenothiazinyl)propionic acid displayed no anticalmodulin activity at concentrations up to 80 microM; at higher concentrations the compound inhibited both basal and calmodulin-stimulated enzyme activity. Other results showed that both chlorpromazine hydrochloride and chlorpromazine methiodide bound to calmodulin in a Ca++-dependent manner. However, the binding of chlorpromazine hydrochloride decreased markedly at alkaline pHs, whereas the binding of chlorpromazine methiodide was not pH-dependent. The fact that the binding of the nondissociable methiodide salt is not pH-dependent, whereas the binding of the hydrochloride salt decreases at pHs at which the drug would be nonionized, indicates that it is the cationic species of the phenothiazine that binds to calmodulin and inhibits its activity.