16] Techniques for measuring the interaction of drugs with calmodulin

Abstract

Two general types of methods can be used to assess the interaction of drugs with calmodulin: (1) determining the effects of drugs on specific calmodulin-dependent processes and (2) measuring the binding of drugs to calmodulin. This latter procedure can be affected by directly measuring the binding of radiolabeled compounds to calmodulin, or, if the radiolabeled compound is unavailable, by determining whether the compound under study can displace a radiolabeled ligand from calmodulin. The effects of drugs on calmodulin activity can be accessed from their ability to inhibit the calmodulin-sensitive form of phosphodiesterase. This form of the enzyme can be isolated from the brain by preparative polyacrylamide disc gel electrophoresis. Investigations into the mechanism by which phenothiazine antipsychotic drugs inhibit calmodulin-dependent phosphodiesterase revealed that the drugs did not interact with phosphodiesterase itself as do many other phosphodiesterase inhibitors, but rather they interacted with the activator of phosphodiesterase. The studies of the characteristics of this interaction showed that the phenothiazines bound directly to calmodulin in a reversible, saturable, calcium-binding manner.In addition, this binding was relatively selective for calmodulin becausethe drugs showed little or no calcium-dependent binding to a variety of other proteins, including several calcium-binding proteins with substantial structural homology with calmodulin.