Abstract

A calmodulin-binding assay was established in rat striatal particulates which were depleted of endogenous calcium and calmodulin by ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) treatment. The binding of 125I-labeled calmodulin to this preparation was saturable and time-dependent. The dependence of calmodulin binding upon temperature and concentration was also demonstrated. Calcium was a prerequisite for calmodulin binding and it facilitated the binding in a dose-dependent manner. Lanthanum, a known calcium antagonists in other tissue systems, mimicked the effect of calcium on calmodulin binding. When both ions were present at low concentrations, their effects on calmodulin binding was additive. Lanthanum, but not calcium, inhibited calmodulin release from a non-EGTA-treated preparation. This difference in action between calcium and lanthanum suggests that they mediate calmodulin binding in an independent manner. Scatchard analysis of calcium-calmodulin binding to rat striatal particulates revealed that there are two populations of binding sites: a higher affinity (apparent KD = 1.3 X 10(-7) M) and a lower affinity (apparent KD = 2.9 X 10(-7) M) binding site. Trifluoperazine, a phenothiazine antipsychotic drug, at 10(-4) M antagonized calmodulin binding only at the higher affinity binding sites. These sites may play an important role in mediating the action of trifluoperazine in the caudate nucleus.