Cooperativity among calmodulin's drug binding sites

The binding of felodipine, a dihydropyridine Ca2+ antagonist, to calmodulin has been studied by equilibrium dialysis and fluorescence techniques. Analysis using the Hill equation gives a Hill coefficient of 2. A plot of bound [felodipine] vs. free [felodipine]2 gives a Bmax of 1.9 mol/mol and a K0.5 of 22 microM. Two calmodulin antagonists, prenylamine and R24571, which have previously been shown to potentiate the fluorescent enhancement observed when felodipine binds to calmodulin [Johnson, J. D. (1983) Biochem. Biophys. Res. Commun. 112, 787], produce a reduction in Hill coefficient to 0.7 and 1.0, respectively, and account for the observed potentiation of felodipine binding. Titrations of felodipine with calmodulin in the absence and presence of prenylamine and R24571 suggest that these drugs decrease the K0.5 of calmodulin for felodipine by 25-fold. Thus, potentiating drugs (prenylamine and R24571) bind to either of the two felodipine binding sites and, through an allosteric mechanism, result in felodipine binding to the remaining site with greatly enhanced affinity. Two types of potentiating drugs are observed. Prenylamine exhibits a Hill coefficient of 0.8 whereas felodipine, R24571, and diltiazem exhibit Hill coefficients of 2 in their potentiation of felodipine binding. Titrations of felodipine and calmodulin with Ca2+ exhibit cooperativity with a Hill coefficient of 4. Half-maximal binding occurs near pCa 6.0. In the presence of R24571, the calcium dependence of felodipine binding is biphasic, now exhibiting a much higher affinity (pCa 7.6) component. A model is presented to explain the relationship of these various allosterically regulated conformers of calmodulin and their interactions and activation with its target proteins.