Metal ions as allosteric regulators of calmodulin.

Previously we have shown that the fluorescence of the dihydropyridine calcium antagonist felodipine provides an accurate means of monitoring the formation of an allosterically potentiated conformer of calmodulin (Mills, J. S., and Johnson, J. D., (1985) Biochemistry 24, 4897-4903). Characteristic of this conformer is the abolition of cooperativity among the two felodipine-binding sites on calmodulin and a 20-fold increase in the apparent affinity of calmodulin for felodipine. In the present study, we find that the metal cations La3+, Tb3+, Pb2+, and Cd2+ are all capable of abolishing the cooperativity (Hill coefficient = 2.0) among the two felodipine-binding sites on calmodulin and can increase the apparent affinity of calmodulin for felodipine by approximately 20-fold. These effects are seen either in the presence or absence of calcium and are half-maximal at 8, 12, 22, and 1000 microM, respectively. Zinc and H+ are capable of producing similar potentiations of felodipine binding (half-maximal at 570 microM, and pH 5.8), but only in the presence of calcium. In each case, the calcium-binding sites of calmodulin must be occupied (by calcium, La3+, Tb3+, Pb2+, or by Cd2+) before these metals can bind to sites which are distinct from the calcium-binding sites to produce the active conformer of calmodulin which exhibits enhanced affinity for felodipine. Mercury and copper can compete with these potentiating metal cations on calmodulin and produce an inactivation of this active calmodulin conformer. These studies suggest that some metals including La3+, Tb3+, Pb2+, Cd2+, Zn2+ and protons are capable of binding to a calcium-calmodulin complex and forming an allosterically active species of calmodulin which cannot be maintained by physiological concentrations of calcium ions alone. Mercury and copper, on the other hand, are capable of inactivating this active calmodulin conformer independent of the presence of calcium on calmodulin. These findings are examined in terms of the mechanism of action of calmodulin and its possible role in heavy metal toxicity.