NOVEL EFFECTS OF CALMODULIN AND CALMODULIN ANTAGONISTS ON THE PLASMA-MEMBRANE (CA2+ + MG2+)-ATPASE FROM RABBIT KIDNEY PROXIMAL TUBULES

In this work we report an unusual pattern of activation by calmodulin on the (Ca2+ + Mg2+)-ATPase from basolateral membranes of kidney proximal tubule cells. The activity of the ATPase depleted to calmodulin is characterized by a high Ca2+ affinity (K(m) = 2.2-3.4-mu-M) and a biphasic dependence on ATP concentration. The preparation responded to the addition of calmodulin by giving rise to a new Ca2+ site of very high affinity (K(m) < 0.05-mu-M). Calmodulin antagonists had diverse effects on ATPase activity. Compound 48/80 inhibited calmodulin-stimulated activity by 70%, whereas calmidazolium did not modify this component. In the absence of calmodulin, 48/80 still acted as an antagonist, increasing the K(m) for Ca2+ to 5.7-mu-M and reducing enzyme turnover by competing with ATP at the low affinity regulatory site. Calmidazolium did not affect Ca2+ affinity, but it did displace ATP from the regulatory site. At fixed Ca2+ (30-mu-M) and ATP (5 mM) concentrations, P(i) protected against 48/80 and potentiated inhibition by calmidazolium. At 25-mu-M ATP, P(i) protected against calmidazolium inhibition. We propose that the effects of ATP and P(i) arise because binding of the drugs to the ATPase occurs mainly on the E2 forms.