Ca2+ binding to sarcoplasmic reticulum ATPase revisited. I. Mechanism of affinity and cooperativity modulation by H+ and Mg2+

Abstract

H+ and Mg2+ are known to inhibit Ca2+ binding to the transport sites of sarcoplasmic reticulum-ATPase. Evaluation of the affinity for the Ca2+ binding sites requires measurement of the amount of Ca2+ bound to ATPase as a function of the free Ca2+ concentration imposed by a Ca2+ chelator. The choice of the chelator is crucial as it determines the accuracy of the free Ca2+ concentration. At pH > 7, the EGTA affinity for Ca2+ is higher than that of ATPase, inducing artifacts that alter the shape of the binding curves. Thus, we have used 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), whose affinity is unchanged at pH > or = 7. Ca2+ binding was studied at equilibrium, from pH 6 to pH 8 and from 0 to 10 mM Mg2+, using EGTA and/or BAPTA and [45Ca]Ca2+. Under all conditions, the stoichiometry was 2 Ca2+/ATPase. At variance with previous studies, the Hill coefficient was 1.1-2 and higher at pH 6 than at pH 8. In addition, it decreased in the presence of Mg2+. The Ca2+ binding curves were analyzed according to a model in which they result from a sequential binding of two Ca2+, each binding step being modified by H+ and Mg2+. The effect of H+ is described by two steps involving two H+ and one H+, with pK 7 and 7.9, respectively. At pH 6, ATPase must lose two H+ for the first Ca2+ to bind and a third H+ for the second Ca2+ to bind. At pH 9, both Ca2+ bind without any H+ exchange. Mg2+ can bind to all species, except to that saturated with Ca2+. The species having lost two H+ has a higher affinity for Mg2+ (< or = 1 mM) than the species having bound three H+ (4 mM). The above model allows us to analyze the effects of H+ and Mg2+ at each Ca2+ binding step and to explain the changes in the apparent affinity and cooperativity.