COOPERATIVE INTERACTION BETWEEN CA2-SITES IN THE HYDROPHILIC LOOP OF THE NA+-CA2+ EXCHANGER( BINDING)

A high affinity Ca2+-binding domain which is located in a middle porti on of the large intracellular loop of the Na+-Ca2+ exchanger contains two highly acidic sequences, each characterized by three consecutive a spartic acid residues (Levitsky DO, Nicoll DA, and Philipson KD (1994) J Biol Chem 269: 22847-22852). This portion of the protein provides s econdary Ca2+ regulation of the exchanger activity. To determine numbe r of Ca2+ binding sites participating in formation of the high affinit y domain, we isolated polypeptides of different lengths encompassing t he domain and measured Ca-45(2+) binding. The fusion proteins containi ng the high affinity domain were obtained in a Ca2+-bound form and as evidenced by shifts in there mobility in SDS-polyacrylamide gels after EGTA treatment. The Ca2+ binding curves obtained after equilibrium di alysis reached saturation at 1 mu M free Ca2+, Kd value being approx. 0.4 mu M. The Ca2+ binding occured in a highly cooperative manner. Upo n saturation, the amount of Ca2+ ion bound varied from 1.3-2.1 mol per mol protein. Proteins with an aspartate in each acidic sequence mutat ed lacked the positive cooperativity, had lower Ca2+ affinity and boun d two to three times less Ca2+. Na+-Ca2+ exchangers of tissues other t han heart though different from the cardiac exchanger by molecular wei ght most likely possess a similar Ca2+ binding site. It is concluded t hat, by analogy with Ca2+ binding proteins of EF-type, the high Ca2+-a ffinity domain of the Na+-Ca2+ exchanger is comprised of at least two binding sites interacting cooperatively.