Cooperative interaction between Ca2+ binding sites in the hydrophilic loop of the Na+-Ca2+ exchanger

A high affinity Ca²⁺-binding domain which is located in a middle portion of the large intracellular loop of the Na⁺-Ca²⁺ exchanger contains two highly acidic sequences, each characterized by three consecutive aspartic acid residues (Levitsky DO, Nicoll DA, and Philipson KD (1994) J Biol Chem 269: 22847–22852). This portion of the protein provides secondary Ca²⁺ regulation of the exchanger activity. To determine number of Ca²⁺ binding sites participating in formation of the high affinity domain, we isolated polypeptides of different lengths encompassing the domain and measured ⁴⁵Ca²⁺ binding. The fusion proteins containing 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 dialysis reached saturation at 1 M free Ca²⁺, Kd value being approx. 0.4 M. The Ca²⁺ binding occured in a highly cooperative manner. Upon saturation, the amount of Ca²⁺ ion bound varied from 1.3–2.1 mot per mot protein. Proteins with an aspartate in each acidic sequence mutated lacked the positive cooperativity, had lower Ca²⁺ affinity and bound two to three times less Ca²⁺. Na⁺-Ca²⁺ exchangers of tissues other than heart though different from the cardiac exchanger by molecular weight most likely possess a similar Ca²⁺ binding site. It is concluded that, by analogy with Ca²⁺ binding proteins of EF-type, the high Ca²⁺-affinity domain of the Na⁺-Ca²⁺ exchanger is comprised of at least two binding sites interacting cooperatively.