Dynamic features of allosteric Ca2+ sensor in tissue-specific NCX variants

The Na+–Ca2+ exchanger (NCX) mediated Ca2+ fluxes are essential for handling Ca2+ homeostasis in many cell-types. Eukaryotic NCX variants contain regulatory CBD1 and CBD2 domains, whereas in distinct variants the Ca2+ binding to Ca3–Ca4 sites of CBD1 results either in sustained activation, inhibition or no effect. CBD2 contains an alternatively spliced segment, which is expressed in a tissue-specific manner although its impact on allosteric regulation remains unclear. Recent studies revealed that the Ca2+ binding to Ca3–Ca4 sites results in interdomain tethering of CBDs, which rigidifies CBDs movements with accompanied slow dissociation of “occluded” Ca2+. Here we investigate the effects of CBD2 variants on Ca2+ occlusion in the two-domain construct (CBD12). Mutational studies revealed that both sites (Ca3 and Ca4) contribute to Ca2+ occlusion, whereas after dissociation of the first Ca2+ ion the second Ca2+ ion becomes occluded. This mechanism is common for the brain, kidney and cardiac splice variants of CBD12, although the occluded Ca2+ exhibits 20–50-fold difference in off-rates among the tested variants. Therefore, the spliced exons on CBD2 affect the rate-limiting step of the occluded Ca2+ dissociation at the primary regulatory sensor to shape dynamic features of allosteric regulation in NCX variants.