Structure-based dynamic arrays in regulatory domains of sodium-calcium exchanger (NCX) isoforms

Mammalian Na⁺/Ca²⁺ exchangers, NCX1 and NCX3, generate splice variants, whereas NCX2 does not. The CBD1 and CBD2 domains form a regulatory tandem (CBD12), where Ca²⁺ binding to CBD1 activates and Ca²⁺ binding to CBD2 (bearing the splicing segment) alleviates the Na⁺-induced inactivation. Here, the NCX2-CBD12, NCX3-CBD12-B, and NCX3-CBD12-AC proteins were analyzed by small-angle X-ray scattering (SAXS) and hydrogen-deuterium exchange mass-spectrometry (HDX-MS) to resolve regulatory variances in the NCX2 and NCX3 variants. SAXS revealed the unified model, according to which the Ca²⁺ binding to CBD12 shifts a dynamic equilibrium without generating new conformational states, and where more rigid conformational states become more populated without any global conformational changes. HDX-MS revealed the differential effects of the B and AC exons on the folding stability of apo CBD1 in NCX3-CBD12, where the dynamic differences become less noticeable in the Ca²⁺-bound state. Therefore, the apo forms predefine incremental changes in backbone dynamics upon Ca²⁺ binding. These observations may account for slower inactivation (caused by slower dissociation of occluded Ca²⁺ from CBD12) in the skeletal vs the brain-expressed NCX2 and NCX3 variants. This may have physiological relevance, since NCX must extrude much higher amounts of Ca²⁺ from the skeletal cell than from the neuron.