Ca 2+ regulation in the Na + /Ca 2+ exchanger features a dual electrostatic switch mechanism

Regulation of ion-transport in the Na+ /Ca2+ exchanger (NCX) occurs via its cytoplasmic Ca2+ -binding domains, CBD1 and CBD2. Here, we present a mechanism for NCX activation and inactivation based on data obtained using NMR, isothermal titration calorimetry (ITC) and small-angle X-ray scattering (SAXS). We initially determined the structure of the Ca2+ -free form of CBD2-AD and the structure of CBD2-BD that represent the two major splice variant classes in NCX1. Although the apo-form of CBD2-AD displays partially disordered Ca2+ -binding sites, those of CBD2-BD are entirely unstructured even in an excess of Ca2+ . Striking differences in the electrostatic potential between the Ca2+ -bound and -free forms strongly suggest that Ca2+ -binding sites in CBD1 and CBD2 form electrostatic switches analogous to C2 -domains. SAXS analysis of a construct containing CBD1 and CBD2 reveals a conformational change mediated by Ca2+ -binding to CBD1. We propose that the electrostatic switch in CBD1 and the associated conformational change are necessary for exchanger activation. The response of the CBD1 switch to intracellular Ca2+ is influenced by the closely located cassette exons. We further propose that Ca2+ -binding to CBD2 induces a second electrostatic switch, required to alleviate Na+ -dependent inactivation of Na+ /Ca2+ exchange. In contrast to CBD1, the electrostatic switch in CBD2 is isoform- and splice variant-specific and allows for tailored exchange activities.