Key residues controlling bidirectional ion movements in Na ⁺ /Ca ²⁺ exchanger

Prokaryotic and eukaryotic Na ⁺ /Ca ²⁺ exchangers (NCX) control Ca ²⁺ homeostasis. NCX orthologs exhibit up to 10 ⁴ -fold differences in their turnover rates (k _cat ), whereas the ratios between the cytosolic (cyt) and extracellular (ext) K _m values (K _int = K _m ^Cyt /K _m ^Ext ) are highly asymmetric and alike (K _int ≤ 0.1) among NCXs. The structural determinants controlling a huge divergence in k _cat at comparable K _int remain unclear, although 11 (out of 12) ion-coordinating residues are highly conserved among NCXs. The crystal structure of the archaeal NCX (NCX_Mj) was explored for testing the mutational effects of pore-allied and loop residues on k _cat and K _int . Among 55 tested residues, 26 mutations affect either k _cat or K _int , where two major groups can be distinguished. The first group of mutations (14 residues) affect k _cat rather than K _int . The majority of these residues (10 out of 14) are located within the extracellular vestibule near the pore center. The second group of mutations (12 residues) affect K _int rather than k _cat , whereas the majority of residues (9 out 12) are randomly dispersed within the extracellular vestibule. In conjunction with computational modeling-simulations and hydrogen-deuterium exchange mass-spectrometry (HDX-MS), the present mutational analysis highlights structural elements that differentially govern the intrinsic asymmetry and transport rates. The key residues, located at specific segments, can affect the characteristic features of local backbone dynamics and thus, the conformational flexibility of ion-transporting helices contributing to critical conformational transitions. The underlying mechanisms might have a physiological relevance for matching the response modes of NCX variants to cell-specific Ca ²⁺ and Na ⁺ signaling.