Residues important for Ca 2+ ion transport in the neuronal K + -dependent Na + -Ca 2+ exchanger (NCKX2).

K+ -dependent Na+ -Ca2+ exchangers (NCKXs) belong to Ca2+ /cation antiporter gene superfamily. NCKX proteins play an important role in Ca2+ homeostasis and are bi-directional plasma membrane Ca2+ -transporters which utilize the inward Na+ and outward K+ gradients to move Ca2+ ions into and out of the cytosol (4Na+ :1Ca2+  + 1 K+ ). In this study, we examined residues in the two regions with the highest degree of homology between the different NCKX isoforms (α-1 and α-2 repeats) to determine which residues are important for Ca2+ coordination. Using fluorescent intracellular Ca2+ -indicating dyes, we measured NCKX-mediated Ca2+ transport in HEK293 cells expressing wildtype or mutant NCKX2 and analyzed shifts in the apparent binding affinity (Km ) of mutant proteins when compared to the wildtype exchanger. Of the 93 residue substitutions tested, 31 were found to show a significant shift in the external Ca2+ ion dependence of which 18 showed an increased affinity to Ca2+ ions and 13 showed a decreased affinity, and, hence, are believed to be important for Ca2+ ion binding and transport. When compared to the crystal structure of the archaeal Na+ -Ca2+ exchanger NCX_Mj and the NCKX2 homology model based on this crystal structure, our biochemical data reveal that these 13 residues are either in direct contact with the Ca2+ ion or lining a Ca2+ transport pathway through the exchanger. Supported by CIHR MOP-81327.