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.
Keywords: Enzyme mutation; Genetic disease; Membrane transport; NCKX; Na(+)-Ca(2+)-K(+)exchange; Sodium-calcium exchange.
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