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

K+ -dependent Na+ -Ca2+ exchangers (NCKXs) play an important role in Ca2+ homeostasis in many tissues. NCKX proteins 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 carried out scanning mutagenesis of all the residues of the highly conserved α-1 and α-2 repeats of NCKX2 to identify residues important for K+ transport. These structural elements are thought to be critical for cation transport. Using fluorescent intracellular Ca2+ -indicating dyes, we measured the K+ dependence of transport carried out by wildtype or mutant NCKX2 proteins expressed in HEK293 cells and analyzed shifts in the apparent binding affinity (Km ) of mutant proteins in comparison with the wildtype exchanger. Of the 93 residue substitutions tested, 34 were found to show a significant shift in the external K+ ion dependence of which 16 showed an increased affinity to K+ ions and 18 showed a decreased affinity and hence are believed to be important for K+ ion binding and transport. We also identified 8 residue substitutions that resulted in a partial loss of K+ dependence. Our biochemical data provide strong support for the cation binding sites identified in a homology model of NCKX2 based on crystal structures reported for distantly related archaeal Na+ -Ca2+ exchanger NCX_Mj. In addition, we compare our results here with our previous studies that report on residues important for Ca2+ and Na+ binding. Supported by CIHR MOP-81327.