Residues important for K+ ion transport in the K+-dependent Na+-Ca2+ 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.