Residues important for K+ ion transport in the K+-dependent Na+-Ca2+ exchanger (NCKX2)

Abstract K + -dependent Na + -Ca 2+ exchangers (NCKXs) play an important role in Ca 2+ homeostasis in many tissues. NCKX proteins are bi-directional plasma membrane Ca 2+ -transporters which utilize the inward Na + and outward K + gradients to move Ca 2+ ions into and out of the cytosol (4Na + :1Ca 2+  + 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 Ca 2+ -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 (K m ) 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 + -Ca 2+ exchanger NCX_Mj. In addition, we compare our results here with our previous studies that report on residues important for Ca 2+ and Na + binding. Supported by CIHR MOP-81327 .