Structure-function studies of novel K+ -dependent Na+/Ca2+ exchangers
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AbstractPlasma membrane K -dependent Na /Ca exchangers (NCKX) are believed to play a crucial role in mediating Ca2+ efflux in a wide range of tissues. This activity is encoded by a multigene family of at least five members that share sequence similarity in two internally homologous domains known as the a-repeats. The a-repeats are believed to form the ion binding sites for translocation. I have explored the structure-function relationship of the rat brain NCKX2 exchanger. Two important Cys residues were identified to be critical for stabilizing protein structure. Using an introduced epitope I demonstrated that the protein C-terminus was extracellularly exposed in intact cells. A new topological model for NCKX2 has been presented, which is different from the initial model for NCKX exchangers. NCKX2 could be labeled with biotin maleimide only following application of (3-mercaptoethanol. NCKX2 activity was also enhanced by (3-mercaptoethanol. A critical residue Cys-395 in the large intracellular loop was found to underlie the redox-dependent labeling and functional stimulation. Although NCKX2 associates as an oligomer, this interaction does not require Cys-395. I have isolated a novel cDNA clone encoding NCKX6 from mouse thymus. NCKX6 is a structurally more divergent, and distantly related, Na+/Ca2+ exchanger member compared with other known exchangers. Identification of many NCKX6 paralogs from lower organisms suggested NCKX6 might have evolved independently from the other NCKX members in the Na /Ca exchanger superfamily. A splice variant with a truncated C-terminal hydrophobic exchanger domain, but not the full-length clone, could reach the plasma membrane when expressed in HEK-293 cells. This NCKX6 isoform exhibited K+-dependent Na+/Ca2+ exchange activity. Immunofluorescence staining using a polyclonal NCKX6-specific antibody demonstrated strong fluorescence labeling at the cell surface in native cells. The full-length NCKX6 and its splice variants are expressed in every tissue examined and their relative expression levels were compared. I have performed a comprehensive bioinformatic analysis of the cation/Ca2+ exchanger superfamily, integrating genomic, functional, structural and evolutionary information. Our data defined five major protein families, each with a distinct evolutionary relationship and unique signature motifs. These findings may provide guides for future studies concerning the structure-to-function relationship and evolutionary origins of the cation/Ca2+ exchangers.
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