| Na+/Ca2+-K+ exchangers (NCKX) constitute an important family of plasma membrane Ca 2+ transport proteins in animal cells; to date, however, relatively little is known of their molecular functional operation. This thesis describes the development of a fluorescence-based assay to measure Na+-dependent changes in intracellular Ca2+, through the use of the alkali cation ionophore gramicidin to control [Na+]i. The assay was used to scan through single amino acid substitution mutant constructs of human NCKX2 for shifts in Na+-affinity, to determine which amino acids/regions of the molecule are involved in Na+ transport/liganding. In the process, a kinetic regulatory feature, herein termed Na+ i-dependent inactivation was discovered; this inactive state was produced upon prolonged (>40 s) exposure of NCKX2 to high intracellular Na+ (>35 mM). |
