Structure-function analysis of CALX1.1, a Na+-Ca2 exchanger from Drosophila -: Mutagenesis of ionic regulatory sites

Cytoplasmic Na+ and Ca2+ regulate the activity of Na+-Ca2+ exchange proteins, in addition to serving as the transported ions, and protein regions involved in these processes have been identified for the canine cardiac Na+-Ca2+ exchanger, NCX1.1. Although protein regions associated with Na-i(+)- and Ca-i(2+)-dependent regulation are highly conserved among cloned Na+-Ca2+ exchangers, it is unknown whether or not the structure-function relationships characteristic of NCX1.1 apply to any other exchangers. Therefore, we studied structure-function relationships in a Na+-Ca2+ exchanger from Drosophila, CALX1.1, which is unique among characterized members of this family of proteins in that mu M levels of Ca-i(2+) inhibit exchange current. Wild-type and mutant CALX1.1 exchangers were expressed in Xenopus oocytes and characterized electrophysiologically using the giant excised patch technique, Mutations within the putative regulatory Ca-i(2+) binding site of CALX1.1, like corresponding alterations in NCX1.1, led to reduced ability (i,e. D516V and D5501) or inability (i.e. G555P) of Ca-i(2+) to inhibit Na+-Ca2+ exchange activity. Similarly, mutations within the putative XIP region of CALX1.1, as in NCX1.1, led to two distinct phenotypes: acceleration (i.e. K306Q) and elimination (i.e. Delta 310-313) of Na-i(+)-dependent inactivation. These results indicate that the respective regulatory roles of the Ca-i(2+) binding site and XIP region are conserved between CALX1.1 and NCX1.1, despite opposite responses to Ca-i(2+), We extended these findings using chimeric constructs of CALX1.1 and NCX1.1 to determine whether or not functional interconversion of Ca-i(2+) regulatory phenotypes was feasible. With one chimera (i,e. CALX:NCX:CALX), substitution of a 193-amino acid segment, from the large intracellular loop of NCX1.1, for the corresponding 177-amino acid segment of CALX1.1 led to an exchanger that was stimulated by Ca-i(2+). This result indicates that the regulatory Ca-i(2+) binding site of NCX1.1 retains function in a CALX1.1 parent transporter and that the substituted segment contains some of the amino acid sequence(s) required for transduction of the Ca-i(2+) binding signal.