STRUCTURE-FUNCTION ANALYSIS OF CALX1.1, A NA-CA-2 EXCHANGER FROM DROSOPHILA - MUTAGENESIS OF IONIC REGULATORY SITES()

Cytoplasmic Na+ and Ca2+ regulate the activity of Na+-Ca2+ exchange pr oteins, in addition to serving as the transported ions, and protein re gions involved in these processes have been identified for the canine cardiac Na+-Ca2+ exchanger, NCX1.1. Although protein regions associate d with Na-i(+)- and Ca-i(2+)-dependent regulation are highly conserved among cloned Na+-Ca2+ exchangers, it is unknown whether or not the st ructure-function relationships characteristic of NCX1.1 apply to any o ther exchangers. Therefore, we studied structure-function relationship s in a Na+-Ca2+ exchanger from Drosophila, CALX1.1, which is unique am ong characterized members of this family of proteins in that mu M leve ls of Ca-i(2+) inhibit exchange current. Wild-type and mutant CALX1.1 exchangers were expressed in Xenopus oocytes and characterized electro physiologically using the giant excised patch technique, Mutations wit hin the putative regulatory Ca-i(2+) binding site of CALX1.1, like cor responding alterations in NCX1.1, led to reduced ability (i,e. D516V a nd D5501) or inability (i.e. G555P) of Ca-i(2+) to inhibit Na+-Ca2+ ex change activity. Similarly, mutations within the putative XIP region o f 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 r oles 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 extend ed these findings using chimeric constructs of CALX1.1 and NCX1.1 to d etermine whether or not functional interconversion of Ca-i(2+) regulat ory phenotypes was feasible. With one chimera (i,e. CALX:NCX:CALX), su bstitution of a 193-amino acid segment, from the large intracellular l oop 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 indi cates that the regulatory Ca-i(2+) binding site of NCX1.1 retains func tion 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.