Functional differences in ionic regulation between alternatively spliced isoforms of the Na+-Ca2+ exchanger from Drosophila melanogaster

Ion transport and regulation were studied in two, alternatively spliced isoforms of the Na+-Ca2+ exchanger from Drosophila melanogaster. These exchangers, designated CALX1.1 and CALX1.2, differ by five amino acids in a region where alternative splicing also occurs in the mammalian Na+-Ca2+ exchanger, NCX1. The CALX isoforms were expressed in Xenopus laevis oocytes and characterized electrophysiologically using the giant, excised patch clamp technique. Outward Na+-Ca2+ exchange currents, where pipette Ca-0(2+) exchanges for bath Na-i(+), were examined in all cases. Although the isoforms exhibited similar transport properties with respect to their Na-i(+) affinities and current-voltage relationships, significant differences were observed in their Na-i(+)- and Ca-i(2+)-dependent regulatory properties. Both isoforms underwent Na-i(+)-dependent inactivation, apparent as a time-dependent decrease in outward exchange current upon Na-i(+) application. We observed a two- to threefold difference in recovery rates from this inactive state and the extent of Na-i(+)-dependent inactivation was approximately twofold greater for CALX1.2 as compared with CALX1.1. Both isoforms showed regulation of Na+-Ca2+ exchange activity by Ca-i(2+), but their responses to regulatory Ca-i(2+) differed markedly. For both isoforms, the application of cytoplasmic Ca-i(2+) led to a decrease in outward exchange currents. This negative regulation by Ca-i(2+) is unique to Na+-Ca2+ exchangers from Drosophila, and contrasts to the positive regulation produced by cytoplasmic Ca2+ for all other characterized Na+-Ca2+ exchangers. For CALX1.1, Ca-i(2+) inhibited peak and steady state currents almost equally, with the extent of inhibition being approximate to 80%. In comparison, the effects of regulatory Ca-i(2+) occurred with much higher affinity for CALX1.2, but the extent of these effects was greatly reduced (approximate to 20-40% inhibition). For both exchangers, the effects of regulatory Ca-i(2+) occurred by a direct mechanism and indirectly through effects on Na-i(+)-induced inactivation. Our results show that regulatory Ca-i(2+) decreases Na-i(+)-induced inactivation of CALX1.2, whereas it stabilizes the Na-i(+)-induced inactive state of CALX1.1. These effects of Ca-i(2+) produce striking differences in regulation between CALX isoforms. Our findings indicate that alternative splicing may play a significant role in tailoring the regulatory profile of CALX isoforms and, possibly, other Na+-Ca2+ exchange proteins.