Functional Differences in Ionic Regulation between Alternatively Spliced Isoforms of the Na(+)-Ca(2+) Exchanger from Drosophila melanogaster

Ion transport and regulation were studied in two, alternatively spliced isoforms of the Na(+)-Ca(2+) 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(+)-Ca(2+) exchanger, NCX1. The CALX isoforms were expressed in Xenopus laevis oocytes and characterized electrophysiologically using the giant, excised patch clamp technique. Outward Na(+)-Ca(2+) exchange currents, where pipette Ca(2+) (o) 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(2+) (i)-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(+)-Ca(2+) exchange activity by Ca(2+) (i), but their responses to regulatory Ca(2+) (i) differed markedly. For both isoforms, the application of cytoplasmic Ca(2+) (i) led to a decrease in outward exchange currents. This negative regulation by Ca(2+) (i) is unique to Na(+)-Ca(2+) exchangers from Drosophila, and contrasts to the positive regulation produced by cytoplasmic Ca(2+) for all other characterized Na(+)-Ca(2+) exchangers. For CALX1.1, Ca(2+) (i) inhibited peak and steady state currents almost equally, with the extent of inhibition being ≈80%. In comparison, the effects of regulatory Ca(2+) (i) occurred with much higher affinity for CALX1.2, but the extent of these effects was greatly reduced (≈20–40% inhibition). For both exchangers, the effects of regulatory Ca(2+) (i) occurred by a direct mechanism and indirectly through effects on Na(+) (i)-induced inactivation. Our results show that regulatory Ca(2+) (i) decreases Na(+) (i)-induced inactivation of CALX1.2, whereas it stabilizes the Na(+) (i)-induced inactive state of CALX1.1. These effects of Ca(2+) (i) 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(+)-Ca(2+) exchange proteins.