J. Gen. Physiol., Volume 111, Number 5, May 1, 1998 691-702

Functional Differences in Ionic Regulation between Alternatively Spliced Isoforms of the Na⁺-Ca²⁺ Exchanger from Drosophila melanogaster

Alexander Omelchenko,^* Christopher Dyck,^* Mark Hnatowich,^* John Buchko,^* Debora A. Nicoll,^§ Kenneth D. Philipson,^§ and Larry V. Hryshko^*

From the ^* Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, Winnipeg, Manitoba, Canada R2H 2A6; and  Department of Physiology, and ^§ Department of Medicine and the Cardiovascular Research Laboratories, University of California, Los Angeles, Los Angeles, California 90025-1760

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 Ca²⁺_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 Ca²⁺_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⁺-Ca²⁺ exchange activity by Ca²⁺_i, but their responses to regulatory Ca²⁺_i differed markedly. For both isoforms, the application of cytoplasmic Ca²⁺_i led to a decrease in outward exchange currents. This negative regulation by Ca²⁺_i 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, Ca²⁺_i inhibited peak and steady state currents almost equally, with the extent of inhibition being 80%. In comparison, the effects of regulatory Ca²⁺_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 Ca²⁺_i occurred by a direct mechanism and indirectly through effects on Na⁺_i-induced inactivation. Our results show that regulatory Ca²⁺_i decreases Na⁺_i-induced inactivation of CALX1.2, whereas it stabilizes the Na⁺_i-induced inactive state of CALX1.1. These effects of Ca²⁺_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⁺-Ca²⁺ exchange proteins.

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