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

• Published in: The Journal of general physiology 1998-05, Vol.111 (5), p.691-702
• Main Authors: Omelchenko, A, Dyck, C, Hnatowich, M, Buchko, J, Nicoll, D A, Philipson, K D, Hryshko, L V
• Format: Article
• Language: English
• Subjects: Alternative Splicing; Amino acids; Anatomy & physiology; Animals; Calcium; Calcium - physiology; Drosophila melanogaster - metabolism; Electric Conductivity; Female; Insects; Ions; Oocytes - metabolism; Sodium; Sodium - physiology; Sodium-Calcium Exchanger - genetics; Sodium-Calcium Exchanger - metabolism; Sodium-Calcium Exchanger - physiology; Xenopus laevis
• ISSN: 0022-1295; 1540-7748
• DOI: 10.1085/jgp.111.5.691

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 approximately 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 ( approximately 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.