title: Functional differences in ionic regulation between alternatively spliced isoforms of the Na+-Ca2+ exchanger from Drosophila melanogaster.
abstract: 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.
authors.affiliation: Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, Winnipeg, Manitoba, Canada R2H 2A6.
authors.forename: A
authors.initials: A
authors.lastname: Omelchenko
authors.forename: C
authors.initials: C
authors.lastname: Dyck
authors.forename: M
authors.initials: M
authors.lastname: Hnatowich
authors.forename: J
authors.initials: J
authors.lastname: Buchko
authors.forename: D A
authors.initials: DA
authors.lastname: Nicoll
authors.forename: K D
authors.initials: KD
authors.lastname: Philipson
authors.forename: L V
authors.initials: LV
authors.lastname: Hryshko
mesh_terms: D017398:Alternative Splicing; D000818:Animals; D002118:Calcium / Q000502:physiology; D004331:Drosophila melanogaster / Q000378:metabolism*; D004553:Electric Conductivity; D005260:Female; D007477:Ions; D009865:Oocytes / Q000378:metabolism; D012964:Sodium / Q000502:physiology; D019831:Sodium-Calcium Exchanger / Q000235:genetics* / Q000378:metabolism* / Q000502:physiology; D014982:Xenopus laevis
chemical_list: D007477:Ions; D019831:Sodium-Calcium Exchanger; D012964:Sodium; D002118:Calcium