Exchanger from Drosophila melanogaster

Ion transport and regulation were studied in two, alternatively spliced isoforms of the Na 1 -Ca 2 1 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 1 -Ca 2 1 exchanger, NCX1. The CALX isoforms were expressed in Xenopus laevis oocytes and characterized electrophysiologically using the giant, excised patch clamp technique. Outward Na 1 -Ca 2 1 exchange currents, where pipette Ca 2 1 o exchanges for bath Na 1 i , were examined in all cases. Although the isoforms exhibited similar transport properties with respect to their Na 1 i affinities and current–voltage relationships, significant differences were observed in their Na 1 i and Ca 2 1 i -dependent regulatory properties. Both isoforms underwent Na 1 i -dependent inactivation, apparent as a time-dependent decrease in outward exchange current upon Na 1 i application. We observed a twoto threefold difference in recovery rates from this inactive state and the extent of Na 1 i -dependent inactivation was approximately twofold greater for CALX1.2 as compared with CALX1.1. Both isoforms showed regulation of Na 1 -Ca 2 1 exchange activity by Ca 2 1 i , but their responses to regulatory Ca 2 1 i differed markedly. For both isoforms, the application of cytoplasmic Ca 2 1 i led to a decrease in outward exchange currents. This negative regulation by Ca 2 1 i is unique to Na 1 -Ca 2 1 exchangers from Drosophila , and contrasts to the positive regulation produced by cytoplasmic Ca 2 1 for all other characterized Na 1 -Ca 2 1 exchangers. For CALX1.1, Ca 2 1 i inhibited peak and steady state currents almost equally, with the extent of inhibition being < 80%. In comparison, the effects of regulatory Ca 2 1 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 1 i occurred by a direct mechanism and indirectly through effects on Na 1 i -induced inactivation. Our results show that regulatory Ca 2 1 i decreases Na 1 i -induced inactivation of CALX1.2, whereas it stabilizes the Na 1 i -induced inactive state of CALX1.1. These effects of Ca 2 1 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 1 -Ca 2 1 exchange proteins. key words: Drosophila melanogaster • sodium-calcium exchange • regulation • alternative splicing i n t r o d u c t i o n Sodium-calcium exchange plays a major role in Ca 2 1 homeostasis in a wide variety of tissues (Lederer et al., 1996). In cardiac muscle, for example, the Na 1 -Ca 2 1 exchanger is the primary mechanism for transarcolemmal Ca 2 1 efflux enabling cardiac relaxation (Bers, 1991). Similarly, in neuronal tissue, the exchanger may play an important role in Ca 2 1 efflux and, thus, excitation–secretion coupling (Blaustein et al., 1996). Calcium removal is accomplished by coupling the energy in the Na 1 electrochemical gradient to the uphill movement of Ca 2 1 . Moreover, as a reversible transporter, the Na 1 -Ca 2 1 exchanger may also mediate Ca 2 1 entry, a role recently postulated for excitation of cardiac muscle (Leblanc and Hume, 1990; Levi et al., 1994). Two forms of ionic regulation have been characterized extensively for the cardiac Na 1 -Ca 2 1 exchanger, NCX1, using the giant, excised patch clamp technique (Hilgemann et al., 1992 a , 1992 b ). These regulatory processes are controlled by Na 1 and Ca 2 1 and are most readily observed for outward Na 1 -Ca 2 1 exchange currents, where extracellular Ca 2 1 exchanges for intracellular Na 1 . Upon application of Na 1 i to evoke an outward exchange current, the current peaks and then decays to lower, steady state levels (Hilgemann, 1990). The extent and rate of current inactivation depends upon the concentration of applied Na 1 i , a process referred to as Na 1 i -induced (I 1 ) 1 inactivation (Hilgemann et al., 1992 a ). Calcium-induced (I 2 ) regulation describes the stimulatory influence of cytoplasmic Ca 2 1 Address correspondence to Larry V. Hryshko, Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, 351 Tache Avenue, Winnipeg, Manitoba, Canada R2H 2A6. Fax: 204-2336723; E-mail: [email protected] 1 Abbreviations used in this paper: I 1 , Na 1 i -induced; I 2 , Ca 2 1 i -induced. on Jne 3, 2017 D ow nladed fom Published May 1, 1998