Omelchenko A et al. (1998), Functional differences in ionic regulation betw...

XB-ART-14785

J Gen Physiol 1998 May 01;1115:691-702. doi: 10.1085/jgp.111.5.691.

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

Omelchenko A , Dyck C , Hnatowich M , Buchko J , Nicoll DA , Philipson KD , Hryshko LV .

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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.

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Species referenced: Xenopus laevis
Genes referenced: slc8a1

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	Figure 2. The Na+i dependence of outward Na+-Ca2+ exchange currents for CALX isoforms. Overlapping current traces for both CALX isoforms are shown. Outward currents were activated in the absence of regulatory Ca2+i by application of the indicated concentrations of Na+i, maintained until a steady state was attained, followed by switching back to a Li+i-based bath solution.
	Figure 3. The Na+i dependence of peak and steady state outward Na+-Ca2+ exchange currents for CALX isoforms. Data from seven patches for each exchanger were normalized to the peak (A) and steady state (B) currents generated by application of 100 mM Na+i in the absence of regulatory Ca2+i. Data were “best-fit” to the Hill equation by least-squares regression analysis.
	Figure 4. The Na+i dependence of Fss, the ratio of steady state to peak currents, for CALX isoforms. The Na+i dependence of the ratio of steady state to peak outward currents for CALX1.1 and CALX1.2 is shown. Values are mean ± SEM of six to seven determinations for CALX1.1 and four to six determinations for CALX1.2.
	Figure 5. The rate of recovery from Na+i-induced inactivation of peak outward Na+-Ca2+ exchange currents for CALX isoforms. The top three panels show representative current traces obtained from paired-pulse experiments with 48-, 12-, and 2-s recovery intervals. Currents were activated by 100 mM Na+i, in the absence of regulatory Ca2+i, and maintained until a steady state level was obtained (i.e., Pulse 1). Current was then deactivated by rapidly switching to 100 mM Li+i. After a recovery period of 0.5–48 s, a second pulse of 100 mM Na+i was delivered (i.e., Pulse 2). The bottom summarizes data obtained from 13 patches of CALX1.1 and 5 of CALX1.2 spanning recovery intervals of 0.5–48 s. Data were “best-fit” to a single-exponential function via least-squares regression analysis.
	Figure 6. The effect of regulatory Ca2+i on steady state Na+-Ca2+ exchange currents for CALX isoforms. Representative data are shown illustrating outward currents generated by CALX1.1 and CALX1.2 in response to the application of 100 mM Na+i. Regulatory Ca2+i (1 μM) was applied in the middle of the current record (near steady state), and then removed as indicated. Note that currents are substantially inhibited by regulatory Ca2+i for CALX1.1 but not for CALX1.2.
	Figure 7. The effect of preincubation with regulatory Ca2+i on Na+-Ca2+ exchange current transients for CALX isoforms. Representative data are shown for outward Na+-Ca2+ exchange currents activated by 100 mM Na+i. Regulatory Ca2+ at the indicated concentrations was present before and during current activation.
	Figure 8. The effect of regulatory Ca2+i on peak and steady state Na+-Ca2+ exchange currents for CALX isoforms. Peak (A) and steady state (B) currents for CALX1.1 (10 patches) and for CALX1.2 (8 patches) generated in experiments in which giant patches were incubated with the indicated concentrations of regulatory Ca2+i (rCai2+), as in Fig. 7, were normalized to the peak or steady state currents obtained in the absence of rCa2+i. Values are mean ± SEM of four to six determinations for CALX1.1 (except at 10 and 30 μM rCai2+, which are from one and two determinations, respectively) and four to eight for CALX1.2 (except at 10 μM rCai2+, which is from a single determination). Data were “best-fit” to a two-parameter, single-exponential function via least-squares regression analysis. Dotted lines represent the estimated minimum values of peak and steady state currents.
	Figure 9. The effect of regulatory Ca2+i on Na+-Ca2+ exchange current transients for CALX isoforms. Representative data are shown for outward Na+-Ca2+ exchange currents activated by 100 mM Na+i. Regulatory Ca2+ at the indicated concentrations was present during current activation only.
	Figure 10. The effect of regulatory Ca2+i on rates of recovery from Na+i-induced inactivation for CALX isoforms. (left) Outward Na+-Ca2+ current traces were obtained from a single patch of each isoform in paired-pulse experiments with 4-s recovery intervals (see Fig. 5) in which the effects of 0 and 300 nM regulatory Ca2+i were assessed. The condition of 0 or 300 nM Ca2+i was initiated before delivery of the first pulse of 100 mM Na+i, and maintained throughout activation, recovery, and activation of the second pulse. Currents were normalized to the height of the first, control Na+i pulse to allow for simple comparison of recovery behavior. (right) Graphs summarize the data from these two patches obtained for the full range of recovery intervals (i.e., 0.5–48 s) in the absence and presence of 300 nM Ca2+i. Data were normalized to the height of the first peak of each pair and “best-fit” to a single-exponential function (Eq. A6) via least-squares regression analysis.

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Andersen, The Journal of General Physiology. 1997, Pubmed