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Ionic regulatory properties of brain and kidney splice variants of the NCX1 Na(+)-Ca(2+) exchanger.
Dyck C
,
Omelchenko A
,
Elias CL
,
Quednau BD
,
Philipson KD
,
Hnatowich M
,
Hryshko LV
.
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Ion transport and regulation of Na(+)-Ca(2+) exchange were examined for two alternatively spliced isoforms of the canine cardiac Na(+)-Ca(2+) exchanger, NCX1.1, to assess the role(s) of the mutually exclusive A and B exons. The exchangers examined, NCX1.3 and NCX1.4, are commonly referred to as the kidney and brain splice variants and differ only in the expression of the BD or AD exons, respectively. Outward Na(+)-Ca(2+) exchange activity was assessed in giant, excised membrane patches from Xenopus laevis oocytes expressing the cloned exchangers, and the characteristics of Na(+)(i)- (i.e., I(1)) and Ca(2+)(i)- (i.e., I(2)) dependent regulation of exchange currents were examined using a variety of experimental protocols. No remarkable differences were observed in the current-voltage relationships of NCX1.3 and NCX1.4, whereas these isoforms differed appreciably in terms of their I(1) and I(2) regulatory properties. Sodium-dependent inactivation of NCX1.3 was considerably more pronounced than that of NCX1.4 and resulted in nearly complete inhibition of steady state currents. This novel feature could be abolished by proteolysis with alpha-chymotrypsin. It appears that expression of the B exon in NCX1.3 imparts a substantially more stable I(1) inactive state of the exchanger than does the A exon of NCX1.4. With respect to I(2) regulation, significant differences were also found between NCX1.3 and NCX1.4. While both exchangers were stimulated by low concentrations of regulatory Ca(2+)(i), NCX1.3 showed a prominent decrease at higher concentrations (>1 microM). This does not appear to be due solely to competition between Ca(2+)(i) and Na(+)(i) at the transport site, as the Ca(2+)(i) affinities of inward currents were nearly identical between the two exchangers. Furthermore, regulatory Ca(2+)(i) had only modest effects on Na(+)(i)-dependent inactivation of NCX1.3, whereas I(1) inactivation of NCX1.4 could be completely eliminated by Ca(2+)(i). Our results establish an important role for the mutually exclusive A and B exons of NCX1 in modulating the characteristics of ionic regulation and provide insight into how alternative splicing tailors the regulatory properties of Na(+)-Ca(2+) exchange to fulfill tissue-specific requirements of Ca(2+) homeostasis.
Figure 3. Na+i dependence of peak and steady state outward Na+–Ca2+ exchange current for NCX1.4 and NCX1.3. Outward currents were obtained as described in Fig. 2, and were normalized to the value obtained at 100 mM Na+i. Data are mean ± SEM of three to six determinations from six patches for NCX1.4, four determinations from four patches for NCX1.3, and three determinations from two patches for NCX1.3 after treatment with 1 mg/ml α-chymotrypsin for ≈60 s.
Figure 1. Sequence comparison of the alternatively spliced regions of NCX1.4 and NCX1.3. Single-letter amino acid code is used and identities are boxed. The alternative splicing region of NCX1 is located within the COOH-terminal third of its large intracellular loop. Expression of exons A and B is mutually exclusive.
Figure 2. Na+i dependence of outward Na+–Ca2+ exchange currents of NCX1.4 and NCX1.3. Representative current traces are shown for NCX1.4 and NCX1.3, and for NCX1.3 after proteolysis for ≈60 s with 1 mg/ml α-chymotrypsin. Transport Ca2+o in the pipette was constant at 8 mM and regulatory Ca2+i was held at 1 μM for 32–48 s before and during acquisition of the current traces. Outward currents were activated by the rapid (i.e., ≈200 ms) application of the indicated concentrations of Na+i to the cytoplasmic surface of the patch. After each current activation event, patches were perfused for 32–48 s with 100 mM Li+i-containing solution plus 1 μM Ca2+i to permit full recovery from inactivation before delivery of the next Na+i pulse.
Figure 4. Na+i dependence of the ratio of steady state to peak current for NCX1.4 and NCX1.3. Currents were obtained as described in Fig. 2. Data are mean ± SEM of 8–20 determinations from 16 patches for NCX1.4, and 4–12 determinations from seven patches for NCX1.3.
Figure 5. IV relationships for NCX1.4 and NCX1.3. Each voltage-clamp increment (10-mV steps from −100 to 100 mV) was initiated from a holding potential of 0 mV. IV records obtained at a during perfusion with 100 mM Li+i-containing solution were subtracted from those obtained at b during perfusion with 100 mM Na+i-containing solution. Regulatory Ca2+i (1 μM) was present throughout the current recordings. Pooled data shown (bottom) are mean ± SEM from four patches for NCX1.4 and three patches for NCX1.3.
Figure 6. Ca2+i regulation of outward Na+–Ca2+ exchange currents for NCX1.4 and NCX1.3. Representative records are shown for NCX1.4 and NCX1.3, where currents were activated by applying 100 mM Na+i in the presence of 1 μM Ca2+i. Regulatory Ca2+i was present for 32–48 s before the application of transport Na+i. Upon approaching steady state current levels, Ca2+i was removed for 16 s, and then reapplied for a further 16-s interval before deactivating exchange current by returning to a Li+i-based perfusing solution. The traces shown are typical of five patches for NCX1.4 and three patches for NCX1.3.
Figure 7. Ca2+i dependence of outward Na+–Ca2+ exchange currents for NCX1.4 and NCX1.3. Currents were activated by applying 100 mM Na+i with regulatory Ca2+i present at the indicated concentrations for 32–48 s before and throughout the current recordings. After a current activation event, patches were perfused with a Li+i-based perfusing solution containing a new [Ca2+]i for 32–48 s to allow for equilibration and recovery from the previous activation/inactivation event.
Figure 9. Representative inward Na+–Ca2+ exchange currents produced by NCX1.3 and NCX1.4 are shown in response to the application of three different Ca2+ concentrations (1, 3, and 10 μM). Pipettes contained 100 mM Na+. Similar results were obtained in four additional patches for NCX1.3 and two additional patches for NCX1.4.
Figure 8. Ca2+i dependence of peak (top) and steady state (middle) outward Na+–Ca2+ exchange currents for NCX1.4 and NCX1.3. Currents were obtained as described in Fig. 7 and normalized to the value obtained at 1 μM regulatory Ca2+i. Data are mean ± SEM of three to seven determinations from seven patches for NCX1.4, and three to five determinations from five patches for NCX1.3. The Ca2+i dependence of the ratio of steady state to peak current for NCX1.4 and NCX1.3 is shown (bottom). Currents were obtained as described in Fig. 7. Data are mean ± SEM of 7–20 determinations from 13 patches for NCX1.4, and 7–19 determinations from 15 patches for NCX1.3.
Figure 10. The effect of regulatory Ca2+i on recovery of NCX1.4 and NCX1.3 from Na+i-dependent inactivation using paired-pulse stimulation. The indicated concentrations of regulatory Ca2+i were present throughout the current measurements. The first pulse was activated by applying 100 mM Na+i for 32 s, followed by a 4-s recovery interval. A second, test pulse was then elicited by reapplication of 100 mM Na+i. The graph (bottom) shows representative results from four patches each of NCX1.4 and NCX1.3 over a range of regulatory [Ca2+]i.
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