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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 - J. Gen. Physiol. (1999)

Bottom Line: With respect to I(2) regulation, significant differences were also found between NCX1.3 and NCX1.4.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, Winnipeg, Manitoba, Canada, R2H 2A6.

ABSTRACT
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.

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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.
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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.

Mentions: The observation that NCX1.3 appears to be inhibited at higher concentrations of regulatory Ca2+ could occur if this exchanger had a higher affinity for Ca2+ at the intracellular transport site. Consequently, lower concentrations of Ca2+ could compete for Na+i and reduce current magnitude. To test this possibility, we examined inward Na+–Ca2+ exchange currents for both NCX1.3 and NCX1.4. Pipettes contained 100 mM Na+ and inward currents were activated by applying different Ca2+ concentrations (0.1–100 μM) to the cytoplasmic surface of the patch. Typical inward current recordings are shown in Fig. 9. We did not observe any major differences for inward Na+–Ca2+ exchange currents produced by NCX1.3 and NCX1.4. The apparent affinities calculated for Ca2+ activation of inward Na+–Ca2+ exchange currents were 9.0 ± 1.9 μM (mean ± SD, n = 5 patches) for NCX1.3 and 8.1 ± 1.4 μM (mean ± SD, n = 3 patches) for NCX1.4. These values are similar to that reported for NCX1 (e.g., 7 μM; Matsuoka et al. 1995). Furthermore, this indicates that the inhibitory effects of higher regulatory Ca2+ concentrations observed for outward currents from NCX1.3 are unlikely to be due to greater competition at the intracellular transport site.


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 - J. Gen. Physiol. (1999)

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.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2230537&req=5

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.
Mentions: The observation that NCX1.3 appears to be inhibited at higher concentrations of regulatory Ca2+ could occur if this exchanger had a higher affinity for Ca2+ at the intracellular transport site. Consequently, lower concentrations of Ca2+ could compete for Na+i and reduce current magnitude. To test this possibility, we examined inward Na+–Ca2+ exchange currents for both NCX1.3 and NCX1.4. Pipettes contained 100 mM Na+ and inward currents were activated by applying different Ca2+ concentrations (0.1–100 μM) to the cytoplasmic surface of the patch. Typical inward current recordings are shown in Fig. 9. We did not observe any major differences for inward Na+–Ca2+ exchange currents produced by NCX1.3 and NCX1.4. The apparent affinities calculated for Ca2+ activation of inward Na+–Ca2+ exchange currents were 9.0 ± 1.9 μM (mean ± SD, n = 5 patches) for NCX1.3 and 8.1 ± 1.4 μM (mean ± SD, n = 3 patches) for NCX1.4. These values are similar to that reported for NCX1 (e.g., 7 μM; Matsuoka et al. 1995). Furthermore, this indicates that the inhibitory effects of higher regulatory Ca2+ concentrations observed for outward currents from NCX1.3 are unlikely to be due to greater competition at the intracellular transport site.

Bottom Line: With respect to I(2) regulation, significant differences were also found between NCX1.3 and NCX1.4.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, Winnipeg, Manitoba, Canada, R2H 2A6.

ABSTRACT
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.

Show MeSH