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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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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.
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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.

Mentions: Fig. 3 summarizes the Na+i dependence of peak and steady state outward Na+–Ca2+ exchange currents derived from pooled data obtained with NCX1.3 and NCX1.4 in the presence of 1 μM regulatory Ca2+i, as above. Currents were normalized to the values obtained at 100 mM Nai+. For both isoforms, peak currents progressively increased with increasing [Na+]i. Estimates of exchanger affinity for Na+i based on peak current measurements provided nearly identical values of Kd (33 ± 5 mM, n = 4 vs. 31 ± 4 mM, n = 6, for NCX1.3 and NCX1.4, respectively). For comparison, the peak current-derived Na+i affinity of the cardiac exchanger, NCX1.1, is ≈27 mM (Matsuoka et al. 1995, Matsuoka et al. 1997). With respect to the Na+i affinity derived from measurements of steady state currents, however, a Kd value of 16 ± 1 mM (n = 6) was obtained for NCX1.4, whereas the NCX1.3 isoform appeared to be nearly Na+i independent over the concentration range examined. Thus, a Kd value could not be estimated. However, after treatment with α-chymotrypsin, a Kd value of 26 ± 1 mM (three determinations from two patches) was estimated for NCX1.3, similar to that of peak currents for both exchangers.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

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.
Mentions: Fig. 3 summarizes the Na+i dependence of peak and steady state outward Na+–Ca2+ exchange currents derived from pooled data obtained with NCX1.3 and NCX1.4 in the presence of 1 μM regulatory Ca2+i, as above. Currents were normalized to the values obtained at 100 mM Nai+. For both isoforms, peak currents progressively increased with increasing [Na+]i. Estimates of exchanger affinity for Na+i based on peak current measurements provided nearly identical values of Kd (33 ± 5 mM, n = 4 vs. 31 ± 4 mM, n = 6, for NCX1.3 and NCX1.4, respectively). For comparison, the peak current-derived Na+i affinity of the cardiac exchanger, NCX1.1, is ≈27 mM (Matsuoka et al. 1995, Matsuoka et al. 1997). With respect to the Na+i affinity derived from measurements of steady state currents, however, a Kd value of 16 ± 1 mM (n = 6) was obtained for NCX1.4, whereas the NCX1.3 isoform appeared to be nearly Na+i independent over the concentration range examined. Thus, a Kd value could not be estimated. However, after treatment with α-chymotrypsin, a Kd value of 26 ± 1 mM (three determinations from two patches) was estimated for NCX1.3, similar to that of peak currents for both exchangers.

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
Related in: MedlinePlus