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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 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.
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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.

Mentions: Fig. 4 illustrates the Na+i dependence of the NCX1 isoforms in terms of their ratios of steady state to peak currents, or Fss values (Omelchenko et al. 1998). This fraction is a sensitive measure of the extent of I1 inactivation and can provide insight into the stability of the I1 inactive complex. Both splice variants exhibit a decrease in Fss with increasing [Na+]i, typical of I1-inactivated Na+–Ca2+ exchangers, and reflecting entry into the I1 state from the three Na+i-loaded configuration of the exchanger (Hilgemann et al. 1992b). However, for [Na+]i ≥ 25 mM, the decrease in Fss is more pronounced for NCX1.3 than for NCX1.4. For example, Fss values calculated from currents acquired in response to the application of 100 mM Na+i were 0.24 ± 0.03 and 0.07 ± 0.01 (n = 20 and 25 determinations from 14 patches) for NCX1.4 and NCX1.3, respectively. Note that for NCX1.3, the extent of I1 inactivation is ≈90% at high [Na+]i, whereas at 10 mM Na+i it is ≈20%. This substantial inactivation of NCX1.3-mediated transport is likely to be responsible for the peculiar nature of the Na+i dependence of its steady state currents, as shown in Fig. 3. That is, the increase in steady state current in response to Na+i appears to be largely offset by the extensive inactivation that occurs as [Na+]i is raised. Consequently, steady state Na+–Ca2+ exchange currents mediated by NCX1.3 do not exhibit a hyperbolic response to rising [Na+]i unless this regulatory mechanism is eliminated by proteolysis with α-chymotrypsin.


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 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.
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Related In: Results  -  Collection

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

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.
Mentions: Fig. 4 illustrates the Na+i dependence of the NCX1 isoforms in terms of their ratios of steady state to peak currents, or Fss values (Omelchenko et al. 1998). This fraction is a sensitive measure of the extent of I1 inactivation and can provide insight into the stability of the I1 inactive complex. Both splice variants exhibit a decrease in Fss with increasing [Na+]i, typical of I1-inactivated Na+–Ca2+ exchangers, and reflecting entry into the I1 state from the three Na+i-loaded configuration of the exchanger (Hilgemann et al. 1992b). However, for [Na+]i ≥ 25 mM, the decrease in Fss is more pronounced for NCX1.3 than for NCX1.4. For example, Fss values calculated from currents acquired in response to the application of 100 mM Na+i were 0.24 ± 0.03 and 0.07 ± 0.01 (n = 20 and 25 determinations from 14 patches) for NCX1.4 and NCX1.3, respectively. Note that for NCX1.3, the extent of I1 inactivation is ≈90% at high [Na+]i, whereas at 10 mM Na+i it is ≈20%. This substantial inactivation of NCX1.3-mediated transport is likely to be responsible for the peculiar nature of the Na+i dependence of its steady state currents, as shown in Fig. 3. That is, the increase in steady state current in response to Na+i appears to be largely offset by the extensive inactivation that occurs as [Na+]i is raised. Consequently, steady state Na+–Ca2+ exchange currents mediated by NCX1.3 do not exhibit a hyperbolic response to rising [Na+]i unless this regulatory mechanism is eliminated by proteolysis with α-chymotrypsin.

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