Limits...
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.

Show MeSH

Related in: MedlinePlus

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.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2230537&req=5

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.

Mentions: Fig. 7 shows representative traces that illustrate the dependence of outward Na+–Ca2+ exchange currents mediated by NCX1.3 and NCX1.4 upon [Ca2+]i. Regulatory Ca2+i, at the indicated concentrations, was present for 32–48 s before, during, and after current activation with 100 mM Na+i. For both isoforms, exchange currents are augmented by Ca2+i. In particular, NCX1.4 behaves similar to the cardiac exchanger, NCX1.1, in that regulatory Ca2+i not only stimulates exchange activity, but also alleviates I1 inactivation. At 10 μM Ca2+i, Na+i-dependent inactivation is nearly eliminated and the current recording adopts a square appearance. With NCX1.3, however, I1 inactivation is still prominent at 10 μM Ca2+i, in sharp distinction to both NCX1.4 and NCX1.1. That is, regulatory Ca2+i is not only incapable of alleviating I1 inactivation for NCX1.3, it appears to inhibit outward current generation. These relationships are illustrated graphically in Fig. 8.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

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.
Mentions: Fig. 7 shows representative traces that illustrate the dependence of outward Na+–Ca2+ exchange currents mediated by NCX1.3 and NCX1.4 upon [Ca2+]i. Regulatory Ca2+i, at the indicated concentrations, was present for 32–48 s before, during, and after current activation with 100 mM Na+i. For both isoforms, exchange currents are augmented by Ca2+i. In particular, NCX1.4 behaves similar to the cardiac exchanger, NCX1.1, in that regulatory Ca2+i not only stimulates exchange activity, but also alleviates I1 inactivation. At 10 μM Ca2+i, Na+i-dependent inactivation is nearly eliminated and the current recording adopts a square appearance. With NCX1.3, however, I1 inactivation is still prominent at 10 μM Ca2+i, in sharp distinction to both NCX1.4 and NCX1.1. That is, regulatory Ca2+i is not only incapable of alleviating I1 inactivation for NCX1.3, it appears to inhibit outward current generation. These relationships are illustrated graphically in Fig. 8.

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