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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 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.
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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.

Mentions: We examined the [Na+]i dependence of peak and steady state outward Na+–Ca2+ exchange currents mediated by NCX1.4 and NCX1.3 to obtain estimates of Na+ transport affinities, as well as the rate and extent of I1 inactivation. Fig. 2 shows representative current traces obtained in response to the rapid (i.e., <200 ms) application of 10–100 mM Na+i to the cytoplasmic surface of the patches in the continuous presence of 1 μM regulatory Ca2+i. Transport Ca2+o in the pipette was constant at 8 mM. After each current activation event, patches were allowed to recover for 32–48 s in Li+i-containing solution plus 1 μM Ca2+i before delivery of the next Na+i pulse. With increasing [Na+]i, the isoforms exhibited similar increases in peak current and in the extent of current inactivation, characteristic of Na+i-dependent, or I1, inactivation (Hilgemann et al. 1992b). In response to the application of 100 mM Na+i at 1-μM regulatory Ca2+i, the rate of inactivation of NCX1.4 was marginally faster than that observed for NCX1.3 (0.29 ± 0.03 s−1, n = 18 vs. 0.22 ± 0.03 s−1, n = 11, respectively, NS). However, the major difference between the splice variants was in steady state current levels produced in response to changes in [Na+]i. Whereas NCX1.4 exhibited a [Na+]i-dependent increase in steady state current levels, similar to that observed with the cardiac Na+–Ca2+ exchanger, NCX1.1 (Matsuoka et al. 1995), steady state currents mediated by NCX1.3 were mainly insensitive to changes in [Na+]i over the concentration range examined (10–100 mM). However, this behavior could be abolished by treatment of the patch with α-chymotrypsin (Fig. 2, bottom), a procedure known to deregulate Na+–Ca2+ exchangers (Hilgemann 1990). After proteolysis, the Na+i dependence of NCX1.3 became hyperbolic, similar to that observed for peak currents with NCX1.4. Thus, ionic regulation alters the apparent Na+i affinity of steady state Na+–Ca2+ exchange currents for NCX1.3.


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 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.
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Related In: Results  -  Collection

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

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.
Mentions: We examined the [Na+]i dependence of peak and steady state outward Na+–Ca2+ exchange currents mediated by NCX1.4 and NCX1.3 to obtain estimates of Na+ transport affinities, as well as the rate and extent of I1 inactivation. Fig. 2 shows representative current traces obtained in response to the rapid (i.e., <200 ms) application of 10–100 mM Na+i to the cytoplasmic surface of the patches in the continuous presence of 1 μM regulatory Ca2+i. Transport Ca2+o in the pipette was constant at 8 mM. After each current activation event, patches were allowed to recover for 32–48 s in Li+i-containing solution plus 1 μM Ca2+i before delivery of the next Na+i pulse. With increasing [Na+]i, the isoforms exhibited similar increases in peak current and in the extent of current inactivation, characteristic of Na+i-dependent, or I1, inactivation (Hilgemann et al. 1992b). In response to the application of 100 mM Na+i at 1-μM regulatory Ca2+i, the rate of inactivation of NCX1.4 was marginally faster than that observed for NCX1.3 (0.29 ± 0.03 s−1, n = 18 vs. 0.22 ± 0.03 s−1, n = 11, respectively, NS). However, the major difference between the splice variants was in steady state current levels produced in response to changes in [Na+]i. Whereas NCX1.4 exhibited a [Na+]i-dependent increase in steady state current levels, similar to that observed with the cardiac Na+–Ca2+ exchanger, NCX1.1 (Matsuoka et al. 1995), steady state currents mediated by NCX1.3 were mainly insensitive to changes in [Na+]i over the concentration range examined (10–100 mM). However, this behavior could be abolished by treatment of the patch with α-chymotrypsin (Fig. 2, bottom), a procedure known to deregulate Na+–Ca2+ exchangers (Hilgemann 1990). After proteolysis, the Na+i dependence of NCX1.3 became hyperbolic, similar to that observed for peak currents with NCX1.4. Thus, ionic regulation alters the apparent Na+i affinity of steady state Na+–Ca2+ exchange currents for NCX1.3.

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