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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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Ca2+i regulation of outward Na+–Ca2+ exchange currents for NCX1.4 and NCX1.3. Representative records are shown for NCX1.4 and NCX1.3, where currents were activated by applying 100 mM Na+i in the presence of 1 μM Ca2+i. Regulatory Ca2+i was present for 32–48 s before the application of transport Na+i. Upon approaching steady state current levels, Ca2+i was removed for 16 s, and then reapplied for a further 16-s interval before deactivating exchange current by returning to a Li+i-based perfusing solution. The traces shown are typical of five patches for NCX1.4 and three patches for NCX1.3.
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Figure 6: Ca2+i regulation of outward Na+–Ca2+ exchange currents for NCX1.4 and NCX1.3. Representative records are shown for NCX1.4 and NCX1.3, where currents were activated by applying 100 mM Na+i in the presence of 1 μM Ca2+i. Regulatory Ca2+i was present for 32–48 s before the application of transport Na+i. Upon approaching steady state current levels, Ca2+i was removed for 16 s, and then reapplied for a further 16-s interval before deactivating exchange current by returning to a Li+i-based perfusing solution. The traces shown are typical of five patches for NCX1.4 and three patches for NCX1.3.

Mentions: Fig. 6 illustrates the effects of removal and reapplication of regulatory Ca2+i on outward Na+–Ca2+ exchange currents mediated by NCX1.3 and NCX1.4. Currents were elicited by applying 100 mM Na+i, with 1 μM Ca2+i present before activation. Regulatory Ca2+i was then removed and reapplied in the middle of the current trace in the continuous presence of 100 mM Na+i. For NCX1.4, Ca2+i removal led to rapid and nearly complete inhibition of outward exchange current, with a half-time for current decay of 0.52 ± 0.05 s (n = 5). Similarly, steady state levels were rapidly restored when Ca2+i was reapplied, with a half-time of 0.49 ± 0.14 s (n = 5). For comparison with NCX1.1, the equivalent protocols yield half-times for loss and reacquisition of steady state current levels of 10.8 and 7.5 s, respectively (Matsuoka et al. 1995). Thus, steady state Na+–Ca2+ exchange currents for NCX1.4 exhibits considerably faster responses to this experimental maneuver than those observed for the cardiac exchanger. In contrast, with NCX1.3, half-times for loss and restoration of steady state current after removal and reapplication of Ca2+i could not be determined due to the negligible steady state currents generated even in the presence of regulatory Ca2+i.


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 regulation of outward Na+–Ca2+ exchange currents for NCX1.4 and NCX1.3. Representative records are shown for NCX1.4 and NCX1.3, where currents were activated by applying 100 mM Na+i in the presence of 1 μM Ca2+i. Regulatory Ca2+i was present for 32–48 s before the application of transport Na+i. Upon approaching steady state current levels, Ca2+i was removed for 16 s, and then reapplied for a further 16-s interval before deactivating exchange current by returning to a Li+i-based perfusing solution. The traces shown are typical of five patches for NCX1.4 and three patches for NCX1.3.
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Related In: Results  -  Collection

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

Figure 6: Ca2+i regulation of outward Na+–Ca2+ exchange currents for NCX1.4 and NCX1.3. Representative records are shown for NCX1.4 and NCX1.3, where currents were activated by applying 100 mM Na+i in the presence of 1 μM Ca2+i. Regulatory Ca2+i was present for 32–48 s before the application of transport Na+i. Upon approaching steady state current levels, Ca2+i was removed for 16 s, and then reapplied for a further 16-s interval before deactivating exchange current by returning to a Li+i-based perfusing solution. The traces shown are typical of five patches for NCX1.4 and three patches for NCX1.3.
Mentions: Fig. 6 illustrates the effects of removal and reapplication of regulatory Ca2+i on outward Na+–Ca2+ exchange currents mediated by NCX1.3 and NCX1.4. Currents were elicited by applying 100 mM Na+i, with 1 μM Ca2+i present before activation. Regulatory Ca2+i was then removed and reapplied in the middle of the current trace in the continuous presence of 100 mM Na+i. For NCX1.4, Ca2+i removal led to rapid and nearly complete inhibition of outward exchange current, with a half-time for current decay of 0.52 ± 0.05 s (n = 5). Similarly, steady state levels were rapidly restored when Ca2+i was reapplied, with a half-time of 0.49 ± 0.14 s (n = 5). For comparison with NCX1.1, the equivalent protocols yield half-times for loss and reacquisition of steady state current levels of 10.8 and 7.5 s, respectively (Matsuoka et al. 1995). Thus, steady state Na+–Ca2+ exchange currents for NCX1.4 exhibits considerably faster responses to this experimental maneuver than those observed for the cardiac exchanger. In contrast, with NCX1.3, half-times for loss and restoration of steady state current after removal and reapplication of Ca2+i could not be determined due to the negligible steady state currents generated even in the presence of regulatory Ca2+i.

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