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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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IV relationships for NCX1.4 and NCX1.3. Each voltage-clamp increment (10-mV steps from −100 to 100 mV) was initiated from a holding potential of 0 mV. IV records obtained at a during perfusion with 100 mM Li+i-containing solution were subtracted from those obtained at b during perfusion with 100 mM Na+i-containing solution. Regulatory Ca2+i (1 μM) was present throughout the current recordings. Pooled data shown (bottom) are mean ± SEM from four patches for NCX1.4 and three patches for NCX1.3.
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Figure 5: IV relationships for NCX1.4 and NCX1.3. Each voltage-clamp increment (10-mV steps from −100 to 100 mV) was initiated from a holding potential of 0 mV. IV records obtained at a during perfusion with 100 mM Li+i-containing solution were subtracted from those obtained at b during perfusion with 100 mM Na+i-containing solution. Regulatory Ca2+i (1 μM) was present throughout the current recordings. Pooled data shown (bottom) are mean ± SEM from four patches for NCX1.4 and three patches for NCX1.3.

Mentions: Fig. 5 illustrates the current–voltage (IV)1 relationships for NCX1.3 and NCX1.4. Outward currents were activated by switching from 100 mM Li+i-containing perfusing solution to 100 mM Na+i, and 1 μM regulatory Ca2+i was present throughout. The IV relationship was determined before (a) and during (b) exchange current activation, and the former values were subtracted from the latter. From a holding potential of 0 mV, 40-ms voltage steps, in 10-mV increments, were applied from −100 to +100 mV, with a return to the holding potential between each step. Pooled data shown in Fig. 5 (bottom) are from three NCX1.3 and four NCX1.4 patches, with currents normalized to the values obtained at 0 mV. Note that a reversal potential is not observed under these conditions as the pipette solution does not contain Na+o. The exchanger isoforms exhibited similar IV relationships to that observed for the cardiac exchanger, NCX1.1 (Matsuoka and Hilgemann 1992). We did not observe significant differences in the voltage dependency of the kidney exchanger, in contrast to an earlier report (Ruknudin et al. 1998).


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)

IV relationships for NCX1.4 and NCX1.3. Each voltage-clamp increment (10-mV steps from −100 to 100 mV) was initiated from a holding potential of 0 mV. IV records obtained at a during perfusion with 100 mM Li+i-containing solution were subtracted from those obtained at b during perfusion with 100 mM Na+i-containing solution. Regulatory Ca2+i (1 μM) was present throughout the current recordings. Pooled data shown (bottom) are mean ± SEM from four patches for NCX1.4 and three patches for NCX1.3.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2230537&req=5

Figure 5: IV relationships for NCX1.4 and NCX1.3. Each voltage-clamp increment (10-mV steps from −100 to 100 mV) was initiated from a holding potential of 0 mV. IV records obtained at a during perfusion with 100 mM Li+i-containing solution were subtracted from those obtained at b during perfusion with 100 mM Na+i-containing solution. Regulatory Ca2+i (1 μM) was present throughout the current recordings. Pooled data shown (bottom) are mean ± SEM from four patches for NCX1.4 and three patches for NCX1.3.
Mentions: Fig. 5 illustrates the current–voltage (IV)1 relationships for NCX1.3 and NCX1.4. Outward currents were activated by switching from 100 mM Li+i-containing perfusing solution to 100 mM Na+i, and 1 μM regulatory Ca2+i was present throughout. The IV relationship was determined before (a) and during (b) exchange current activation, and the former values were subtracted from the latter. From a holding potential of 0 mV, 40-ms voltage steps, in 10-mV increments, were applied from −100 to +100 mV, with a return to the holding potential between each step. Pooled data shown in Fig. 5 (bottom) are from three NCX1.3 and four NCX1.4 patches, with currents normalized to the values obtained at 0 mV. Note that a reversal potential is not observed under these conditions as the pipette solution does not contain Na+o. The exchanger isoforms exhibited similar IV relationships to that observed for the cardiac exchanger, NCX1.1 (Matsuoka and Hilgemann 1992). We did not observe significant differences in the voltage dependency of the kidney exchanger, in contrast to an earlier report (Ruknudin et al. 1998).

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