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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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The effect of regulatory Ca2+i on recovery of NCX1.4 and NCX1.3 from Na+i-dependent inactivation using paired-pulse stimulation. The indicated concentrations of regulatory Ca2+i were present throughout the current measurements. The first pulse was activated by applying 100 mM Na+i for 32 s, followed by a 4-s recovery interval. A second, test pulse was then elicited by reapplication of 100 mM Na+i. The graph (bottom) shows representative results from four patches each of NCX1.4 and NCX1.3 over a range of regulatory [Ca2+]i.
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Figure 10: The effect of regulatory Ca2+i on recovery of NCX1.4 and NCX1.3 from Na+i-dependent inactivation using paired-pulse stimulation. The indicated concentrations of regulatory Ca2+i were present throughout the current measurements. The first pulse was activated by applying 100 mM Na+i for 32 s, followed by a 4-s recovery interval. A second, test pulse was then elicited by reapplication of 100 mM Na+i. The graph (bottom) shows representative results from four patches each of NCX1.4 and NCX1.3 over a range of regulatory [Ca2+]i.

Mentions: Fig. 10 illustrates representative current recordings obtained for paired-pulse experiments conducted at two different regulatory Ca2+ concentrations (0.3 and 10 μM Ca2+i). In each case, currents were activated by 100 mM Na+i and transported Ca2+o in the pipette was constant at 8 mM. Regulatory Ca2+i, at the indicated concentration, was present throughout the entire paired-pulse trials. For NCX1.4, the second test pulse is substantially reduced after a 4-s interval at 0.3 μM Ca2+i, whereas the two pulses are nearly identical in magnitude at 10 μM Ca2+i. This behavior illustrates the ability of Ca2+i to accelerate exit from, and/or reduce entry into, the I1 inactive state, and is typical of NCX1.1 (Hilgemann et al. 1992a). With NCX1.3, however, I1 inactivation is only weakly affected by regulatory Ca2+i, and substantial inactivation is observed for paired-pulses even at 10 μM. This difference is shown graphically (Fig. 10, bottom) for representative data over a range of regulatory [Ca2+]i's for a 4-s inter-pulse interval. The parameter chosen to evaluate recovery, (Ipeak − Iss, pulse 2)/(Ipeak − Iss, pulse 1), was used so that recovery values would fall between 0 and 100%. That is, steady state currents are subtracted so that only the portion of current that inactivates is analyzed in terms of its recovery. This behavior was confirmed in a total of four patches each for NCX1.3 and NCX1.4.


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)

The effect of regulatory Ca2+i on recovery of NCX1.4 and NCX1.3 from Na+i-dependent inactivation using paired-pulse stimulation. The indicated concentrations of regulatory Ca2+i were present throughout the current measurements. The first pulse was activated by applying 100 mM Na+i for 32 s, followed by a 4-s recovery interval. A second, test pulse was then elicited by reapplication of 100 mM Na+i. The graph (bottom) shows representative results from four patches each of NCX1.4 and NCX1.3 over a range of regulatory [Ca2+]i.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2230537&req=5

Figure 10: The effect of regulatory Ca2+i on recovery of NCX1.4 and NCX1.3 from Na+i-dependent inactivation using paired-pulse stimulation. The indicated concentrations of regulatory Ca2+i were present throughout the current measurements. The first pulse was activated by applying 100 mM Na+i for 32 s, followed by a 4-s recovery interval. A second, test pulse was then elicited by reapplication of 100 mM Na+i. The graph (bottom) shows representative results from four patches each of NCX1.4 and NCX1.3 over a range of regulatory [Ca2+]i.
Mentions: Fig. 10 illustrates representative current recordings obtained for paired-pulse experiments conducted at two different regulatory Ca2+ concentrations (0.3 and 10 μM Ca2+i). In each case, currents were activated by 100 mM Na+i and transported Ca2+o in the pipette was constant at 8 mM. Regulatory Ca2+i, at the indicated concentration, was present throughout the entire paired-pulse trials. For NCX1.4, the second test pulse is substantially reduced after a 4-s interval at 0.3 μM Ca2+i, whereas the two pulses are nearly identical in magnitude at 10 μM Ca2+i. This behavior illustrates the ability of Ca2+i to accelerate exit from, and/or reduce entry into, the I1 inactive state, and is typical of NCX1.1 (Hilgemann et al. 1992a). With NCX1.3, however, I1 inactivation is only weakly affected by regulatory Ca2+i, and substantial inactivation is observed for paired-pulses even at 10 μM. This difference is shown graphically (Fig. 10, bottom) for representative data over a range of regulatory [Ca2+]i's for a 4-s inter-pulse interval. The parameter chosen to evaluate recovery, (Ipeak − Iss, pulse 2)/(Ipeak − Iss, pulse 1), was used so that recovery values would fall between 0 and 100%. That is, steady state currents are subtracted so that only the portion of current that inactivates is analyzed in terms of its recovery. This behavior was confirmed in a total of four patches each for NCX1.3 and NCX1.4.

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