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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.

Show MeSH
Sequence comparison of the alternatively spliced regions of NCX1.4 and NCX1.3. Single-letter amino acid code is used and identities are boxed. The alternative splicing region of NCX1 is located within the COOH-terminal third of its large intracellular loop. Expression of exons A and B is mutually exclusive.
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Figure 1: Sequence comparison of the alternatively spliced regions of NCX1.4 and NCX1.3. Single-letter amino acid code is used and identities are boxed. The alternative splicing region of NCX1 is located within the COOH-terminal third of its large intracellular loop. Expression of exons A and B is mutually exclusive.

Mentions: Inspection of the aligned sequences of exons A (encoding 35 amino acids) and B (encoding 34 amino acids) in Fig. 1 reveals substantial similarity between NCX1.3 and NCX1.4. 13 identities are found and the exons are ≈63% similar with respect to conservative substitutions, with the greatest similarity occurring towards their NH2 termini. Charge reversal occurs at two positions, substitution of charged for neutral residues is observed at six, polar residues coincide with hydrophobics at three, and the overall electric charge of exon A is −2, whereas exon B is +1. Exon B encodes a cysteine residue at position 585 (Nicoll et al. 1990), whereas no cysteines are found in any other exons of the alternatively spliced region of NCX1. Although the specific contribution of dissimilar amino acids between NCX1.3 and NCX1.4 has not been determined, the net effect of interchanging exons A and B is substantially altered ionic regulatory behavior.


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)

Sequence comparison of the alternatively spliced regions of NCX1.4 and NCX1.3. Single-letter amino acid code is used and identities are boxed. The alternative splicing region of NCX1 is located within the COOH-terminal third of its large intracellular loop. Expression of exons A and B is mutually exclusive.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Sequence comparison of the alternatively spliced regions of NCX1.4 and NCX1.3. Single-letter amino acid code is used and identities are boxed. The alternative splicing region of NCX1 is located within the COOH-terminal third of its large intracellular loop. Expression of exons A and B is mutually exclusive.
Mentions: Inspection of the aligned sequences of exons A (encoding 35 amino acids) and B (encoding 34 amino acids) in Fig. 1 reveals substantial similarity between NCX1.3 and NCX1.4. 13 identities are found and the exons are ≈63% similar with respect to conservative substitutions, with the greatest similarity occurring towards their NH2 termini. Charge reversal occurs at two positions, substitution of charged for neutral residues is observed at six, polar residues coincide with hydrophobics at three, and the overall electric charge of exon A is −2, whereas exon B is +1. Exon B encodes a cysteine residue at position 585 (Nicoll et al. 1990), whereas no cysteines are found in any other exons of the alternatively spliced region of NCX1. Although the specific contribution of dissimilar amino acids between NCX1.3 and NCX1.4 has not been determined, the net effect of interchanging exons A and B is substantially altered ionic regulatory behavior.

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