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Calmodulin Interacts with the Sodium/Calcium Exchanger NCX1 to Regulate Activity.

Chou AC, Ju YT, Pan CY - PLoS ONE (2015)

Bottom Line: Mutating the first two conserved a.a. in NCX1.1 decreased exchange activity; mutating the 3rd or 4th conserved a.a. residues did not alter exchange activity, but CaM co-expression suppressed activity.Mutating the 2nd and 3rd conserved a.a. residues in NCX1.3 decreased exchange activity.Taken together, our results demonstrate that CaM senses changes in [Ca2+]i and binds to the cytoplasmic loop of NCX1 to regulate exchange activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Science, National Taiwan University, Taipei, Taiwan.

ABSTRACT
Changes in intracellular Ca2+ concentrations ([Ca2+]i) are an important signal for various physiological activities. The Na+/Ca2+ exchangers (NCX) at the plasma membrane transport Ca2+ into or out of the cell according to the electrochemical gradients of Na+ and Ca2+ to modulate [Ca2+]i homeostasis. Calmodulin (CaM) senses [Ca2+]i changes and relays Ca2+ signals by binding to target proteins such as channels and transporters. However, it is not clear how calmodulin modulates NCX activity. Using CaM as a bait, we pulled down the intracellular loops subcloned from the NCX1 splice variants NCX1.1 and NCX1.3. This interaction requires both Ca2+ and a putative CaM-binding segment (CaMS). To determine whether CaM modulates NCX activity, we co-expressed NCX1 splice variants with CaM or CaM1234 (a Ca2+-binding deficient mutant) in HEK293T cells and measured the increase in [Ca2+]i contributed by the influx of Ca2+ through NCX. Deleting the CaMS from NCX1.1 and NCX1.3 attenuated exchange activity and decreased membrane localization. Without the mutually exclusive exon, the exchange activity was decreased and could be partially rescued by CaM1234. Point-mutations at any of the 4 conserved a.a. residues in the CaMS had differential effects in NCX1.1 and NCX1.3. Mutating the first two conserved a.a. in NCX1.1 decreased exchange activity; mutating the 3rd or 4th conserved a.a. residues did not alter exchange activity, but CaM co-expression suppressed activity. Mutating the 2nd and 3rd conserved a.a. residues in NCX1.3 decreased exchange activity. Taken together, our results demonstrate that CaM senses changes in [Ca2+]i and binds to the cytoplasmic loop of NCX1 to regulate exchange activity.

No MeSH data available.


Related in: MedlinePlus

Mutations in the conserved a.a. residues of the NCX1.3 CaMS affect exchange activity.The conserved 1st, 5th, 8th, and 14th a.a. residues of the CaMS of NCX1.3 was independently mutated from F, V, L, and L, to A (F1A), A (V5A), D (L8D), and D (L14D), respectively. We measured the reverse-mode exchange activity of HEK293T cells expressing constructs with or without co-expression of CaM or CaM1234. A. Merged images of cells expressing various NCX1.1 mutants stained with phalloidin (Red), DAPI (Blue), and an antibody against the V5 epitope tag (Green) to visualize actin filaments, nuclear DNA, and the exchanger, respectively. Scale bar: 10 μm. B. The [Ca2+]i responses of single HEK293T cells expressing various constructs. The black lines under each trace indicate the period of NMG perfusion. C. Average [Ca2+]i elevations in cells expressing various NCX1.3 mutants with or without co-expression of CaM or CaM1234. The digits in each column indicate the number of cells in each group. Data shown are the mean ± SEM pooled from three different batches of cells and analyzed by a one-way ANOVA with Fisher's post hoc test (***: p < 0.001 when compared with the corresponding group expressing only the NCX1.3 mutant).
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pone.0138856.g007: Mutations in the conserved a.a. residues of the NCX1.3 CaMS affect exchange activity.The conserved 1st, 5th, 8th, and 14th a.a. residues of the CaMS of NCX1.3 was independently mutated from F, V, L, and L, to A (F1A), A (V5A), D (L8D), and D (L14D), respectively. We measured the reverse-mode exchange activity of HEK293T cells expressing constructs with or without co-expression of CaM or CaM1234. A. Merged images of cells expressing various NCX1.1 mutants stained with phalloidin (Red), DAPI (Blue), and an antibody against the V5 epitope tag (Green) to visualize actin filaments, nuclear DNA, and the exchanger, respectively. Scale bar: 10 μm. B. The [Ca2+]i responses of single HEK293T cells expressing various constructs. The black lines under each trace indicate the period of NMG perfusion. C. Average [Ca2+]i elevations in cells expressing various NCX1.3 mutants with or without co-expression of CaM or CaM1234. The digits in each column indicate the number of cells in each group. Data shown are the mean ± SEM pooled from three different batches of cells and analyzed by a one-way ANOVA with Fisher's post hoc test (***: p < 0.001 when compared with the corresponding group expressing only the NCX1.3 mutant).

Mentions: The 4 conserved a.a. residues of the CaMS in NCX1.3 were individually mutated as F1A, V5A, L8D, and L14D to characterize the roles of these a.a. residues in modulating NCX1.3 activity (Fig 7). Immunostaining revealed that these NCX1.3 mutants were present at the cell boundary and that they overlapped with F-actin staining. The line intensity profiles also suggested a plasma membrane distribution for these mutants (S4 Fig). These mutants all displayed rNCX activity upon NMG perfusion, but NCX1.3V5A and NCX1.3L8D had lower [Ca2+]i elevations than those of NCX1.3F1A and NCX1.3L14D. The average [Ca2+]i changes in cells expressing NCX1.3F1A and NCX1.3L14D were 1160 ± 158 (n = 16) and 829 ± 90 (n = 49) nM, respectively, similar to that of NCX1.3 (902 ± 100 nM, Fig 4C). In contrast, the NCX1.3V5A and NCX1.3L8D had a significantly smaller [Ca2+]i changes of 550 ± 56 (n = 45, p < 0,01) and 587 ± 59 (n = 51, p < 0,01) nM, respectively, compared with the wild type (Fig 4C). CaM and CaM1234 co-expression did not affect the exchange activity of these mutants, except for NCX1.3L8D, whose activity was slightly, but not significantly attenuated to 400 ± 78 nM (n = 25, p = 0.07) by CaM and significantly increased to 1013 ± 142 nM (n = 29, p < 0.001) by CaM1234. These results demonstrate that these conserved a.a. residues in the CaMS have differential effects on CaM-mediated regulation.


Calmodulin Interacts with the Sodium/Calcium Exchanger NCX1 to Regulate Activity.

Chou AC, Ju YT, Pan CY - PLoS ONE (2015)

Mutations in the conserved a.a. residues of the NCX1.3 CaMS affect exchange activity.The conserved 1st, 5th, 8th, and 14th a.a. residues of the CaMS of NCX1.3 was independently mutated from F, V, L, and L, to A (F1A), A (V5A), D (L8D), and D (L14D), respectively. We measured the reverse-mode exchange activity of HEK293T cells expressing constructs with or without co-expression of CaM or CaM1234. A. Merged images of cells expressing various NCX1.1 mutants stained with phalloidin (Red), DAPI (Blue), and an antibody against the V5 epitope tag (Green) to visualize actin filaments, nuclear DNA, and the exchanger, respectively. Scale bar: 10 μm. B. The [Ca2+]i responses of single HEK293T cells expressing various constructs. The black lines under each trace indicate the period of NMG perfusion. C. Average [Ca2+]i elevations in cells expressing various NCX1.3 mutants with or without co-expression of CaM or CaM1234. The digits in each column indicate the number of cells in each group. Data shown are the mean ± SEM pooled from three different batches of cells and analyzed by a one-way ANOVA with Fisher's post hoc test (***: p < 0.001 when compared with the corresponding group expressing only the NCX1.3 mutant).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4589332&req=5

pone.0138856.g007: Mutations in the conserved a.a. residues of the NCX1.3 CaMS affect exchange activity.The conserved 1st, 5th, 8th, and 14th a.a. residues of the CaMS of NCX1.3 was independently mutated from F, V, L, and L, to A (F1A), A (V5A), D (L8D), and D (L14D), respectively. We measured the reverse-mode exchange activity of HEK293T cells expressing constructs with or without co-expression of CaM or CaM1234. A. Merged images of cells expressing various NCX1.1 mutants stained with phalloidin (Red), DAPI (Blue), and an antibody against the V5 epitope tag (Green) to visualize actin filaments, nuclear DNA, and the exchanger, respectively. Scale bar: 10 μm. B. The [Ca2+]i responses of single HEK293T cells expressing various constructs. The black lines under each trace indicate the period of NMG perfusion. C. Average [Ca2+]i elevations in cells expressing various NCX1.3 mutants with or without co-expression of CaM or CaM1234. The digits in each column indicate the number of cells in each group. Data shown are the mean ± SEM pooled from three different batches of cells and analyzed by a one-way ANOVA with Fisher's post hoc test (***: p < 0.001 when compared with the corresponding group expressing only the NCX1.3 mutant).
Mentions: The 4 conserved a.a. residues of the CaMS in NCX1.3 were individually mutated as F1A, V5A, L8D, and L14D to characterize the roles of these a.a. residues in modulating NCX1.3 activity (Fig 7). Immunostaining revealed that these NCX1.3 mutants were present at the cell boundary and that they overlapped with F-actin staining. The line intensity profiles also suggested a plasma membrane distribution for these mutants (S4 Fig). These mutants all displayed rNCX activity upon NMG perfusion, but NCX1.3V5A and NCX1.3L8D had lower [Ca2+]i elevations than those of NCX1.3F1A and NCX1.3L14D. The average [Ca2+]i changes in cells expressing NCX1.3F1A and NCX1.3L14D were 1160 ± 158 (n = 16) and 829 ± 90 (n = 49) nM, respectively, similar to that of NCX1.3 (902 ± 100 nM, Fig 4C). In contrast, the NCX1.3V5A and NCX1.3L8D had a significantly smaller [Ca2+]i changes of 550 ± 56 (n = 45, p < 0,01) and 587 ± 59 (n = 51, p < 0,01) nM, respectively, compared with the wild type (Fig 4C). CaM and CaM1234 co-expression did not affect the exchange activity of these mutants, except for NCX1.3L8D, whose activity was slightly, but not significantly attenuated to 400 ± 78 nM (n = 25, p = 0.07) by CaM and significantly increased to 1013 ± 142 nM (n = 29, p < 0.001) by CaM1234. These results demonstrate that these conserved a.a. residues in the CaMS have differential effects on CaM-mediated regulation.

Bottom Line: Mutating the first two conserved a.a. in NCX1.1 decreased exchange activity; mutating the 3rd or 4th conserved a.a. residues did not alter exchange activity, but CaM co-expression suppressed activity.Mutating the 2nd and 3rd conserved a.a. residues in NCX1.3 decreased exchange activity.Taken together, our results demonstrate that CaM senses changes in [Ca2+]i and binds to the cytoplasmic loop of NCX1 to regulate exchange activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Science, National Taiwan University, Taipei, Taiwan.

ABSTRACT
Changes in intracellular Ca2+ concentrations ([Ca2+]i) are an important signal for various physiological activities. The Na+/Ca2+ exchangers (NCX) at the plasma membrane transport Ca2+ into or out of the cell according to the electrochemical gradients of Na+ and Ca2+ to modulate [Ca2+]i homeostasis. Calmodulin (CaM) senses [Ca2+]i changes and relays Ca2+ signals by binding to target proteins such as channels and transporters. However, it is not clear how calmodulin modulates NCX activity. Using CaM as a bait, we pulled down the intracellular loops subcloned from the NCX1 splice variants NCX1.1 and NCX1.3. This interaction requires both Ca2+ and a putative CaM-binding segment (CaMS). To determine whether CaM modulates NCX activity, we co-expressed NCX1 splice variants with CaM or CaM1234 (a Ca2+-binding deficient mutant) in HEK293T cells and measured the increase in [Ca2+]i contributed by the influx of Ca2+ through NCX. Deleting the CaMS from NCX1.1 and NCX1.3 attenuated exchange activity and decreased membrane localization. Without the mutually exclusive exon, the exchange activity was decreased and could be partially rescued by CaM1234. Point-mutations at any of the 4 conserved a.a. residues in the CaMS had differential effects in NCX1.1 and NCX1.3. Mutating the first two conserved a.a. in NCX1.1 decreased exchange activity; mutating the 3rd or 4th conserved a.a. residues did not alter exchange activity, but CaM co-expression suppressed activity. Mutating the 2nd and 3rd conserved a.a. residues in NCX1.3 decreased exchange activity. Taken together, our results demonstrate that CaM senses changes in [Ca2+]i and binds to the cytoplasmic loop of NCX1 to regulate exchange activity.

No MeSH data available.


Related in: MedlinePlus