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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

Each of the conserved a.a. residues in the NCX1.1 CaMS has differential effects on exchange activity.The conserved 1st, 5th, 8th, and 14th a.a. residues of the CaMS in NCX1.1 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 these 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. Representative [Ca2+]i responses from single HEK293T cells expressing various constructs. The line under each trace indicates the period of NMG perfusion. C. Average [Ca2+]i elevations in cells expressing the various NCX1.1 mutants with or without co-expression of CaM or CaM1234. The digits in each column indicate the number of cells in each group. Data 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 < 0.05, ***: p < 0.001 when compared with the corresponding group expressing only the NCX1.1 mutant).
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pone.0138856.g006: Each of the conserved a.a. residues in the NCX1.1 CaMS has differential effects on exchange activity.The conserved 1st, 5th, 8th, and 14th a.a. residues of the CaMS in NCX1.1 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 these 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. Representative [Ca2+]i responses from single HEK293T cells expressing various constructs. The line under each trace indicates the period of NMG perfusion. C. Average [Ca2+]i elevations in cells expressing the various NCX1.1 mutants with or without co-expression of CaM or CaM1234. The digits in each column indicate the number of cells in each group. Data 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 < 0.05, ***: p < 0.001 when compared with the corresponding group expressing only the NCX1.1 mutant).

Mentions: To further characterize the importance of the CaMS in CaM-mediated regulation, we mutated the 4 conserved a.a. residues (Fig 1B) in the CaMS from F, V, K, and L to A, A, D, and D (named F1A, V5A, L8D, and L14D) [25] and monitored rNCX activity (Fig 6). Immunostaining for the V5 epitope at the C-termini of these mutants showed a concentrated distribution at the cell boundary, overlapping with F-actin. The line intensity profiles implied that these mutants were located at the cell membrane (S4 Fig). These mutants also showed rNCX activity; however, NCX1.1F1A and NCX1.1V5A had lower [Ca2+]i elevation levels compared with the other two mutants. The averages show that mutants NCX1.1F1A and NCX1.1V5A had a significantly reduced change in [Ca2+]i to 559 ± 74 (n = 26, p < 0.01) and 566 ± 90 (n = 37, p < 0.01) nM, respectively, compared with NCX1.1 (971 ± 71 nM, Fig 4B). CaM and CaM1234 co-expression did not affect [Ca2+]i changes in cells expressing NCX1.1F1A, with [Ca2+]i levels of 547 ± 59 (n = 68) and 428 ± 48 (n = 37) nM, respectively, or NCX1.1V5A, with [Ca2+]i levels of 503 ± 73 (n = 43) and 539 ± 49 nM (n = 44), respectively.


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

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

Each of the conserved a.a. residues in the NCX1.1 CaMS has differential effects on exchange activity.The conserved 1st, 5th, 8th, and 14th a.a. residues of the CaMS in NCX1.1 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 these 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. Representative [Ca2+]i responses from single HEK293T cells expressing various constructs. The line under each trace indicates the period of NMG perfusion. C. Average [Ca2+]i elevations in cells expressing the various NCX1.1 mutants with or without co-expression of CaM or CaM1234. The digits in each column indicate the number of cells in each group. Data 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 < 0.05, ***: p < 0.001 when compared with the corresponding group expressing only the NCX1.1 mutant).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0138856.g006: Each of the conserved a.a. residues in the NCX1.1 CaMS has differential effects on exchange activity.The conserved 1st, 5th, 8th, and 14th a.a. residues of the CaMS in NCX1.1 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 these 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. Representative [Ca2+]i responses from single HEK293T cells expressing various constructs. The line under each trace indicates the period of NMG perfusion. C. Average [Ca2+]i elevations in cells expressing the various NCX1.1 mutants with or without co-expression of CaM or CaM1234. The digits in each column indicate the number of cells in each group. Data 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 < 0.05, ***: p < 0.001 when compared with the corresponding group expressing only the NCX1.1 mutant).
Mentions: To further characterize the importance of the CaMS in CaM-mediated regulation, we mutated the 4 conserved a.a. residues (Fig 1B) in the CaMS from F, V, K, and L to A, A, D, and D (named F1A, V5A, L8D, and L14D) [25] and monitored rNCX activity (Fig 6). Immunostaining for the V5 epitope at the C-termini of these mutants showed a concentrated distribution at the cell boundary, overlapping with F-actin. The line intensity profiles implied that these mutants were located at the cell membrane (S4 Fig). These mutants also showed rNCX activity; however, NCX1.1F1A and NCX1.1V5A had lower [Ca2+]i elevation levels compared with the other two mutants. The averages show that mutants NCX1.1F1A and NCX1.1V5A had a significantly reduced change in [Ca2+]i to 559 ± 74 (n = 26, p < 0.01) and 566 ± 90 (n = 37, p < 0.01) nM, respectively, compared with NCX1.1 (971 ± 71 nM, Fig 4B). CaM and CaM1234 co-expression did not affect [Ca2+]i changes in cells expressing NCX1.1F1A, with [Ca2+]i levels of 547 ± 59 (n = 68) and 428 ± 48 (n = 37) nM, respectively, or NCX1.1V5A, with [Ca2+]i levels of 503 ± 73 (n = 43) and 539 ± 49 nM (n = 44), respectively.

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