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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

Deletion of exon A/B attenuates exchange activity.To activate the rNCX activity in HEK293T cells expressing NCX1.4 or NCX1D, we treated them with ouabain and then perfused with NMG buffer. The [Ca2+]i was calibrated based on the changes in fura-2 fluorescence intensities. A. Localization of NCX1.4 and NCX1D. Cells expressing NCX1.4 (upper row) or NCX1D (lower row) were stained with V5 antibody (Green), phalloidin (Red), and DAPI (Blue) to visualize the exchanger, actin filaments, and nuclear DNA, respectively. The intensity profiles plotted on the right indicate intensity of the red and green fluorescence signals along the arrows indicated in the corresponding merged images. Scale bar: 10 μm. B. Representative [Ca2+]i response traces from cells expressing NCX1.4 or NCX1D. The lines under each trace indicate the period of NMG perfusion. C. Average [Ca2+]i changes in cells expressing NCX1.4 and NCX1D with co-expression of CaM or CaM1234. Data presented 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 compared with the group without co-expression).
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pone.0138856.g005: Deletion of exon A/B attenuates exchange activity.To activate the rNCX activity in HEK293T cells expressing NCX1.4 or NCX1D, we treated them with ouabain and then perfused with NMG buffer. The [Ca2+]i was calibrated based on the changes in fura-2 fluorescence intensities. A. Localization of NCX1.4 and NCX1D. Cells expressing NCX1.4 (upper row) or NCX1D (lower row) were stained with V5 antibody (Green), phalloidin (Red), and DAPI (Blue) to visualize the exchanger, actin filaments, and nuclear DNA, respectively. The intensity profiles plotted on the right indicate intensity of the red and green fluorescence signals along the arrows indicated in the corresponding merged images. Scale bar: 10 μm. B. Representative [Ca2+]i response traces from cells expressing NCX1.4 or NCX1D. The lines under each trace indicate the period of NMG perfusion. C. Average [Ca2+]i changes in cells expressing NCX1.4 and NCX1D with co-expression of CaM or CaM1234. Data presented 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 compared with the group without co-expression).

Mentions: The difference between NCX1.1 and 1.3 are the mutually exclusive exons A and B, which are important for the Ca2+-chelating ability of the CBD2 motif [11,14]. To characterize the role of these exons in CaM-related regulatory effects, we constructed two NCX1 splice variants containing exons A and D (NCX1.4) or only exon D (NCX1D) for use in rNCX activity assays (Fig 5). Immunostaining shows that both NCX1.4 and NCX1D localize to the plasma membrane compared with the distribution of F-actin. Line intensity profiles confirm that the distributions of NCX1.4 and NCX1D were concentrated at the cell membrane. During NMG perfusion, cells expressing NCX1.4 and NCX1D showed elevated [Ca2+]i; however, NCX1D had a smaller [Ca2+]i elevation than that of NCX1.4. The average [Ca2+]i change in cells expressing NCX1.4 was 850 ± 87 nM (n = 15); CaM and CaM1234 co-expression did not affect [Ca2+]i, with [Ca2+]i reaching 790 ± 119 (n = 12) and 836 ± 81 (n = 43) nM, respectively. In cells expressing NCX1D, the average [Ca2+]i elevation was 387 ± 61 nM (n = 34, p < 0.001), which was significantly less than that in cells expressing NCX1.4. CaM co-expression did not affect the exchange activity of NCX1D (478 ± 43 nM, n = 27), but CaM1234 overexpression enhanced the [Ca2+]i changes to 707 ± 101 nM (n = 25, p < 0.05). These results suggest that exons A and B support exchange activity and are involved in the CaM-mediated modulation of NCX1 activity.


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

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

Deletion of exon A/B attenuates exchange activity.To activate the rNCX activity in HEK293T cells expressing NCX1.4 or NCX1D, we treated them with ouabain and then perfused with NMG buffer. The [Ca2+]i was calibrated based on the changes in fura-2 fluorescence intensities. A. Localization of NCX1.4 and NCX1D. Cells expressing NCX1.4 (upper row) or NCX1D (lower row) were stained with V5 antibody (Green), phalloidin (Red), and DAPI (Blue) to visualize the exchanger, actin filaments, and nuclear DNA, respectively. The intensity profiles plotted on the right indicate intensity of the red and green fluorescence signals along the arrows indicated in the corresponding merged images. Scale bar: 10 μm. B. Representative [Ca2+]i response traces from cells expressing NCX1.4 or NCX1D. The lines under each trace indicate the period of NMG perfusion. C. Average [Ca2+]i changes in cells expressing NCX1.4 and NCX1D with co-expression of CaM or CaM1234. Data presented 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 compared with the group without co-expression).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0138856.g005: Deletion of exon A/B attenuates exchange activity.To activate the rNCX activity in HEK293T cells expressing NCX1.4 or NCX1D, we treated them with ouabain and then perfused with NMG buffer. The [Ca2+]i was calibrated based on the changes in fura-2 fluorescence intensities. A. Localization of NCX1.4 and NCX1D. Cells expressing NCX1.4 (upper row) or NCX1D (lower row) were stained with V5 antibody (Green), phalloidin (Red), and DAPI (Blue) to visualize the exchanger, actin filaments, and nuclear DNA, respectively. The intensity profiles plotted on the right indicate intensity of the red and green fluorescence signals along the arrows indicated in the corresponding merged images. Scale bar: 10 μm. B. Representative [Ca2+]i response traces from cells expressing NCX1.4 or NCX1D. The lines under each trace indicate the period of NMG perfusion. C. Average [Ca2+]i changes in cells expressing NCX1.4 and NCX1D with co-expression of CaM or CaM1234. Data presented 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 compared with the group without co-expression).
Mentions: The difference between NCX1.1 and 1.3 are the mutually exclusive exons A and B, which are important for the Ca2+-chelating ability of the CBD2 motif [11,14]. To characterize the role of these exons in CaM-related regulatory effects, we constructed two NCX1 splice variants containing exons A and D (NCX1.4) or only exon D (NCX1D) for use in rNCX activity assays (Fig 5). Immunostaining shows that both NCX1.4 and NCX1D localize to the plasma membrane compared with the distribution of F-actin. Line intensity profiles confirm that the distributions of NCX1.4 and NCX1D were concentrated at the cell membrane. During NMG perfusion, cells expressing NCX1.4 and NCX1D showed elevated [Ca2+]i; however, NCX1D had a smaller [Ca2+]i elevation than that of NCX1.4. The average [Ca2+]i change in cells expressing NCX1.4 was 850 ± 87 nM (n = 15); CaM and CaM1234 co-expression did not affect [Ca2+]i, with [Ca2+]i reaching 790 ± 119 (n = 12) and 836 ± 81 (n = 43) nM, respectively. In cells expressing NCX1D, the average [Ca2+]i elevation was 387 ± 61 nM (n = 34, p < 0.001), which was significantly less than that in cells expressing NCX1.4. CaM co-expression did not affect the exchange activity of NCX1D (478 ± 43 nM, n = 27), but CaM1234 overexpression enhanced the [Ca2+]i changes to 707 ± 101 nM (n = 25, p < 0.05). These results suggest that exons A and B support exchange activity and are involved in the CaM-mediated modulation of NCX1 activity.

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