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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 the CaMS reduces exchange activity.HEK293T cells expressing NCX1 and mutants were treated with ouabain to elevate the intracellular Na+ concentration. Cells were then locally perfused with NMG buffer for 1 min to induce reverse-mode exchange activity. The [Ca2+]i was calibrated based on changes in fura-2 fluorescence intensities. A. Representative [Ca2+]i traces in single cells expressing different constructs. The black lines under each trace indicate the period of NMG perfusion. Cells transfected with a plasmid missing the exchanger were used as a control. B. The average elevation of [Ca2+]i in cells expressing wild-type NCX1.1 (WT, empty columns) and NCX1.1ΔCaMS (ΔCaMS, filled columns) with CaM or CaM1234 co-expression. C. Average [Ca2+]i responses in cells expressing NCX1.3 WT (empty columns) and NCX1.1ΔCaMS (filled columns) with CaM or CaM1234 co-expression. The digits in each column indicate the sample number. Data are the mean ± SEM pooled from three different sets of cells and analyzed by a one-way ANOVA with Fisher's post hoc test (*: p < 0.05, ***: p < 0.001 compared with WT).
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pone.0138856.g004: Deletion of the CaMS reduces exchange activity.HEK293T cells expressing NCX1 and mutants were treated with ouabain to elevate the intracellular Na+ concentration. Cells were then locally perfused with NMG buffer for 1 min to induce reverse-mode exchange activity. The [Ca2+]i was calibrated based on changes in fura-2 fluorescence intensities. A. Representative [Ca2+]i traces in single cells expressing different constructs. The black lines under each trace indicate the period of NMG perfusion. Cells transfected with a plasmid missing the exchanger were used as a control. B. The average elevation of [Ca2+]i in cells expressing wild-type NCX1.1 (WT, empty columns) and NCX1.1ΔCaMS (ΔCaMS, filled columns) with CaM or CaM1234 co-expression. C. Average [Ca2+]i responses in cells expressing NCX1.3 WT (empty columns) and NCX1.1ΔCaMS (filled columns) with CaM or CaM1234 co-expression. The digits in each column indicate the sample number. Data are the mean ± SEM pooled from three different sets of cells and analyzed by a one-way ANOVA with Fisher's post hoc test (*: p < 0.05, ***: p < 0.001 compared with WT).

Mentions: To determine the importance of the CaMS on NCX1 activity, we expressed the splice variants in HEK293T cells and monitored [Ca2+]i elevations induced by rNCX activity. To reverse the Na+ gradient, we treated the cells with ouabain to inhibit the Na+-pump and locally perfused a single cell with NMG buffer, in which Na+ was substituted with NMG. Fig 4A shows that during NMG perfusion, [Ca2+]i rose quickly and then remained steady in cells expressing either NCX1.1 or NCX1.3; after the perfusion period, [Ca2+]i gradually returned to basal levels. Despite receiving the same treatment, cells expressing only the vector did not show an elevation in [Ca2+]i. Cells expressing NCX1.1ΔCaMS or NCX1.3ΔCaMS showed a similar [Ca2+]i response pattern, but the elevation was smaller compared with cells expressing the wild type. The average [Ca2+]i change in cells expressing NCX1.1 and NCX1.3 was 971 ± 71 (n = 53) and 913 ± 55 (n = 75) nM, respectively. Without the CaMS, the [Ca2+]i increases were significantly reduced to 694 ± 77 (n = 25, p < 0.05) and 462 ± 67 (n = 32 p < 0.001) nM in cells expressing NCX1.1ΔCaMS and NCX1.3ΔCaMS, respectively (Fig 4B and 4C).


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

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

Deletion of the CaMS reduces exchange activity.HEK293T cells expressing NCX1 and mutants were treated with ouabain to elevate the intracellular Na+ concentration. Cells were then locally perfused with NMG buffer for 1 min to induce reverse-mode exchange activity. The [Ca2+]i was calibrated based on changes in fura-2 fluorescence intensities. A. Representative [Ca2+]i traces in single cells expressing different constructs. The black lines under each trace indicate the period of NMG perfusion. Cells transfected with a plasmid missing the exchanger were used as a control. B. The average elevation of [Ca2+]i in cells expressing wild-type NCX1.1 (WT, empty columns) and NCX1.1ΔCaMS (ΔCaMS, filled columns) with CaM or CaM1234 co-expression. C. Average [Ca2+]i responses in cells expressing NCX1.3 WT (empty columns) and NCX1.1ΔCaMS (filled columns) with CaM or CaM1234 co-expression. The digits in each column indicate the sample number. Data are the mean ± SEM pooled from three different sets of cells and analyzed by a one-way ANOVA with Fisher's post hoc test (*: p < 0.05, ***: p < 0.001 compared with WT).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4589332&req=5

pone.0138856.g004: Deletion of the CaMS reduces exchange activity.HEK293T cells expressing NCX1 and mutants were treated with ouabain to elevate the intracellular Na+ concentration. Cells were then locally perfused with NMG buffer for 1 min to induce reverse-mode exchange activity. The [Ca2+]i was calibrated based on changes in fura-2 fluorescence intensities. A. Representative [Ca2+]i traces in single cells expressing different constructs. The black lines under each trace indicate the period of NMG perfusion. Cells transfected with a plasmid missing the exchanger were used as a control. B. The average elevation of [Ca2+]i in cells expressing wild-type NCX1.1 (WT, empty columns) and NCX1.1ΔCaMS (ΔCaMS, filled columns) with CaM or CaM1234 co-expression. C. Average [Ca2+]i responses in cells expressing NCX1.3 WT (empty columns) and NCX1.1ΔCaMS (filled columns) with CaM or CaM1234 co-expression. The digits in each column indicate the sample number. Data are the mean ± SEM pooled from three different sets of cells and analyzed by a one-way ANOVA with Fisher's post hoc test (*: p < 0.05, ***: p < 0.001 compared with WT).
Mentions: To determine the importance of the CaMS on NCX1 activity, we expressed the splice variants in HEK293T cells and monitored [Ca2+]i elevations induced by rNCX activity. To reverse the Na+ gradient, we treated the cells with ouabain to inhibit the Na+-pump and locally perfused a single cell with NMG buffer, in which Na+ was substituted with NMG. Fig 4A shows that during NMG perfusion, [Ca2+]i rose quickly and then remained steady in cells expressing either NCX1.1 or NCX1.3; after the perfusion period, [Ca2+]i gradually returned to basal levels. Despite receiving the same treatment, cells expressing only the vector did not show an elevation in [Ca2+]i. Cells expressing NCX1.1ΔCaMS or NCX1.3ΔCaMS showed a similar [Ca2+]i response pattern, but the elevation was smaller compared with cells expressing the wild type. The average [Ca2+]i change in cells expressing NCX1.1 and NCX1.3 was 971 ± 71 (n = 53) and 913 ± 55 (n = 75) nM, respectively. Without the CaMS, the [Ca2+]i increases were significantly reduced to 694 ± 77 (n = 25, p < 0.05) and 462 ± 67 (n = 32 p < 0.001) nM in cells expressing NCX1.1ΔCaMS and NCX1.3ΔCaMS, respectively (Fig 4B and 4C).

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