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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

The CaMS affects the membrane localization of NCX1 splice variants.A. HEK293T cells expressing NCX1.1 or NCX1.3, with or without the CaMS (ΔCaMS), were permeabilized and stained with an antibody against the V5 epitope (Green) to label the exchanger. Phalloidin (Red) and DAPI (Blue) were used to visualize actin filaments and nuclear DNA, respectively. Images were obtained on a Leica SP5 confocal microscope. The intensity profiles plotted on the right indicate the red and green fluorescence intensity along the arrows in the corresponding merged images. B. Immunostaining of non-permeabilized HEK293T cells. Left, cells co-expressing NCX1 splice variants (Red) and GFP-GPI (Green). Right, PDM image of the boxed region shown to the left. The grey scale in each image represents the PDM value in each pixel. Scale bar: 10 μm
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pone.0138856.g003: The CaMS affects the membrane localization of NCX1 splice variants.A. HEK293T cells expressing NCX1.1 or NCX1.3, with or without the CaMS (ΔCaMS), were permeabilized and stained with an antibody against the V5 epitope (Green) to label the exchanger. Phalloidin (Red) and DAPI (Blue) were used to visualize actin filaments and nuclear DNA, respectively. Images were obtained on a Leica SP5 confocal microscope. The intensity profiles plotted on the right indicate the red and green fluorescence intensity along the arrows in the corresponding merged images. B. Immunostaining of non-permeabilized HEK293T cells. Left, cells co-expressing NCX1 splice variants (Red) and GFP-GPI (Green). Right, PDM image of the boxed region shown to the left. The grey scale in each image represents the PDM value in each pixel. Scale bar: 10 μm

Mentions: To characterize whether CaMS affects the localization of NCX1 splice variants, we expressed NCX1.1 and NCX1.3 with or without CaMS in HEK293T cells and stained the cells with an antibody against the V5 epitope (Fig 3). After fixation and permeabilization, the confocal images show that wild-type NCX1.1 and NCX1.3 are largely present at the plasma membrane compared with phalloidin staining, which illustrated the distribution of F-actin to the subplasmalemmal region. The line intensity profiles indicated that both NCX1.1 and NCX1.3 had an overlapping distribution with F-actin at the cell boundary. NCX1 mutants that lack the CaMS (NCX1.1ΔCaMS and NCX1.3ΔCaMS) localized to both the plasma membrane and the cytosolic region. Compared with the F-actin distribution shown by the line intensity profiles, both mutants without the CaMS localized mostly to the cytosolic side of the plasma membrane. To further confirm the membrane localization, we co-expressed the NCX1 splice variants and mutants with GFP-GPI, which encodes a GPI-anchored form of GFP that localized to the extracellular face of the plasma membrane, in HEK293T cells. We then stained the V5 epitope in non-permeabilized cells (Fig 3B). Both NCX1.1 and NCX1.3 were present at the membrane surface and overlapped with GFP-GPI. Under the same exposure settings, both mutants without CaMS displayed lower membrane surface expression levels than those of the corresponding wild-type proteins. The PDM images showed that the distributions of both NCX1.1 and NCX1.3 correlated well with GFP-GPI at the plasma membrane; in contrast, a lower correlation was observed in cells expressing NCX1.1ΔCaMS or NCX1.3ΔCaMS. In permeabilized cells, the fluorescence signals of the stained V5 and GFP-GPI were concentrated at the plasma membrane for NCX1.1 and NCX1.3; in contrast, the fluorescence signals in permeabilized cells expressing NCX1.1ΔCaMS or NCX1.3ΔCaMS were mostly distributed in a cytosolic region near the membrane (S2 Fig). Therefore, both F-actin and GFP-GPI staining revealed that NCX1 splice variants without the CaMS appeared to be localize to the cytosolic side of the membrane and partly on the cell membrane. These results suggest that CaMS plays an important role in the targeting of NCX1 splice variants to the plasma membrane.


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

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

The CaMS affects the membrane localization of NCX1 splice variants.A. HEK293T cells expressing NCX1.1 or NCX1.3, with or without the CaMS (ΔCaMS), were permeabilized and stained with an antibody against the V5 epitope (Green) to label the exchanger. Phalloidin (Red) and DAPI (Blue) were used to visualize actin filaments and nuclear DNA, respectively. Images were obtained on a Leica SP5 confocal microscope. The intensity profiles plotted on the right indicate the red and green fluorescence intensity along the arrows in the corresponding merged images. B. Immunostaining of non-permeabilized HEK293T cells. Left, cells co-expressing NCX1 splice variants (Red) and GFP-GPI (Green). Right, PDM image of the boxed region shown to the left. The grey scale in each image represents the PDM value in each pixel. Scale bar: 10 μm
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Related In: Results  -  Collection

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pone.0138856.g003: The CaMS affects the membrane localization of NCX1 splice variants.A. HEK293T cells expressing NCX1.1 or NCX1.3, with or without the CaMS (ΔCaMS), were permeabilized and stained with an antibody against the V5 epitope (Green) to label the exchanger. Phalloidin (Red) and DAPI (Blue) were used to visualize actin filaments and nuclear DNA, respectively. Images were obtained on a Leica SP5 confocal microscope. The intensity profiles plotted on the right indicate the red and green fluorescence intensity along the arrows in the corresponding merged images. B. Immunostaining of non-permeabilized HEK293T cells. Left, cells co-expressing NCX1 splice variants (Red) and GFP-GPI (Green). Right, PDM image of the boxed region shown to the left. The grey scale in each image represents the PDM value in each pixel. Scale bar: 10 μm
Mentions: To characterize whether CaMS affects the localization of NCX1 splice variants, we expressed NCX1.1 and NCX1.3 with or without CaMS in HEK293T cells and stained the cells with an antibody against the V5 epitope (Fig 3). After fixation and permeabilization, the confocal images show that wild-type NCX1.1 and NCX1.3 are largely present at the plasma membrane compared with phalloidin staining, which illustrated the distribution of F-actin to the subplasmalemmal region. The line intensity profiles indicated that both NCX1.1 and NCX1.3 had an overlapping distribution with F-actin at the cell boundary. NCX1 mutants that lack the CaMS (NCX1.1ΔCaMS and NCX1.3ΔCaMS) localized to both the plasma membrane and the cytosolic region. Compared with the F-actin distribution shown by the line intensity profiles, both mutants without the CaMS localized mostly to the cytosolic side of the plasma membrane. To further confirm the membrane localization, we co-expressed the NCX1 splice variants and mutants with GFP-GPI, which encodes a GPI-anchored form of GFP that localized to the extracellular face of the plasma membrane, in HEK293T cells. We then stained the V5 epitope in non-permeabilized cells (Fig 3B). Both NCX1.1 and NCX1.3 were present at the membrane surface and overlapped with GFP-GPI. Under the same exposure settings, both mutants without CaMS displayed lower membrane surface expression levels than those of the corresponding wild-type proteins. The PDM images showed that the distributions of both NCX1.1 and NCX1.3 correlated well with GFP-GPI at the plasma membrane; in contrast, a lower correlation was observed in cells expressing NCX1.1ΔCaMS or NCX1.3ΔCaMS. In permeabilized cells, the fluorescence signals of the stained V5 and GFP-GPI were concentrated at the plasma membrane for NCX1.1 and NCX1.3; in contrast, the fluorescence signals in permeabilized cells expressing NCX1.1ΔCaMS or NCX1.3ΔCaMS were mostly distributed in a cytosolic region near the membrane (S2 Fig). Therefore, both F-actin and GFP-GPI staining revealed that NCX1 splice variants without the CaMS appeared to be localize to the cytosolic side of the membrane and partly on the cell membrane. These results suggest that CaMS plays an important role in the targeting of NCX1 splice variants to the plasma membrane.

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