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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

Schematic illustration of NCX1.A. Predicted secondary structure of NCX1. NCX has 10 transmembrane segments and the intracellular loop between the 5th and 6th transmembrane segments contains several modulatory regions including the exchanger inhibitory peptide (XIP), 2 α2-repeats (shaded regions), Ca2+ binding domains (CBD1 and CBD2), an alternative splicing site (AS), and the predicted CaM binding segment (CaMS). B. Sequence alignment of the CaM binding motif. The alignment includes the conserved 1-5-8-14 CaM binding sequences identified in human calcineurin (a.a. 405–424), human plasma membrane Ca2+-ATPase 4b (a.a. 1089–1108), human L-type Ca2+ channel (a.a. 1601–1620), and human small conductance Ca2+-activated K+ channel (a.a. 424–443), bovine/human NCX1.1 (a.a. 716–735), human NCX2 (a.a. 666–685), and human NCX3 (a.a. 663–681). Arrows indicates the conserved residues. C. The bovine NCX1 constructs. Empty box indicates the XIP (a.a. 216–270) and boxes filled with different patterns indicate the AS region with different exons. The filled circle indicates the putative CaMS and the empty circle at the C-terminus represents the V5 epitope tag. The number indicates the a.a residue in the NCX1.1. D. Immunostaining of NCX1.1 and NCX1.3 in non-permeabilized cells. HEK293T cells expressing NCX1.1 and NCX1.3 were stained with an antibody against the V5 epitope tagged at the C-terminus of these splice variants. Images were obtained using a Leica SP5 confocal microscope. Scale bar: 10 μm
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pone.0138856.g001: Schematic illustration of NCX1.A. Predicted secondary structure of NCX1. NCX has 10 transmembrane segments and the intracellular loop between the 5th and 6th transmembrane segments contains several modulatory regions including the exchanger inhibitory peptide (XIP), 2 α2-repeats (shaded regions), Ca2+ binding domains (CBD1 and CBD2), an alternative splicing site (AS), and the predicted CaM binding segment (CaMS). B. Sequence alignment of the CaM binding motif. The alignment includes the conserved 1-5-8-14 CaM binding sequences identified in human calcineurin (a.a. 405–424), human plasma membrane Ca2+-ATPase 4b (a.a. 1089–1108), human L-type Ca2+ channel (a.a. 1601–1620), and human small conductance Ca2+-activated K+ channel (a.a. 424–443), bovine/human NCX1.1 (a.a. 716–735), human NCX2 (a.a. 666–685), and human NCX3 (a.a. 663–681). Arrows indicates the conserved residues. C. The bovine NCX1 constructs. Empty box indicates the XIP (a.a. 216–270) and boxes filled with different patterns indicate the AS region with different exons. The filled circle indicates the putative CaMS and the empty circle at the C-terminus represents the V5 epitope tag. The number indicates the a.a residue in the NCX1.1. D. Immunostaining of NCX1.1 and NCX1.3 in non-permeabilized cells. HEK293T cells expressing NCX1.1 and NCX1.3 were stained with an antibody against the V5 epitope tagged at the C-terminus of these splice variants. Images were obtained using a Leica SP5 confocal microscope. Scale bar: 10 μm

Mentions: NCX1 is composed of 970 a.a. residues and is predicted to have 10 transmembrane segments, with a large intracellular loop between the 5th and 6th transmembrane segments (TMs) (Fig 1A). The crystal structure of an archaebacterial NCX analogue (NCX_Mj, from Methanococcus jannaschii) was recently resolved, revealing 10 TMs [7,8]. A reexamination of the hydropathy profile of mammalian NCX1 indicates that this protein likely also has 10 TMs [9]. At the N-terminal of the intracellular loop, an XIP (exchanger inhibitory peptide) segment attenuates the Na+-dependent inactivation of NCX1 [10]. This is followed by a catenin-like domain whose function is not yet clear. In the C-half of the loop, there are two Ca2+ binding domains, CBD1 (Kd ~ 0.2 μM) and CBD2 (Kd ~ 5 μM), related to the activation of exchange activity [11,12,13]. The C-terminal of CBD2 contains an alternative splicing region that determines the stability of CBD2 [14].


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

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

Schematic illustration of NCX1.A. Predicted secondary structure of NCX1. NCX has 10 transmembrane segments and the intracellular loop between the 5th and 6th transmembrane segments contains several modulatory regions including the exchanger inhibitory peptide (XIP), 2 α2-repeats (shaded regions), Ca2+ binding domains (CBD1 and CBD2), an alternative splicing site (AS), and the predicted CaM binding segment (CaMS). B. Sequence alignment of the CaM binding motif. The alignment includes the conserved 1-5-8-14 CaM binding sequences identified in human calcineurin (a.a. 405–424), human plasma membrane Ca2+-ATPase 4b (a.a. 1089–1108), human L-type Ca2+ channel (a.a. 1601–1620), and human small conductance Ca2+-activated K+ channel (a.a. 424–443), bovine/human NCX1.1 (a.a. 716–735), human NCX2 (a.a. 666–685), and human NCX3 (a.a. 663–681). Arrows indicates the conserved residues. C. The bovine NCX1 constructs. Empty box indicates the XIP (a.a. 216–270) and boxes filled with different patterns indicate the AS region with different exons. The filled circle indicates the putative CaMS and the empty circle at the C-terminus represents the V5 epitope tag. The number indicates the a.a residue in the NCX1.1. D. Immunostaining of NCX1.1 and NCX1.3 in non-permeabilized cells. HEK293T cells expressing NCX1.1 and NCX1.3 were stained with an antibody against the V5 epitope tagged at the C-terminus of these splice variants. Images were obtained using a Leica SP5 confocal microscope. Scale bar: 10 μm
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Related In: Results  -  Collection

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pone.0138856.g001: Schematic illustration of NCX1.A. Predicted secondary structure of NCX1. NCX has 10 transmembrane segments and the intracellular loop between the 5th and 6th transmembrane segments contains several modulatory regions including the exchanger inhibitory peptide (XIP), 2 α2-repeats (shaded regions), Ca2+ binding domains (CBD1 and CBD2), an alternative splicing site (AS), and the predicted CaM binding segment (CaMS). B. Sequence alignment of the CaM binding motif. The alignment includes the conserved 1-5-8-14 CaM binding sequences identified in human calcineurin (a.a. 405–424), human plasma membrane Ca2+-ATPase 4b (a.a. 1089–1108), human L-type Ca2+ channel (a.a. 1601–1620), and human small conductance Ca2+-activated K+ channel (a.a. 424–443), bovine/human NCX1.1 (a.a. 716–735), human NCX2 (a.a. 666–685), and human NCX3 (a.a. 663–681). Arrows indicates the conserved residues. C. The bovine NCX1 constructs. Empty box indicates the XIP (a.a. 216–270) and boxes filled with different patterns indicate the AS region with different exons. The filled circle indicates the putative CaMS and the empty circle at the C-terminus represents the V5 epitope tag. The number indicates the a.a residue in the NCX1.1. D. Immunostaining of NCX1.1 and NCX1.3 in non-permeabilized cells. HEK293T cells expressing NCX1.1 and NCX1.3 were stained with an antibody against the V5 epitope tagged at the C-terminus of these splice variants. Images were obtained using a Leica SP5 confocal microscope. Scale bar: 10 μm
Mentions: NCX1 is composed of 970 a.a. residues and is predicted to have 10 transmembrane segments, with a large intracellular loop between the 5th and 6th transmembrane segments (TMs) (Fig 1A). The crystal structure of an archaebacterial NCX analogue (NCX_Mj, from Methanococcus jannaschii) was recently resolved, revealing 10 TMs [7,8]. A reexamination of the hydropathy profile of mammalian NCX1 indicates that this protein likely also has 10 TMs [9]. At the N-terminal of the intracellular loop, an XIP (exchanger inhibitory peptide) segment attenuates the Na+-dependent inactivation of NCX1 [10]. This is followed by a catenin-like domain whose function is not yet clear. In the C-half of the loop, there are two Ca2+ binding domains, CBD1 (Kd ~ 0.2 μM) and CBD2 (Kd ~ 5 μM), related to the activation of exchange activity [11,12,13]. The C-terminal of CBD2 contains an alternative splicing region that determines the stability of CBD2 [14].

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