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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

CaM interacts with the NCX1 cytosolic loop.Purified GST-CaM or GST-CaM1234 was used as bait to pull down intracellular loops subcloned from NCX1.1 or NCX1.3 with (A. NCX1.1CL: a.a. 288–805 and B. NCX1.3CL: a.a. 288–769) or without the CaMS (C. NCX1.1CLΔCaMS and D. NCX1.3CLΔCaMS). During some of the binding reaction, Ca2+ (2 mM) was included in the buffer and it was omitted in other reactions. The interacting proteins were verified by Western blots with antibodies against the GST or V5 epitope. M indicates the m.w marker lane.
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pone.0138856.g002: CaM interacts with the NCX1 cytosolic loop.Purified GST-CaM or GST-CaM1234 was used as bait to pull down intracellular loops subcloned from NCX1.1 or NCX1.3 with (A. NCX1.1CL: a.a. 288–805 and B. NCX1.3CL: a.a. 288–769) or without the CaMS (C. NCX1.1CLΔCaMS and D. NCX1.3CLΔCaMS). During some of the binding reaction, Ca2+ (2 mM) was included in the buffer and it was omitted in other reactions. The interacting proteins were verified by Western blots with antibodies against the GST or V5 epitope. M indicates the m.w marker lane.

Mentions: To determine whether NCX1 interacts with CaM, we used GST-CaM as the bait to pull down the cytoplasmic loops of NCX1.1 (NCX1.1CL: a.a. 288–805) and NCX1.3 (NCX1.3CL: a.a. 288–769) expressed in HEK293T cells (Figs 1C and 2). Western blots show that the antibody against the V5 epitope stains a protein with a molecular weight similar to the expected size (~70 kD) of NCX1.1CL or NCX1.3CL in the presence of Ca2+. In the absence of Ca2+ or CaM, no stained band was visualized. Neither GST-CaM nor GST-CaM1234 interacted with the cytoplasmic loop without the CaMS (NCX1.1CLΔCaMS and NCX1.3CLΔCaMS). These results suggest that CaM interacts specifically with the NCX1 cytoplasmic loop via the CaMS in a Ca2+-dependent manner.


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

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

CaM interacts with the NCX1 cytosolic loop.Purified GST-CaM or GST-CaM1234 was used as bait to pull down intracellular loops subcloned from NCX1.1 or NCX1.3 with (A. NCX1.1CL: a.a. 288–805 and B. NCX1.3CL: a.a. 288–769) or without the CaMS (C. NCX1.1CLΔCaMS and D. NCX1.3CLΔCaMS). During some of the binding reaction, Ca2+ (2 mM) was included in the buffer and it was omitted in other reactions. The interacting proteins were verified by Western blots with antibodies against the GST or V5 epitope. M indicates the m.w marker lane.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0138856.g002: CaM interacts with the NCX1 cytosolic loop.Purified GST-CaM or GST-CaM1234 was used as bait to pull down intracellular loops subcloned from NCX1.1 or NCX1.3 with (A. NCX1.1CL: a.a. 288–805 and B. NCX1.3CL: a.a. 288–769) or without the CaMS (C. NCX1.1CLΔCaMS and D. NCX1.3CLΔCaMS). During some of the binding reaction, Ca2+ (2 mM) was included in the buffer and it was omitted in other reactions. The interacting proteins were verified by Western blots with antibodies against the GST or V5 epitope. M indicates the m.w marker lane.
Mentions: To determine whether NCX1 interacts with CaM, we used GST-CaM as the bait to pull down the cytoplasmic loops of NCX1.1 (NCX1.1CL: a.a. 288–805) and NCX1.3 (NCX1.3CL: a.a. 288–769) expressed in HEK293T cells (Figs 1C and 2). Western blots show that the antibody against the V5 epitope stains a protein with a molecular weight similar to the expected size (~70 kD) of NCX1.1CL or NCX1.3CL in the presence of Ca2+. In the absence of Ca2+ or CaM, no stained band was visualized. Neither GST-CaM nor GST-CaM1234 interacted with the cytoplasmic loop without the CaMS (NCX1.1CLΔCaMS and NCX1.3CLΔCaMS). These results suggest that CaM interacts specifically with the NCX1 cytoplasmic loop via the CaMS in a Ca2+-dependent manner.

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