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Activation of the cardiac Na(+)-Ca(2+) exchanger by sorcin via the interaction of the respective Ca(2+)-binding domains.

Zamparelli C, Macquaide N, Colotti G, Verzili D, Seidler T, Smith GL, Chiancone E - J. Mol. Cell. Cardiol. (2010)

Bottom Line: To investigate the importance of this region in the interaction with NCX1, three variants were examined: W105G and W99G, mutated respectively near EF3 and EF2, and E124A that does not bind Ca(2+) due to a mutation at EF3.Downregulation of sorcin decreased and supplementation with wt sorcin (3muM) increased NCX activity in isolated cardiomyocytes.The relative stimulatory effects of the sorcin variants were: W105G>wt sorcin>Sorcin Calcium Binding Domain (SCBD)>W99G>E124A.

View Article: PubMed Central - PubMed

Affiliation: C.N.R. Institute of Molecular Biology and Pathology, Department of Biochemical Sciences A. Rossi Fanelli, Sapienza University of Rome, 00185 Rome, Italy. emilia.chiancone@uniroma1.it

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SPR measurement of the interaction between immobilized wild type sorcin and the NCX1 Ca2+-binding domains, CBD1 and CBD2, at different protein concentrations. The increase in RU relative to baseline indicates complex formation; the plateau region represents the steady-state phase of the interaction, whereas the decrease in RU represents dissociation of CBD1 and CBD2 from immobilized sorcin after injection of buffer. The buffer was 10 mM HEPES, 0.15 M NaCl, and 0.005% surfactant P-20 (pH 7.4), containing 50 μM CaCl2. The temperature was 25 °C. The sensorgrams shown are the average of two experiments. (A) CBD1 injected at protein concentrations of 0.45, 1.8, 4.5, 9, 18, 27, and 35 μM, from bottom to top. (B) CBD2 injected at protein concentrations of 0.50, 1.1, 4.5, 10, and 22 μM, from bottom to top. (C) Scatchard plot: the plateau signal at steady state (Req) measured at different CBD1 and CBD2 concentrations is plotted as a function of Req/C. CBD1: Δ, CBD2: □.
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fig5: SPR measurement of the interaction between immobilized wild type sorcin and the NCX1 Ca2+-binding domains, CBD1 and CBD2, at different protein concentrations. The increase in RU relative to baseline indicates complex formation; the plateau region represents the steady-state phase of the interaction, whereas the decrease in RU represents dissociation of CBD1 and CBD2 from immobilized sorcin after injection of buffer. The buffer was 10 mM HEPES, 0.15 M NaCl, and 0.005% surfactant P-20 (pH 7.4), containing 50 μM CaCl2. The temperature was 25 °C. The sensorgrams shown are the average of two experiments. (A) CBD1 injected at protein concentrations of 0.45, 1.8, 4.5, 9, 18, 27, and 35 μM, from bottom to top. (B) CBD2 injected at protein concentrations of 0.50, 1.1, 4.5, 10, and 22 μM, from bottom to top. (C) Scatchard plot: the plateau signal at steady state (Req) measured at different CBD1 and CBD2 concentrations is plotted as a function of Req/C. CBD1: Δ, CBD2: □.

Mentions: Surface plasmon resonance experiments were performed to characterize the binding reaction quantitatively. In a first series of experiments, sorcin was immobilized onto the chip. The changes in refractive index (RU) that occur during injection of the CBD1 and CBD2 solutions at different concentrations in the presence of 50 μM CaCl2 are depicted in Figs. 5(A), (B) and the Scatchard analysis of the data is presented in Fig. 5(C). The equilibrium and kinetic parameters that describe the interaction of immobilized sorcin with the two NCX1 domains are rather similar. In particular, the equilibrium dissociation constant pertaining to CBD1 and CBD2 correspond to KD = 7 ± 2 μM and 3 ± 1.7 μM, respectively. As expected, the interaction with both CBD1 and CBD2 depends on calcium concentration. At nanomolar calcium (a concentration achieved by addition of 2 mM EDTA) no interaction takes place as shown by the extremely small Req values (data not shown) while at 20 and 50 μM there is interaction and the time courses are alike. Experiments with negative control preparations ruled out the occurrence of aspecific binding (Supplementary Fig. S4).


Activation of the cardiac Na(+)-Ca(2+) exchanger by sorcin via the interaction of the respective Ca(2+)-binding domains.

Zamparelli C, Macquaide N, Colotti G, Verzili D, Seidler T, Smith GL, Chiancone E - J. Mol. Cell. Cardiol. (2010)

SPR measurement of the interaction between immobilized wild type sorcin and the NCX1 Ca2+-binding domains, CBD1 and CBD2, at different protein concentrations. The increase in RU relative to baseline indicates complex formation; the plateau region represents the steady-state phase of the interaction, whereas the decrease in RU represents dissociation of CBD1 and CBD2 from immobilized sorcin after injection of buffer. The buffer was 10 mM HEPES, 0.15 M NaCl, and 0.005% surfactant P-20 (pH 7.4), containing 50 μM CaCl2. The temperature was 25 °C. The sensorgrams shown are the average of two experiments. (A) CBD1 injected at protein concentrations of 0.45, 1.8, 4.5, 9, 18, 27, and 35 μM, from bottom to top. (B) CBD2 injected at protein concentrations of 0.50, 1.1, 4.5, 10, and 22 μM, from bottom to top. (C) Scatchard plot: the plateau signal at steady state (Req) measured at different CBD1 and CBD2 concentrations is plotted as a function of Req/C. CBD1: Δ, CBD2: □.
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Related In: Results  -  Collection

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Show All Figures
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fig5: SPR measurement of the interaction between immobilized wild type sorcin and the NCX1 Ca2+-binding domains, CBD1 and CBD2, at different protein concentrations. The increase in RU relative to baseline indicates complex formation; the plateau region represents the steady-state phase of the interaction, whereas the decrease in RU represents dissociation of CBD1 and CBD2 from immobilized sorcin after injection of buffer. The buffer was 10 mM HEPES, 0.15 M NaCl, and 0.005% surfactant P-20 (pH 7.4), containing 50 μM CaCl2. The temperature was 25 °C. The sensorgrams shown are the average of two experiments. (A) CBD1 injected at protein concentrations of 0.45, 1.8, 4.5, 9, 18, 27, and 35 μM, from bottom to top. (B) CBD2 injected at protein concentrations of 0.50, 1.1, 4.5, 10, and 22 μM, from bottom to top. (C) Scatchard plot: the plateau signal at steady state (Req) measured at different CBD1 and CBD2 concentrations is plotted as a function of Req/C. CBD1: Δ, CBD2: □.
Mentions: Surface plasmon resonance experiments were performed to characterize the binding reaction quantitatively. In a first series of experiments, sorcin was immobilized onto the chip. The changes in refractive index (RU) that occur during injection of the CBD1 and CBD2 solutions at different concentrations in the presence of 50 μM CaCl2 are depicted in Figs. 5(A), (B) and the Scatchard analysis of the data is presented in Fig. 5(C). The equilibrium and kinetic parameters that describe the interaction of immobilized sorcin with the two NCX1 domains are rather similar. In particular, the equilibrium dissociation constant pertaining to CBD1 and CBD2 correspond to KD = 7 ± 2 μM and 3 ± 1.7 μM, respectively. As expected, the interaction with both CBD1 and CBD2 depends on calcium concentration. At nanomolar calcium (a concentration achieved by addition of 2 mM EDTA) no interaction takes place as shown by the extremely small Req values (data not shown) while at 20 and 50 μM there is interaction and the time courses are alike. Experiments with negative control preparations ruled out the occurrence of aspecific binding (Supplementary Fig. S4).

Bottom Line: To investigate the importance of this region in the interaction with NCX1, three variants were examined: W105G and W99G, mutated respectively near EF3 and EF2, and E124A that does not bind Ca(2+) due to a mutation at EF3.Downregulation of sorcin decreased and supplementation with wt sorcin (3muM) increased NCX activity in isolated cardiomyocytes.The relative stimulatory effects of the sorcin variants were: W105G>wt sorcin>Sorcin Calcium Binding Domain (SCBD)>W99G>E124A.

View Article: PubMed Central - PubMed

Affiliation: C.N.R. Institute of Molecular Biology and Pathology, Department of Biochemical Sciences A. Rossi Fanelli, Sapienza University of Rome, 00185 Rome, Italy. emilia.chiancone@uniroma1.it

Show MeSH
Related in: MedlinePlus