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SERCA1 truncated proteins unable to pump calcium reduce the endoplasmic reticulum calcium concentration and induce apoptosis.

Chami M, Gozuacik D, Lagorce D, Brini M, Falson P, Peaucellier G, Pinton P, Lecoeur H, Gougeon ML, le Maire M, Rizzuto R, Bréchot C, Paterlini-Bréchot P - J. Cell Biol. (2001)

Bottom Line: Using ER-targeted aequorin (erAEQ), we have found that S1T proteins reduce ER calcium and reverse elevation of ER calcium loading induced by SERCA1 and SERCA2b.Finally, when overexpressed in liver-derived cells, S1T proteins significantly induce apoptosis.These data reveal a further mechanism modulating Ca(2+) accumulation into the ER of nonmuscle cells and highlight the relevance of S1T proteins to the control of apoptosis.

View Article: PubMed Central - PubMed

Affiliation: The French Institute of Health and Medical Research Institut National de la Santé et de la Recherche Médicale (INSERM/Pasteur U370)/Necker Faculty Institute of Medicine, 75015 Paris, France.

ABSTRACT
By pumping calcium from the cytosol to the ER, sarco/endoplasmic reticulum calcium ATPases (SERCAs) play a major role in the control of calcium signaling. We describe two SERCA1 splice variants (S1Ts) characterized by exon 4 and/or exon 11 splicing, encoding COOH terminally truncated proteins, having only one of the seven calcium-binding residues, and thus unable to pump calcium. As shown by semiquantitative RT-PCR, S1T transcripts are differentially expressed in several adult and fetal human tissues, but not in skeletal muscle and heart. S1T proteins expression was detected by Western blot in nontransfected cell lines. In transiently transfected cells, S1T homodimers were revealed by Western blot using mildly denaturing conditions. S1T proteins were shown, by confocal scanning microscopy, to colocalize with endogenous SERCA2b into the ER membrane. Using ER-targeted aequorin (erAEQ), we have found that S1T proteins reduce ER calcium and reverse elevation of ER calcium loading induced by SERCA1 and SERCA2b. Our results also show that SERCA1 variants increase ER calcium leakage and are consistent with the hypothesis of a cation channel formed by S1T homodimers. Finally, when overexpressed in liver-derived cells, S1T proteins significantly induce apoptosis. These data reveal a further mechanism modulating Ca(2+) accumulation into the ER of nonmuscle cells and highlight the relevance of S1T proteins to the control of apoptosis.

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Expression of endogenous SERCA2b in cells overexpressing S1T (A), relationship between [Ca2+]er and leakage rate in control cells, S1T+4-, and S1T−4-expressing cells (B and C), and dimerization of S1T+4 proteins under mildly denaturing conditions (D). (A) Expression of endogenous SERCA2b is similar in S1T+4-transfected as compared with nontransfected cells. Western blot analysis of the microsomal fraction of S1T+4 transiently transfected and nontransfected COS7 cells. The same extracts were run in parallel (SDS-PAGE). Corresponding membranes were hybridized with anti-SERCA1 (79B) and anti-SERCA2 (IID8) antibodies. (B) S1T proteins increase ER calcium leakage from the ER. Dependence on [Ca2+]er of the Ca2+ leak rate from the ER. Transfection, depletion of Ca2+ stores, and aequorin reconstitution were carried out as described in the legend to Fig. 5; after the steady state [Ca2+]er was reached, Ca2+ release was initiated by treating the cells with 50 μM tBuBHQ. Based on the experimental trace, the maximum rates of Ca2+ release (measured from the first derivative) at different [Ca2+]er values were calculated and plotted for S1T-transfected and control cells. The plot contains data obtained from independent experiments (n = 21 for controls, n = 9 for S1T−4, and n = 11 for S1T+4). Due to the mixing time in the luminometer chamber, the kinetics of [Ca2+]er decrease are sigmoidal, and the maximum rate is obtained 2–3 s after the addition of tBuBHQ. Accordingly, we considered the maximum rates as the best approximation for the initial rate of [Ca2+]er reduction. Fitting of the curve was performed using Microsoft Excel software. (C) [Ca2+]er measurements performed in HuH7 cells nontransfected (control) and transfected with the S1T+4 or S1T−4 construct, having comparable steady state calcium levels. The kinetics of Ca2+ efflux is clearly more rapid in S1T-transfected than in nontransfected cells. (D) S1T+4 proteins form dimers (92 kD) under mildly denaturing conditions. HuH7 cells were transiently transfected with S1T+4- (right) and SERCA2b-expressing (left) constructs. Western blots were performed with heated samples treated (+U) or not (−U) with urea. A 92-kD band corresponding to S1T+4 dimers was detected for the S1T+4 sample without urea treatment (−U). Note the absence of a 151-kD band that would have been consistent with heterodimers S1T+4–SERCA2b. A 210-kD band corresponding to SERCA2b dimers was present in the SERCA2b sample without urea (−U). SR, see legend to Fig. 3.
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Figure 7: Expression of endogenous SERCA2b in cells overexpressing S1T (A), relationship between [Ca2+]er and leakage rate in control cells, S1T+4-, and S1T−4-expressing cells (B and C), and dimerization of S1T+4 proteins under mildly denaturing conditions (D). (A) Expression of endogenous SERCA2b is similar in S1T+4-transfected as compared with nontransfected cells. Western blot analysis of the microsomal fraction of S1T+4 transiently transfected and nontransfected COS7 cells. The same extracts were run in parallel (SDS-PAGE). Corresponding membranes were hybridized with anti-SERCA1 (79B) and anti-SERCA2 (IID8) antibodies. (B) S1T proteins increase ER calcium leakage from the ER. Dependence on [Ca2+]er of the Ca2+ leak rate from the ER. Transfection, depletion of Ca2+ stores, and aequorin reconstitution were carried out as described in the legend to Fig. 5; after the steady state [Ca2+]er was reached, Ca2+ release was initiated by treating the cells with 50 μM tBuBHQ. Based on the experimental trace, the maximum rates of Ca2+ release (measured from the first derivative) at different [Ca2+]er values were calculated and plotted for S1T-transfected and control cells. The plot contains data obtained from independent experiments (n = 21 for controls, n = 9 for S1T−4, and n = 11 for S1T+4). Due to the mixing time in the luminometer chamber, the kinetics of [Ca2+]er decrease are sigmoidal, and the maximum rate is obtained 2–3 s after the addition of tBuBHQ. Accordingly, we considered the maximum rates as the best approximation for the initial rate of [Ca2+]er reduction. Fitting of the curve was performed using Microsoft Excel software. (C) [Ca2+]er measurements performed in HuH7 cells nontransfected (control) and transfected with the S1T+4 or S1T−4 construct, having comparable steady state calcium levels. The kinetics of Ca2+ efflux is clearly more rapid in S1T-transfected than in nontransfected cells. (D) S1T+4 proteins form dimers (92 kD) under mildly denaturing conditions. HuH7 cells were transiently transfected with S1T+4- (right) and SERCA2b-expressing (left) constructs. Western blots were performed with heated samples treated (+U) or not (−U) with urea. A 92-kD band corresponding to S1T+4 dimers was detected for the S1T+4 sample without urea treatment (−U). Note the absence of a 151-kD band that would have been consistent with heterodimers S1T+4–SERCA2b. A 210-kD band corresponding to SERCA2b dimers was present in the SERCA2b sample without urea (−U). SR, see legend to Fig. 3.

Mentions: Online supplementary Figure B shows the indication of the time interval needed to obtain a decrease of [Ca2+]er from 120 to 57 μM in the profiles depicted in C of Fig. 7. This time interval is 165 s in nontransfected cells (control) and 28 and 37 s in S1T+4- and S1T−4-transfected cells, respectively. Supplementary material is available at http://www.jcb.org/cgi/content/full/153/6/1301/DC1.


SERCA1 truncated proteins unable to pump calcium reduce the endoplasmic reticulum calcium concentration and induce apoptosis.

Chami M, Gozuacik D, Lagorce D, Brini M, Falson P, Peaucellier G, Pinton P, Lecoeur H, Gougeon ML, le Maire M, Rizzuto R, Bréchot C, Paterlini-Bréchot P - J. Cell Biol. (2001)

Expression of endogenous SERCA2b in cells overexpressing S1T (A), relationship between [Ca2+]er and leakage rate in control cells, S1T+4-, and S1T−4-expressing cells (B and C), and dimerization of S1T+4 proteins under mildly denaturing conditions (D). (A) Expression of endogenous SERCA2b is similar in S1T+4-transfected as compared with nontransfected cells. Western blot analysis of the microsomal fraction of S1T+4 transiently transfected and nontransfected COS7 cells. The same extracts were run in parallel (SDS-PAGE). Corresponding membranes were hybridized with anti-SERCA1 (79B) and anti-SERCA2 (IID8) antibodies. (B) S1T proteins increase ER calcium leakage from the ER. Dependence on [Ca2+]er of the Ca2+ leak rate from the ER. Transfection, depletion of Ca2+ stores, and aequorin reconstitution were carried out as described in the legend to Fig. 5; after the steady state [Ca2+]er was reached, Ca2+ release was initiated by treating the cells with 50 μM tBuBHQ. Based on the experimental trace, the maximum rates of Ca2+ release (measured from the first derivative) at different [Ca2+]er values were calculated and plotted for S1T-transfected and control cells. The plot contains data obtained from independent experiments (n = 21 for controls, n = 9 for S1T−4, and n = 11 for S1T+4). Due to the mixing time in the luminometer chamber, the kinetics of [Ca2+]er decrease are sigmoidal, and the maximum rate is obtained 2–3 s after the addition of tBuBHQ. Accordingly, we considered the maximum rates as the best approximation for the initial rate of [Ca2+]er reduction. Fitting of the curve was performed using Microsoft Excel software. (C) [Ca2+]er measurements performed in HuH7 cells nontransfected (control) and transfected with the S1T+4 or S1T−4 construct, having comparable steady state calcium levels. The kinetics of Ca2+ efflux is clearly more rapid in S1T-transfected than in nontransfected cells. (D) S1T+4 proteins form dimers (92 kD) under mildly denaturing conditions. HuH7 cells were transiently transfected with S1T+4- (right) and SERCA2b-expressing (left) constructs. Western blots were performed with heated samples treated (+U) or not (−U) with urea. A 92-kD band corresponding to S1T+4 dimers was detected for the S1T+4 sample without urea treatment (−U). Note the absence of a 151-kD band that would have been consistent with heterodimers S1T+4–SERCA2b. A 210-kD band corresponding to SERCA2b dimers was present in the SERCA2b sample without urea (−U). SR, see legend to Fig. 3.
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Figure 7: Expression of endogenous SERCA2b in cells overexpressing S1T (A), relationship between [Ca2+]er and leakage rate in control cells, S1T+4-, and S1T−4-expressing cells (B and C), and dimerization of S1T+4 proteins under mildly denaturing conditions (D). (A) Expression of endogenous SERCA2b is similar in S1T+4-transfected as compared with nontransfected cells. Western blot analysis of the microsomal fraction of S1T+4 transiently transfected and nontransfected COS7 cells. The same extracts were run in parallel (SDS-PAGE). Corresponding membranes were hybridized with anti-SERCA1 (79B) and anti-SERCA2 (IID8) antibodies. (B) S1T proteins increase ER calcium leakage from the ER. Dependence on [Ca2+]er of the Ca2+ leak rate from the ER. Transfection, depletion of Ca2+ stores, and aequorin reconstitution were carried out as described in the legend to Fig. 5; after the steady state [Ca2+]er was reached, Ca2+ release was initiated by treating the cells with 50 μM tBuBHQ. Based on the experimental trace, the maximum rates of Ca2+ release (measured from the first derivative) at different [Ca2+]er values were calculated and plotted for S1T-transfected and control cells. The plot contains data obtained from independent experiments (n = 21 for controls, n = 9 for S1T−4, and n = 11 for S1T+4). Due to the mixing time in the luminometer chamber, the kinetics of [Ca2+]er decrease are sigmoidal, and the maximum rate is obtained 2–3 s after the addition of tBuBHQ. Accordingly, we considered the maximum rates as the best approximation for the initial rate of [Ca2+]er reduction. Fitting of the curve was performed using Microsoft Excel software. (C) [Ca2+]er measurements performed in HuH7 cells nontransfected (control) and transfected with the S1T+4 or S1T−4 construct, having comparable steady state calcium levels. The kinetics of Ca2+ efflux is clearly more rapid in S1T-transfected than in nontransfected cells. (D) S1T+4 proteins form dimers (92 kD) under mildly denaturing conditions. HuH7 cells were transiently transfected with S1T+4- (right) and SERCA2b-expressing (left) constructs. Western blots were performed with heated samples treated (+U) or not (−U) with urea. A 92-kD band corresponding to S1T+4 dimers was detected for the S1T+4 sample without urea treatment (−U). Note the absence of a 151-kD band that would have been consistent with heterodimers S1T+4–SERCA2b. A 210-kD band corresponding to SERCA2b dimers was present in the SERCA2b sample without urea (−U). SR, see legend to Fig. 3.
Mentions: Online supplementary Figure B shows the indication of the time interval needed to obtain a decrease of [Ca2+]er from 120 to 57 μM in the profiles depicted in C of Fig. 7. This time interval is 165 s in nontransfected cells (control) and 28 and 37 s in S1T+4- and S1T−4-transfected cells, respectively. Supplementary material is available at http://www.jcb.org/cgi/content/full/153/6/1301/DC1.

Bottom Line: Using ER-targeted aequorin (erAEQ), we have found that S1T proteins reduce ER calcium and reverse elevation of ER calcium loading induced by SERCA1 and SERCA2b.Finally, when overexpressed in liver-derived cells, S1T proteins significantly induce apoptosis.These data reveal a further mechanism modulating Ca(2+) accumulation into the ER of nonmuscle cells and highlight the relevance of S1T proteins to the control of apoptosis.

View Article: PubMed Central - PubMed

Affiliation: The French Institute of Health and Medical Research Institut National de la Santé et de la Recherche Médicale (INSERM/Pasteur U370)/Necker Faculty Institute of Medicine, 75015 Paris, France.

ABSTRACT
By pumping calcium from the cytosol to the ER, sarco/endoplasmic reticulum calcium ATPases (SERCAs) play a major role in the control of calcium signaling. We describe two SERCA1 splice variants (S1Ts) characterized by exon 4 and/or exon 11 splicing, encoding COOH terminally truncated proteins, having only one of the seven calcium-binding residues, and thus unable to pump calcium. As shown by semiquantitative RT-PCR, S1T transcripts are differentially expressed in several adult and fetal human tissues, but not in skeletal muscle and heart. S1T proteins expression was detected by Western blot in nontransfected cell lines. In transiently transfected cells, S1T homodimers were revealed by Western blot using mildly denaturing conditions. S1T proteins were shown, by confocal scanning microscopy, to colocalize with endogenous SERCA2b into the ER membrane. Using ER-targeted aequorin (erAEQ), we have found that S1T proteins reduce ER calcium and reverse elevation of ER calcium loading induced by SERCA1 and SERCA2b. Our results also show that SERCA1 variants increase ER calcium leakage and are consistent with the hypothesis of a cation channel formed by S1T homodimers. Finally, when overexpressed in liver-derived cells, S1T proteins significantly induce apoptosis. These data reveal a further mechanism modulating Ca(2+) accumulation into the ER of nonmuscle cells and highlight the relevance of S1T proteins to the control of apoptosis.

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Related in: MedlinePlus