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Transient receptor potential (TRP) and Cch1-Yam8 channels play key roles in the regulation of cytoplasmic Ca2+ in fission yeast.

Ma Y, Sugiura R, Koike A, Ebina H, Sio SO, Kuno T - PLoS ONE (2011)

Bottom Line: We found that the extracellularly added Ca(2+) caused a dose-dependent increase in the cytoplasmic Ca(2+) level and resulted in a burst-like peak.We also found that extracellularly added NaCl plus FK506 caused a synergistic cytosolic Ca(2+) increase that is dependent on the inhibition of calcineurin activity, but not on the inhibition of Prz1.These results suggest that Pmk1 MAPK and calcineurin positively and negatively regulate the Cch1-Yam8 complex, respectively, via modulating the balance between phosphorylation and dyphosphorylation state.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Pharmacology and Pharmacogenomics, Department of Biochemistry and Molecular Biology, Kobe University Graduate School of Medicine, Kobe, Japan. mayan@med.kobe-u.ac.jp

ABSTRACT
The regulation of cytoplasmic Ca(2+) is crucial for various cellular processes. Here, we examined the cytoplasmic Ca(2+) levels in living fission yeast cells by a highly sensitive bioluminescence resonance energy transfer-based assay using GFP-aequorin fusion protein linked by 19 amino acid. We monitored the cytoplasmic Ca(2+) level and its change caused by extracellular stimulants such as CaCl(2) or NaCl plus FK506 (calcineurin inhibitor). We found that the extracellularly added Ca(2+) caused a dose-dependent increase in the cytoplasmic Ca(2+) level and resulted in a burst-like peak. The overexpression of two transient receptor potential (TRP) channel homologues, Trp1322 or Pkd2, markedly enhanced this response. Interestingly, the burst-like peak upon TRP overexpression was completely abolished by gene deletion of calcineurin and was dramatically decreased by gene deletion of Prz1, a downstream transcription factor activated by calcineurin. Furthermore, 1 hour treatment with FK506 failed to suppress the burst-like peak. These results suggest that the burst-like Ca(2+) peak is dependent on the transcriptional activity of Prz1, but not on the direct TRP dephosphorylation. We also found that extracellularly added NaCl plus FK506 caused a synergistic cytosolic Ca(2+) increase that is dependent on the inhibition of calcineurin activity, but not on the inhibition of Prz1. The synergistic Ca(2+) increase is abolished by the addition of the Ca(2+) chelator BAPTA into the media, and is also abolished by deletion of the gene encoding a subunit of the Cch1-Yam8 Ca(2+) channel complex, indicating that the synergistic increase is caused by the Ca(2+) influx from the extracellular medium via the Cch1-Yam8 complex. Furthermore, deletion of Pmk1 MAPK abolished the Ca(2+) influx, and overexpression of the constitutively active Pek1 MAPKK enhanced the influx. These results suggest that Pmk1 MAPK and calcineurin positively and negatively regulate the Cch1-Yam8 complex, respectively, via modulating the balance between phosphorylation and dyphosphorylation state.

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NaCl plus FK506 caused a synergistic cytoplasmic Ca2+ increase via the Cch1-Yam8 channel complex.(A) The synergistic increase in the cytoplasmic Ca2+ level is derived from the extracellular medium. The experiment was performed as described in Figure 4, except that prior to the addition of FK506, various concentrations of BAPTA (0.25, 0.5, 1, and 2 mM) were added to chelate Ca2+ in the EMM medium. The histogram was calculated as described in the legend of Figure 4. (B) The cch1 and yam8 deletion abolished the synergistic increase in the cytoplasmic Ca2+ level. The Δyam8, Δcch1, or Δyam8Δcch1 cells harboring pKB6892 were cultured and assayed as described in Figure 4. The histogram was calculated as described in the legend of Figure 4. (C) The pmk1 deletion abolished the synergistic increase in the cytoplasmic Ca2+ level. The Δpmk1 cells harboring pKB6892 were cultured and assayed as described in Figure 4. The histogram was calculated as described in the legend of Figure 4. (D) Overexpression of the constitutively active Pek1 MAPKK stimulates the Cch1-Yam8-mediated Ca2+ influx. The wild-type or Δcch1 cells integrated with chromosomal pREP1-GST-Pek1DD were transformed with pKB6892, and the transformants were cultured in EMM containing 4 µM thiamine for 12 hours. Then the cells were collected and washed three times with EMM without thiamine. The washed cells were divided into two portions, one portion was cultured in EMM containing 4 µM thiamine, and the other portion was cultured in EMM without thiamine, and the cells were grown to exponential phase. The cytoplasmic Ca2+ level was monitored as described in Figure 4. “Promoter OFF” indicates that the expression of Pek1DD was repressed by the addition of 4 µM thiamine to the medium and “Promoter ON” indicates that the expression of Pek1DD was induced by the removal of thiamine from the medium. The histogram was calculated as described in the legend of Figure 4. The data represent the means ± standard deviations of RLU taken at 240 min from three independent experiments, and each sample was analyzed in duplicate.
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pone-0022421-g005: NaCl plus FK506 caused a synergistic cytoplasmic Ca2+ increase via the Cch1-Yam8 channel complex.(A) The synergistic increase in the cytoplasmic Ca2+ level is derived from the extracellular medium. The experiment was performed as described in Figure 4, except that prior to the addition of FK506, various concentrations of BAPTA (0.25, 0.5, 1, and 2 mM) were added to chelate Ca2+ in the EMM medium. The histogram was calculated as described in the legend of Figure 4. (B) The cch1 and yam8 deletion abolished the synergistic increase in the cytoplasmic Ca2+ level. The Δyam8, Δcch1, or Δyam8Δcch1 cells harboring pKB6892 were cultured and assayed as described in Figure 4. The histogram was calculated as described in the legend of Figure 4. (C) The pmk1 deletion abolished the synergistic increase in the cytoplasmic Ca2+ level. The Δpmk1 cells harboring pKB6892 were cultured and assayed as described in Figure 4. The histogram was calculated as described in the legend of Figure 4. (D) Overexpression of the constitutively active Pek1 MAPKK stimulates the Cch1-Yam8-mediated Ca2+ influx. The wild-type or Δcch1 cells integrated with chromosomal pREP1-GST-Pek1DD were transformed with pKB6892, and the transformants were cultured in EMM containing 4 µM thiamine for 12 hours. Then the cells were collected and washed three times with EMM without thiamine. The washed cells were divided into two portions, one portion was cultured in EMM containing 4 µM thiamine, and the other portion was cultured in EMM without thiamine, and the cells were grown to exponential phase. The cytoplasmic Ca2+ level was monitored as described in Figure 4. “Promoter OFF” indicates that the expression of Pek1DD was repressed by the addition of 4 µM thiamine to the medium and “Promoter ON” indicates that the expression of Pek1DD was induced by the removal of thiamine from the medium. The histogram was calculated as described in the legend of Figure 4. The data represent the means ± standard deviations of RLU taken at 240 min from three independent experiments, and each sample was analyzed in duplicate.

Mentions: To investigate whether the increase in the cytoplasmic Ca2+ level is due to the Ca2+ influx from the extracellular medium or due to the release from an internal store, we examined the effect of the rapid Ca2+ chelator BAPTA (1, 2-bis (o-aminophenoxy) ethane-N, N, N′, N′-tetraacetic acid). As shown in Figure 5A, the synergistic increase was inhibited by the addition of BAPTA in a dose-dependent manner, indicating that the increase in the cytoplasmic Ca2+ level is dependent on the influx across the Ca2+ channel that exists on the plasma membrane.


Transient receptor potential (TRP) and Cch1-Yam8 channels play key roles in the regulation of cytoplasmic Ca2+ in fission yeast.

Ma Y, Sugiura R, Koike A, Ebina H, Sio SO, Kuno T - PLoS ONE (2011)

NaCl plus FK506 caused a synergistic cytoplasmic Ca2+ increase via the Cch1-Yam8 channel complex.(A) The synergistic increase in the cytoplasmic Ca2+ level is derived from the extracellular medium. The experiment was performed as described in Figure 4, except that prior to the addition of FK506, various concentrations of BAPTA (0.25, 0.5, 1, and 2 mM) were added to chelate Ca2+ in the EMM medium. The histogram was calculated as described in the legend of Figure 4. (B) The cch1 and yam8 deletion abolished the synergistic increase in the cytoplasmic Ca2+ level. The Δyam8, Δcch1, or Δyam8Δcch1 cells harboring pKB6892 were cultured and assayed as described in Figure 4. The histogram was calculated as described in the legend of Figure 4. (C) The pmk1 deletion abolished the synergistic increase in the cytoplasmic Ca2+ level. The Δpmk1 cells harboring pKB6892 were cultured and assayed as described in Figure 4. The histogram was calculated as described in the legend of Figure 4. (D) Overexpression of the constitutively active Pek1 MAPKK stimulates the Cch1-Yam8-mediated Ca2+ influx. The wild-type or Δcch1 cells integrated with chromosomal pREP1-GST-Pek1DD were transformed with pKB6892, and the transformants were cultured in EMM containing 4 µM thiamine for 12 hours. Then the cells were collected and washed three times with EMM without thiamine. The washed cells were divided into two portions, one portion was cultured in EMM containing 4 µM thiamine, and the other portion was cultured in EMM without thiamine, and the cells were grown to exponential phase. The cytoplasmic Ca2+ level was monitored as described in Figure 4. “Promoter OFF” indicates that the expression of Pek1DD was repressed by the addition of 4 µM thiamine to the medium and “Promoter ON” indicates that the expression of Pek1DD was induced by the removal of thiamine from the medium. The histogram was calculated as described in the legend of Figure 4. The data represent the means ± standard deviations of RLU taken at 240 min from three independent experiments, and each sample was analyzed in duplicate.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3139647&req=5

pone-0022421-g005: NaCl plus FK506 caused a synergistic cytoplasmic Ca2+ increase via the Cch1-Yam8 channel complex.(A) The synergistic increase in the cytoplasmic Ca2+ level is derived from the extracellular medium. The experiment was performed as described in Figure 4, except that prior to the addition of FK506, various concentrations of BAPTA (0.25, 0.5, 1, and 2 mM) were added to chelate Ca2+ in the EMM medium. The histogram was calculated as described in the legend of Figure 4. (B) The cch1 and yam8 deletion abolished the synergistic increase in the cytoplasmic Ca2+ level. The Δyam8, Δcch1, or Δyam8Δcch1 cells harboring pKB6892 were cultured and assayed as described in Figure 4. The histogram was calculated as described in the legend of Figure 4. (C) The pmk1 deletion abolished the synergistic increase in the cytoplasmic Ca2+ level. The Δpmk1 cells harboring pKB6892 were cultured and assayed as described in Figure 4. The histogram was calculated as described in the legend of Figure 4. (D) Overexpression of the constitutively active Pek1 MAPKK stimulates the Cch1-Yam8-mediated Ca2+ influx. The wild-type or Δcch1 cells integrated with chromosomal pREP1-GST-Pek1DD were transformed with pKB6892, and the transformants were cultured in EMM containing 4 µM thiamine for 12 hours. Then the cells were collected and washed three times with EMM without thiamine. The washed cells were divided into two portions, one portion was cultured in EMM containing 4 µM thiamine, and the other portion was cultured in EMM without thiamine, and the cells were grown to exponential phase. The cytoplasmic Ca2+ level was monitored as described in Figure 4. “Promoter OFF” indicates that the expression of Pek1DD was repressed by the addition of 4 µM thiamine to the medium and “Promoter ON” indicates that the expression of Pek1DD was induced by the removal of thiamine from the medium. The histogram was calculated as described in the legend of Figure 4. The data represent the means ± standard deviations of RLU taken at 240 min from three independent experiments, and each sample was analyzed in duplicate.
Mentions: To investigate whether the increase in the cytoplasmic Ca2+ level is due to the Ca2+ influx from the extracellular medium or due to the release from an internal store, we examined the effect of the rapid Ca2+ chelator BAPTA (1, 2-bis (o-aminophenoxy) ethane-N, N, N′, N′-tetraacetic acid). As shown in Figure 5A, the synergistic increase was inhibited by the addition of BAPTA in a dose-dependent manner, indicating that the increase in the cytoplasmic Ca2+ level is dependent on the influx across the Ca2+ channel that exists on the plasma membrane.

Bottom Line: We found that the extracellularly added Ca(2+) caused a dose-dependent increase in the cytoplasmic Ca(2+) level and resulted in a burst-like peak.We also found that extracellularly added NaCl plus FK506 caused a synergistic cytosolic Ca(2+) increase that is dependent on the inhibition of calcineurin activity, but not on the inhibition of Prz1.These results suggest that Pmk1 MAPK and calcineurin positively and negatively regulate the Cch1-Yam8 complex, respectively, via modulating the balance between phosphorylation and dyphosphorylation state.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Pharmacology and Pharmacogenomics, Department of Biochemistry and Molecular Biology, Kobe University Graduate School of Medicine, Kobe, Japan. mayan@med.kobe-u.ac.jp

ABSTRACT
The regulation of cytoplasmic Ca(2+) is crucial for various cellular processes. Here, we examined the cytoplasmic Ca(2+) levels in living fission yeast cells by a highly sensitive bioluminescence resonance energy transfer-based assay using GFP-aequorin fusion protein linked by 19 amino acid. We monitored the cytoplasmic Ca(2+) level and its change caused by extracellular stimulants such as CaCl(2) or NaCl plus FK506 (calcineurin inhibitor). We found that the extracellularly added Ca(2+) caused a dose-dependent increase in the cytoplasmic Ca(2+) level and resulted in a burst-like peak. The overexpression of two transient receptor potential (TRP) channel homologues, Trp1322 or Pkd2, markedly enhanced this response. Interestingly, the burst-like peak upon TRP overexpression was completely abolished by gene deletion of calcineurin and was dramatically decreased by gene deletion of Prz1, a downstream transcription factor activated by calcineurin. Furthermore, 1 hour treatment with FK506 failed to suppress the burst-like peak. These results suggest that the burst-like Ca(2+) peak is dependent on the transcriptional activity of Prz1, but not on the direct TRP dephosphorylation. We also found that extracellularly added NaCl plus FK506 caused a synergistic cytosolic Ca(2+) increase that is dependent on the inhibition of calcineurin activity, but not on the inhibition of Prz1. The synergistic Ca(2+) increase is abolished by the addition of the Ca(2+) chelator BAPTA into the media, and is also abolished by deletion of the gene encoding a subunit of the Cch1-Yam8 Ca(2+) channel complex, indicating that the synergistic increase is caused by the Ca(2+) influx from the extracellular medium via the Cch1-Yam8 complex. Furthermore, deletion of Pmk1 MAPK abolished the Ca(2+) influx, and overexpression of the constitutively active Pek1 MAPKK enhanced the influx. These results suggest that Pmk1 MAPK and calcineurin positively and negatively regulate the Cch1-Yam8 complex, respectively, via modulating the balance between phosphorylation and dyphosphorylation state.

Show MeSH
Related in: MedlinePlus