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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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The role of Trp1322 in the regulation of calcium homeostasis in fission yeast.(A) The overexpression of Trp1322 and Pkd2 abolished the synergistic effect of NaCl plus FK506. The KP3028 cells were transformed with the control vector, pREP1-GFP-Trp1322 or pREP1-Pkd2-GFP, then were cultured as described in Figure 2A, and were assayed as described in Figure 4. The histogram was calculated as described in the legend of Figure 4. (B) When Trp1322 was overexpressed, the high basal CDRE-reporter activity was significantly decreased by the addition of NaCl. Note the marked activation of the reporter activity by the same stimuli in non-induced cells (Promoter OFF, left pannel). The KP2755 (h− leu1 arg1 3×CDRE::luc(R2.2)::arg1+) cells were transformed with pREP1-GFP-Trp1322. The assay was performed as described in Figure 3B. The data shown are representative of multiple experiments. (C) The overexpression of trp1322+ gene suppressed the salts sensitivity of Δppb1 cells. The Δppb1 cells transformed with the control vector, pREP1-GFP-Trp1322 or pREP1-Pkd2-GFP were streaked onto the plates as indicated and then incubated for 4 days at 27°C. (D) The effect of the overexpression of Pkd2 or Trp1322 on the cytosolic Ca2+ level upon the addition of extracellular calcium in Δppb1 and Δprz1 cells. The KP3028 (wild-type), KP3750 (Δppb1) or KP3688 (Δprz1) cells transformed with the control vector, pREP1-GFP-Trp1322 or pREP1-Pkd2-GFP, respectively were cultured and assayed as described in Figure 2A. The RLU ratio was determined by dividing the peak RLU at 0 mM CaCl2 by the peak RLU at 1.25, 2.5, 5, and 10 mM CaCl2, respectively. The data represent the means ± standard deviations of RLU ratio from three independent experiments, and each sample was analyzed in duplicate. (E) The deletion of trp1322+ gene suppressed the synergistic effect of NaCl plus FK506. The Δtrp1322 cells harboring pKB6892 were cultured and assayed as described in Figure 4. 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. (F) Overexpression of the constitutively active Pek1 MAPKK also stimulates Ca2+ influx in Δtrp1322 cells. The Δtrp1322 cells integrated with the chromosomal pREP1-GST-Pek1DD were transformed with pKB6892, and the transformants were cultured in EMM with the addition of 4 µM thiamine for 12 hours. Then the assay was performed as described in the legend of Figure 5D. 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-g007: The role of Trp1322 in the regulation of calcium homeostasis in fission yeast.(A) The overexpression of Trp1322 and Pkd2 abolished the synergistic effect of NaCl plus FK506. The KP3028 cells were transformed with the control vector, pREP1-GFP-Trp1322 or pREP1-Pkd2-GFP, then were cultured as described in Figure 2A, and were assayed as described in Figure 4. The histogram was calculated as described in the legend of Figure 4. (B) When Trp1322 was overexpressed, the high basal CDRE-reporter activity was significantly decreased by the addition of NaCl. Note the marked activation of the reporter activity by the same stimuli in non-induced cells (Promoter OFF, left pannel). The KP2755 (h− leu1 arg1 3×CDRE::luc(R2.2)::arg1+) cells were transformed with pREP1-GFP-Trp1322. The assay was performed as described in Figure 3B. The data shown are representative of multiple experiments. (C) The overexpression of trp1322+ gene suppressed the salts sensitivity of Δppb1 cells. The Δppb1 cells transformed with the control vector, pREP1-GFP-Trp1322 or pREP1-Pkd2-GFP were streaked onto the plates as indicated and then incubated for 4 days at 27°C. (D) The effect of the overexpression of Pkd2 or Trp1322 on the cytosolic Ca2+ level upon the addition of extracellular calcium in Δppb1 and Δprz1 cells. The KP3028 (wild-type), KP3750 (Δppb1) or KP3688 (Δprz1) cells transformed with the control vector, pREP1-GFP-Trp1322 or pREP1-Pkd2-GFP, respectively were cultured and assayed as described in Figure 2A. The RLU ratio was determined by dividing the peak RLU at 0 mM CaCl2 by the peak RLU at 1.25, 2.5, 5, and 10 mM CaCl2, respectively. The data represent the means ± standard deviations of RLU ratio from three independent experiments, and each sample was analyzed in duplicate. (E) The deletion of trp1322+ gene suppressed the synergistic effect of NaCl plus FK506. The Δtrp1322 cells harboring pKB6892 were cultured and assayed as described in Figure 4. 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. (F) Overexpression of the constitutively active Pek1 MAPKK also stimulates Ca2+ influx in Δtrp1322 cells. The Δtrp1322 cells integrated with the chromosomal pREP1-GST-Pek1DD were transformed with pKB6892, and the transformants were cultured in EMM with the addition of 4 µM thiamine for 12 hours. Then the assay was performed as described in the legend of Figure 5D. 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: We then examined the effect of NaCl plus FK506 on the cells overexpressing Trp1322 or Pkd2 in wild-type cells. Overexpression of these two TRP channels showed high basal cytoplasmic Ca2+ levels in the absence of NaCl or FK506 (Figure 7A, control). In cells overexpressing Trp1322 or Pkd2 when NaCl alone was added to the medium, the high basal cytoplasmic Ca2+ level was decreased, while in cells harboring the vector the cytoplasmic Ca2+ level was markedly elevated (Figure 7A, +NaCl). Consistently, in wild-type cells when Trp1322 was overexpressed the high basal CDRE-reporter activity was significantly lowered by the addition of NaCl (Figure 7B, promoter ON). Surprisingly, when the cells overexpressing Trp1322 were treated with NaCl plus FK506, no marked increase in the cytoplasmic Ca2+ level was observed (Figure 7A, wt+Trp1322, NaCl+FK). These results suggest that the overexpression of Trp1322 may inhibit the Ca2+ influx via the Cch1-Yam8 channel complex. Consistently in Δppb1 cells, the overexpression of Trp1322 significantly suppressed the NaCl and MgCl2 sensitivity (Figure 7C).


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)

The role of Trp1322 in the regulation of calcium homeostasis in fission yeast.(A) The overexpression of Trp1322 and Pkd2 abolished the synergistic effect of NaCl plus FK506. The KP3028 cells were transformed with the control vector, pREP1-GFP-Trp1322 or pREP1-Pkd2-GFP, then were cultured as described in Figure 2A, and were assayed as described in Figure 4. The histogram was calculated as described in the legend of Figure 4. (B) When Trp1322 was overexpressed, the high basal CDRE-reporter activity was significantly decreased by the addition of NaCl. Note the marked activation of the reporter activity by the same stimuli in non-induced cells (Promoter OFF, left pannel). The KP2755 (h− leu1 arg1 3×CDRE::luc(R2.2)::arg1+) cells were transformed with pREP1-GFP-Trp1322. The assay was performed as described in Figure 3B. The data shown are representative of multiple experiments. (C) The overexpression of trp1322+ gene suppressed the salts sensitivity of Δppb1 cells. The Δppb1 cells transformed with the control vector, pREP1-GFP-Trp1322 or pREP1-Pkd2-GFP were streaked onto the plates as indicated and then incubated for 4 days at 27°C. (D) The effect of the overexpression of Pkd2 or Trp1322 on the cytosolic Ca2+ level upon the addition of extracellular calcium in Δppb1 and Δprz1 cells. The KP3028 (wild-type), KP3750 (Δppb1) or KP3688 (Δprz1) cells transformed with the control vector, pREP1-GFP-Trp1322 or pREP1-Pkd2-GFP, respectively were cultured and assayed as described in Figure 2A. The RLU ratio was determined by dividing the peak RLU at 0 mM CaCl2 by the peak RLU at 1.25, 2.5, 5, and 10 mM CaCl2, respectively. The data represent the means ± standard deviations of RLU ratio from three independent experiments, and each sample was analyzed in duplicate. (E) The deletion of trp1322+ gene suppressed the synergistic effect of NaCl plus FK506. The Δtrp1322 cells harboring pKB6892 were cultured and assayed as described in Figure 4. 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. (F) Overexpression of the constitutively active Pek1 MAPKK also stimulates Ca2+ influx in Δtrp1322 cells. The Δtrp1322 cells integrated with the chromosomal pREP1-GST-Pek1DD were transformed with pKB6892, and the transformants were cultured in EMM with the addition of 4 µM thiamine for 12 hours. Then the assay was performed as described in the legend of Figure 5D. 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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Related In: Results  -  Collection

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

pone-0022421-g007: The role of Trp1322 in the regulation of calcium homeostasis in fission yeast.(A) The overexpression of Trp1322 and Pkd2 abolished the synergistic effect of NaCl plus FK506. The KP3028 cells were transformed with the control vector, pREP1-GFP-Trp1322 or pREP1-Pkd2-GFP, then were cultured as described in Figure 2A, and were assayed as described in Figure 4. The histogram was calculated as described in the legend of Figure 4. (B) When Trp1322 was overexpressed, the high basal CDRE-reporter activity was significantly decreased by the addition of NaCl. Note the marked activation of the reporter activity by the same stimuli in non-induced cells (Promoter OFF, left pannel). The KP2755 (h− leu1 arg1 3×CDRE::luc(R2.2)::arg1+) cells were transformed with pREP1-GFP-Trp1322. The assay was performed as described in Figure 3B. The data shown are representative of multiple experiments. (C) The overexpression of trp1322+ gene suppressed the salts sensitivity of Δppb1 cells. The Δppb1 cells transformed with the control vector, pREP1-GFP-Trp1322 or pREP1-Pkd2-GFP were streaked onto the plates as indicated and then incubated for 4 days at 27°C. (D) The effect of the overexpression of Pkd2 or Trp1322 on the cytosolic Ca2+ level upon the addition of extracellular calcium in Δppb1 and Δprz1 cells. The KP3028 (wild-type), KP3750 (Δppb1) or KP3688 (Δprz1) cells transformed with the control vector, pREP1-GFP-Trp1322 or pREP1-Pkd2-GFP, respectively were cultured and assayed as described in Figure 2A. The RLU ratio was determined by dividing the peak RLU at 0 mM CaCl2 by the peak RLU at 1.25, 2.5, 5, and 10 mM CaCl2, respectively. The data represent the means ± standard deviations of RLU ratio from three independent experiments, and each sample was analyzed in duplicate. (E) The deletion of trp1322+ gene suppressed the synergistic effect of NaCl plus FK506. The Δtrp1322 cells harboring pKB6892 were cultured and assayed as described in Figure 4. 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. (F) Overexpression of the constitutively active Pek1 MAPKK also stimulates Ca2+ influx in Δtrp1322 cells. The Δtrp1322 cells integrated with the chromosomal pREP1-GST-Pek1DD were transformed with pKB6892, and the transformants were cultured in EMM with the addition of 4 µM thiamine for 12 hours. Then the assay was performed as described in the legend of Figure 5D. 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: We then examined the effect of NaCl plus FK506 on the cells overexpressing Trp1322 or Pkd2 in wild-type cells. Overexpression of these two TRP channels showed high basal cytoplasmic Ca2+ levels in the absence of NaCl or FK506 (Figure 7A, control). In cells overexpressing Trp1322 or Pkd2 when NaCl alone was added to the medium, the high basal cytoplasmic Ca2+ level was decreased, while in cells harboring the vector the cytoplasmic Ca2+ level was markedly elevated (Figure 7A, +NaCl). Consistently, in wild-type cells when Trp1322 was overexpressed the high basal CDRE-reporter activity was significantly lowered by the addition of NaCl (Figure 7B, promoter ON). Surprisingly, when the cells overexpressing Trp1322 were treated with NaCl plus FK506, no marked increase in the cytoplasmic Ca2+ level was observed (Figure 7A, wt+Trp1322, NaCl+FK). These results suggest that the overexpression of Trp1322 may inhibit the Ca2+ influx via the Cch1-Yam8 channel complex. Consistently in Δppb1 cells, the overexpression of Trp1322 significantly suppressed the NaCl and MgCl2 sensitivity (Figure 7C).

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