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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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TRP channels play key roles in the regulation of cytoplasmic Ca2+ in fission yeast.(A) The overexpression of trp1322+ and pkd2+ markedly enhanced the response to extracellularly added Ca2+. The KP3028 cells were transformed with a control vector, pREP1-GFP-Trp1322, or pREP1-Pkd2-GFP. The transformants were grown to exponential phase in the absence of thiamine for 36 hours to express GFP-19-AEQ, Trp1322 or Pkd2. The monitoring of the cytoplasmic Ca2+ level was performed as described in Figure 2A. The data shown are representative of multiple experiments. (B) The CDRE-reporter activity in the cells overexpressing Trp1322. The KP2755 (h− leu1 arg1 3×CDRE::luc(R2.2)::arg1+) cells were transformed with pREP1-GFP-Trp1322. The transformants were grown to exponential phase in the presence (Promoter OFF) or absence (Promoter ON, overexpression) of thiamine for 20 hours. The CDRE assay was performed as described in Materials and Methods. The data shown are representative of multiple experiments. (C) Intracellular localization of the two TRP channels. The wild-type cells harboring GFP-Trp1322 or Trp663-GFP were grown to early log phase in EMM containing 4 µM thiamine, and then were examined under the fluorescence microscope. Bar, 10 µm. (D) Pkd2 plays roles in inducing Ca2+ release from the Golgi. The KP3028 or KP3025 cells were transformed with pREP1-GFP-Trp1322 or pREP1-Pkd2-GFP, then were cultured and assayed as described in Figure 2A.
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pone-0022421-g003: TRP channels play key roles in the regulation of cytoplasmic Ca2+ in fission yeast.(A) The overexpression of trp1322+ and pkd2+ markedly enhanced the response to extracellularly added Ca2+. The KP3028 cells were transformed with a control vector, pREP1-GFP-Trp1322, or pREP1-Pkd2-GFP. The transformants were grown to exponential phase in the absence of thiamine for 36 hours to express GFP-19-AEQ, Trp1322 or Pkd2. The monitoring of the cytoplasmic Ca2+ level was performed as described in Figure 2A. The data shown are representative of multiple experiments. (B) The CDRE-reporter activity in the cells overexpressing Trp1322. The KP2755 (h− leu1 arg1 3×CDRE::luc(R2.2)::arg1+) cells were transformed with pREP1-GFP-Trp1322. The transformants were grown to exponential phase in the presence (Promoter OFF) or absence (Promoter ON, overexpression) of thiamine for 20 hours. The CDRE assay was performed as described in Materials and Methods. The data shown are representative of multiple experiments. (C) Intracellular localization of the two TRP channels. The wild-type cells harboring GFP-Trp1322 or Trp663-GFP were grown to early log phase in EMM containing 4 µM thiamine, and then were examined under the fluorescence microscope. Bar, 10 µm. (D) Pkd2 plays roles in inducing Ca2+ release from the Golgi. The KP3028 or KP3025 cells were transformed with pREP1-GFP-Trp1322 or pREP1-Pkd2-GFP, then were cultured and assayed as described in Figure 2A.

Mentions: The TRP channel superfamily consists of a diverse group of cation channels, and plays critical roles in response to external stimuli [1]–[3]. Blast searches against Sanger Center S. pombe databases led to the identification of three open reading frames which are as follows, SPCC1322.03 (named as trp1322+ in this study), SPCC663.14c (named as trp663+ in this study), and SPAC1F7.03 (Pkd2) [8] that encode TRP-like ion channels. We subcloned each of the three TRP channels in pREP1 vector and examined the effect of their overexpression on the cytoplasmic Ca2+ levels in wild-type cells. The results showed that the overexpression of Trp1322 or Pkd2 markedly induced an approximately 10∼20 fold burst-like increase in the cytoplasmic Ca2+ levels when stimulated by the extracellularly added CaCl2 as compared with the control vector (Figure 3A). In contrast, the overexpression of Trp663 induced a cytoplasmic Ca2+ increase similar to that of the control vector (Figure 3A).


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)

TRP channels play key roles in the regulation of cytoplasmic Ca2+ in fission yeast.(A) The overexpression of trp1322+ and pkd2+ markedly enhanced the response to extracellularly added Ca2+. The KP3028 cells were transformed with a control vector, pREP1-GFP-Trp1322, or pREP1-Pkd2-GFP. The transformants were grown to exponential phase in the absence of thiamine for 36 hours to express GFP-19-AEQ, Trp1322 or Pkd2. The monitoring of the cytoplasmic Ca2+ level was performed as described in Figure 2A. The data shown are representative of multiple experiments. (B) The CDRE-reporter activity in the cells overexpressing Trp1322. The KP2755 (h− leu1 arg1 3×CDRE::luc(R2.2)::arg1+) cells were transformed with pREP1-GFP-Trp1322. The transformants were grown to exponential phase in the presence (Promoter OFF) or absence (Promoter ON, overexpression) of thiamine for 20 hours. The CDRE assay was performed as described in Materials and Methods. The data shown are representative of multiple experiments. (C) Intracellular localization of the two TRP channels. The wild-type cells harboring GFP-Trp1322 or Trp663-GFP were grown to early log phase in EMM containing 4 µM thiamine, and then were examined under the fluorescence microscope. Bar, 10 µm. (D) Pkd2 plays roles in inducing Ca2+ release from the Golgi. The KP3028 or KP3025 cells were transformed with pREP1-GFP-Trp1322 or pREP1-Pkd2-GFP, then were cultured and assayed as described in Figure 2A.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3139647&req=5

pone-0022421-g003: TRP channels play key roles in the regulation of cytoplasmic Ca2+ in fission yeast.(A) The overexpression of trp1322+ and pkd2+ markedly enhanced the response to extracellularly added Ca2+. The KP3028 cells were transformed with a control vector, pREP1-GFP-Trp1322, or pREP1-Pkd2-GFP. The transformants were grown to exponential phase in the absence of thiamine for 36 hours to express GFP-19-AEQ, Trp1322 or Pkd2. The monitoring of the cytoplasmic Ca2+ level was performed as described in Figure 2A. The data shown are representative of multiple experiments. (B) The CDRE-reporter activity in the cells overexpressing Trp1322. The KP2755 (h− leu1 arg1 3×CDRE::luc(R2.2)::arg1+) cells were transformed with pREP1-GFP-Trp1322. The transformants were grown to exponential phase in the presence (Promoter OFF) or absence (Promoter ON, overexpression) of thiamine for 20 hours. The CDRE assay was performed as described in Materials and Methods. The data shown are representative of multiple experiments. (C) Intracellular localization of the two TRP channels. The wild-type cells harboring GFP-Trp1322 or Trp663-GFP were grown to early log phase in EMM containing 4 µM thiamine, and then were examined under the fluorescence microscope. Bar, 10 µm. (D) Pkd2 plays roles in inducing Ca2+ release from the Golgi. The KP3028 or KP3025 cells were transformed with pREP1-GFP-Trp1322 or pREP1-Pkd2-GFP, then were cultured and assayed as described in Figure 2A.
Mentions: The TRP channel superfamily consists of a diverse group of cation channels, and plays critical roles in response to external stimuli [1]–[3]. Blast searches against Sanger Center S. pombe databases led to the identification of three open reading frames which are as follows, SPCC1322.03 (named as trp1322+ in this study), SPCC663.14c (named as trp663+ in this study), and SPAC1F7.03 (Pkd2) [8] that encode TRP-like ion channels. We subcloned each of the three TRP channels in pREP1 vector and examined the effect of their overexpression on the cytoplasmic Ca2+ levels in wild-type cells. The results showed that the overexpression of Trp1322 or Pkd2 markedly induced an approximately 10∼20 fold burst-like increase in the cytoplasmic Ca2+ levels when stimulated by the extracellularly added CaCl2 as compared with the control vector (Figure 3A). In contrast, the overexpression of Trp663 induced a cytoplasmic Ca2+ increase similar to that of the control vector (Figure 3A).

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