title: Transient receptor potential (TRP) and Cch1-Yam8 channels play key roles in the regulation of cytoplasmic Ca2+ in fission yeast.
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.
authors.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
authors.forename: Yan
authors.initials: Y
authors.lastname: Ma
authors.forename: Reiko
authors.initials: R
authors.lastname: Sugiura
authors.forename: Atsushi
authors.initials: A
authors.lastname: Koike
authors.forename: Hidemine
authors.initials: H
authors.lastname: Ebina
authors.forename: Susie O
authors.initials: SO
authors.lastname: Sio
authors.forename: Takayoshi
authors.initials: T
authors.lastname: Kuno
mesh_terms: D000331:Aequorin / Q000378:metabolism; D019703:Calcineurin / Q000378:metabolism; D065095:Calcineurin Inhibitors; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism*; D002122:Calcium Chloride / Q000494:pharmacology; D002463:Cell Membrane Permeability / Q000187:drug effects; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism*; D005110:Extracellular Space / Q000187:drug effects / Q000378:metabolism; D055786:Gene Knockout Techniques; D005800:Genes, Fungal / Q000235:genetics; D006056:Golgi Apparatus / Q000187:drug effects / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D015151:Immunoblotting; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012568:Schizosaccharomyces / Q000166:cytology / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins / Q000378:metabolism*; D012965:Sodium Chloride / Q000494:pharmacology; D016559:Tacrolimus / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects; D050051:Transient Receptor Potential Channels / Q000378:metabolism*
chemical_list: D065095:Calcineurin Inhibitors; D015220:Calcium Channels; C568273:Cch1 protein, S pombe; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D029702:Schizosaccharomyces pombe Proteins; D050051:Transient Receptor Potential Channels; C405644:YAM8 protein, S pombe; D049452:Green Fluorescent Proteins; D012965:Sodium Chloride; D000331:Aequorin; D019703:Calcineurin; D002122:Calcium Chloride; D002118:Calcium; D016559:Tacrolimus