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

PLoS One. 2011;6(7):e22421. doi: 10.1371/journal.pone.0022421. Epub 2011 Jul 19.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aequorin / metabolism
  • Calcineurin / metabolism
  • Calcineurin Inhibitors
  • Calcium / metabolism*
  • Calcium Channels / metabolism*
  • Calcium Chloride / pharmacology
  • Cell Membrane Permeability / drug effects
  • Cytoplasm / drug effects
  • Cytoplasm / metabolism*
  • Extracellular Space / drug effects
  • Extracellular Space / metabolism
  • Gene Knockout Techniques
  • Genes, Fungal / genetics
  • Golgi Apparatus / drug effects
  • Golgi Apparatus / metabolism
  • Green Fluorescent Proteins / metabolism
  • Immunoblotting
  • Membrane Glycoproteins / metabolism*
  • Microscopy, Fluorescence
  • Recombinant Fusion Proteins / metabolism
  • Schizosaccharomyces / cytology
  • Schizosaccharomyces / drug effects
  • Schizosaccharomyces / genetics
  • Schizosaccharomyces / metabolism*
  • Schizosaccharomyces pombe Proteins / metabolism*
  • Sodium Chloride / pharmacology
  • Tacrolimus / pharmacology
  • Transcription, Genetic / drug effects
  • Transient Receptor Potential Channels / metabolism*

Substances

  • Calcineurin Inhibitors
  • Calcium Channels
  • Cch1 protein, S pombe
  • Membrane Glycoproteins
  • Recombinant Fusion Proteins
  • Schizosaccharomyces pombe Proteins
  • Transient Receptor Potential Channels
  • YAM8 protein, S pombe
  • Green Fluorescent Proteins
  • Sodium Chloride
  • Aequorin
  • Calcineurin
  • Calcium Chloride
  • Calcium
  • Tacrolimus