Transient Receptor Potential (TRP) and Cch1-Yam8 Channels Play Key Roles in the Regulation of Cytoplasmic Ca in Fission Yeast

The regulation of cytoplasmic Ca is crucial for various cellular processes. Here, we examined the cytoplasmic Ca 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 level and its change caused by extracellular stimulants such as CaCl2 or NaCl plus FK506 (calcineurin inhibitor). We found that the extracellularly added Ca 2+ caused a dose-dependent increase in the cytoplasmic Ca 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 burstlike 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 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 increase that is dependent on the inhibition of calcineurin activity, but not on the inhibition of Prz1. The synergistic Ca increase is abolished by the addition of the Ca chelator BAPTA into the media, and is also abolished by deletion of the gene encoding a subunit of the Cch1-Yam8 Ca channel complex, indicating that the synergistic increase is caused by the Ca influx from the extracellular medium via the Cch1-Yam8 complex. Furthermore, deletion of Pmk1 MAPK abolished the Ca 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 Cch1Yam8 complex, respectively, via modulating the balance between phosphorylation and dyphosphorylation state. Citation: Ma Y, Sugiura R, Koike A, Ebina H, Sio SO, et al. (2011) Transient Receptor Potential (TRP) and Cch1-Yam8 Channels Play Key Roles in the Regulation of Cytoplasmic Ca in Fission Yeast. PLoS ONE 6(7): e22421. doi:10.1371/journal.pone. 0022421 Editor: Juan Mata, University of Cambridge, United Kingdom Received March 14, 2011; Accepted June 23, 2011; Published July 19, 2011 Copyright: 2011 Ma et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Financial support for this study was provided by Grant-in-Aid for Scientific Research, on Scientific Research (B) to Takayoshi Kuno (No. 22390048) and on Grant-in-Aid for Young Scientists (B) to Yan Ma (No. 21790241), from Japan Society for the Promotion of Science (http://www.jsps.go.jp/english/e-grants/ grants.html). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: mayan@med.kobe-u.ac. jp