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Published 5 June 2006. doi:10.1083/jcb.200512141
The Rockefeller University Press, 0021-9525 $8.00
JCB, Volume 173, Number 5, 755-765
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Article

Cytosolic inositol 1,4,5-trisphosphate dynamics during intracellular calcium oscillations in living cells



Toru Matsu-ura1, Takayuki Michikawa1,2,3, Takafumi Inoue2,3, Atsushi Miyawaki4, Manabu Yoshida3,5, and Katsuhiko Mikoshiba1,2,3

1 Laboratory for Developmental Neurobiology, Brain Science Institute, RIKEN, Saitama 351-0198, Japan
2 Division of Molecular Neurobiology, Department of Basic Medical Sciences, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan
3 Calcium Oscillation Project, International Cooperative Research Project—Solution Oriented Research for Science and Technology, Japan Science and Technology Agency, Saitama 332-0012, Japan
4 Laboratory for Cell Function and Dynamics, Advanced Technology Development Center, Brain Science Institute, RIKEN, Saitama 351-0198, Japan
5 Misaki Marine Biological Station, Graduate School of Science, University of Tokyo, Kanagawa, 238-0225, Japan

Correspondence to Takayuki Michikawa: takamich{at}ims.u-tokyo.ac.jp; or Katsuhiko Mikoshiba: mikosiba{at}ims.u-tokyo.ac.jp

We developed genetically encoded fluorescent inositol 1,4,5-trisphosphate (IP3) sensors that do not severely interfere with intracellular Ca2+ dynamics and used them to monitor the spatiotemporal dynamics of both cytosolic IP3 and Ca2+ in single HeLa cells after stimulation of exogenously expressed metabotropic glutamate receptor 5a or endogenous histamine receptors. IP3 started to increase at a relatively constant rate before the pacemaker Ca2+ rise, and the subsequent abrupt Ca2+ rise was not accompanied by any acceleration in the rate of increase in IP3. Cytosolic [IP3] did not return to its basal level during the intervals between Ca2+ spikes, and IP3 gradually accumulated in the cytosol with a little or no fluctuations during cytosolic Ca2+ oscillations. These results indicate that the Ca2+-induced regenerative IP3 production is not a driving force of the upstroke of Ca2+ spikes and that the apparent IP3 sensitivity for Ca2+ spike generation progressively decreases during Ca2+ oscillations.

Abbreviations used in this paper: [Ca2+]c, cytosolic Ca2+ concentration; C-PHD, cyan fluorescent protein–fused PHD; ECFP, enhanced cyan fluorescent protein; IP3, inositol 1,4,5-trisphosphate; [IP3]c, cytosolic IP3 concentration; IP3R, IP3 receptor; IRIS, IP3R-based IP3 sensor; mGluR, metabotropic glutamate receptor; PHD, pleckstrin homology domain; V-PHD, Venus-fused PHD.


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