期刊
JOURNAL OF PHYSIOLOGY-LONDON
卷 586, 期 12, 页码 2855-2871出版社
WILEY
DOI: 10.1113/jphysiol.2008.153791
关键词
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资金
- British Heart Foundation Funding Source: Medline
- Wellcome Trust Funding Source: Medline
Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P-2) is a key component in signal transduction, being a precursor to other signalling molecules and itself associated with roles in signal transduction and cell biology. Tubby is a membrane bound transcription factor whose dysfunction results in obesity in mice. It contains a domain that selectively binds PtdIns(4,5)P-2. We have investigated the use of a fluorescently tagged version of this domain to monitor changes in PtdIns(4,5)P-2 concentration in living cells and compared it to the pleckstrin homology domain of PLC delta 1. Our results show that selected mutants of this domain report receptor-mediated changes in cellular PtdIns(4,5)P-2. In contrast to the pleckstrin homology domain of PLC delta 1 it does not have a significant affinity for inositol 1,4,5-trisphosphate (IP3). Using a selected mutant, we examine the regulation of ATP-sensitive K+ channels via a G(q/11)-coupled receptor. These experiments reveal a correlation between reporter translocation and the onset of current inhibition whilst the recovery of current after agonist removal is delayed when compared to the reporter. Furthermore our studies reveal the importance of Ca2+ in determining the overall activity of phospholipase C in living cells. This probe may be valuable in examining changes in PtdIns(4,5)P-2 distinct from those of IP3 in intact cells in a variety of physiological settings.
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