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J Physiol Volume 586, Number 12, 2855-2871, June 15, 2008 DOI: 10.1113/jphysiol.2008.153791
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CELLULAR

Monitoring changes in membrane phosphatidylinositol 4,5-bisphosphate in living cells using a domain from the transcription factor tubby

 Kathryn V. Quinn1, Philippe Behe1 and Andrew Tinker1

1 BHF Laboratories and Department of Medicine, University College London, 5 University Street, London WC1E 6JJ, UK

Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) 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)P2. We have investigated the use of a fluorescently tagged version of this domain to monitor changes in PtdIns(4,5)P2 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)P2. 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 Gq/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)P2 distinct from those of IP3 in intact cells in a variety of physiological settings.

(Received 10 March 2008; accepted after revision 16 April 2008; first published online 17 April 2008)
Corresponding author A. Tinker: BHF Laboratories and Department of Medicine, University College London, 5 University Street, London WC1E 6JJ, UK. Email: a.tinker{at}ucl.ac.uk






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