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¹ Department of Physiology and Biophysics University of Washington School of Medicine, Seattle, WA 98195, USA

Activation of phospholipase C (PLC) through G-protein-coupled receptors produces a large number of second messengers and regulates many physiological processes. Many membrane proteins including ion channels require the phosphoinositide phosphatidylinositol 4,5-bisphosphate (PIP₂) to function. Activation of PLC can shut down their activity if it depletes the PIP₂ pool strongly. Such a mechanism accounts for the muscarinic suppression of current in KCNQ channels. We describe a variety of methods used to show that these channels require PIP₂ and that current in the channels is suppressed when receptor-activated PLC depletes PIP₂. The methods include observing translocation of lipid-sensitive protein domains, overexpression of enzymes of phosphoinositide metabolism, engineering these enzymes to move to the plasma membrane in response to a chemical signal, and direct chemical analysis of phospholipids. These approaches are general and can be used to test for PIP₂ requirements of other membrane proteins.

(Received 16 March 2007; accepted after revision 4 April 2007; first published online 5 April 2007)
Corresponding author Bertil Hille, Department of Physiology and Biophysics, University of Washington School of Medicine, G-424 Health Sciences Building, Box 357290, Seattle, WA 98195-7290, USA. Email: hille{at}u.washington.edu

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