Regulation of KCNQ channels by manipulation of phosphoinositides

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.