PT - JOURNAL ARTICLE AU - Susan Oldfield AU - Jane Hancock AU - Angharad Mason AU - Sally A. Hobson AU - David Wynick AU - Eamonn Kelly AU - Andrew D. Randall AU - Neil V. Marrion TI - Receptor-Mediated Suppression of Potassium Currents Requires Colocalization within Lipid Rafts AID - 10.1124/mol.109.058008 DP - 2009 Dec 01 TA - Molecular Pharmacology PG - 1279--1289 VI - 76 IP - 6 4099 - http://molpharm.aspetjournals.org/content/76/6/1279.short 4100 - http://molpharm.aspetjournals.org/content/76/6/1279.full SO - Mol Pharmacol2009 Dec 01; 76 AB - Expression of KCNQ2/3 (Kv7.2 and -7.3) heteromers underlies the neuronal M current, a current that is suppressed by activation of a variety of receptors that couple to the hydrolysis of phosphatidylinositol 4,5-bisphosphate. Expression of Kv7.2/7.3 channels in human embryonic kidney (HEK) 293 cells produced a noninactivating potassium current characteristic of M current. Muscarinic receptors endogenous to HEK293 cells were identified as being M3 by pharmacology and Western blotting, producing a rise of intracellular calcium ([Ca2+]i) upon activation. Activation of these endogenous muscarinic receptors however, failed to suppress expressed Kv7.2/7.3 current. Current suppression was reconstituted by coexpression of HA-tagged muscarinic m1 or m3 receptors. Examination of membrane fractions showed that both expressed receptors and Kv7.2 and -7.3 channel subunits resided within lipid rafts. Disruption of lipid rafts by pretreatment of cells expressing either m1 or m3 muscarinic receptors with methyl-β-cyclodextrin produced a loss of localization of proteins within lipid raft membrane fractions. This pretreatment also abolished both the increase of [Ca2+]i and suppression of expressed Kv7.2/7.3 current evoked by activation of expressed m1 or m3 muscarinic receptors. A similar loss of muscarinic receptor-mediated suppression of M current native to rat dorsal root ganglion neurons was observed after incubating dissociated cells with methyl-β-cyclodextrin. These data suggested that lipid rafts colocalized both muscarinic receptors and channel subunits to enable receptor-mediated suppression of channel activity, a spatial colocalization that enables specificity of coupling between receptor and ion channel.© 2009 The American Society for Pharmacology and Experimental Therapeutics