Receptor-mediated suppression of potassium currents requires colocalization within lipid rafts.
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-beta-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-beta-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.
Oldfield
Susan
S
Department of Physiology and Pharmacology, School of Medical Sciences, University of Bristol, Bristol, BS81TD, UK.
Hancock
Jane
J
Mason
Angharad
A
Hobson
Sally A
SA
Wynick
David
D
Kelly
Eamonn
E
Randall
Andrew D
AD
Marrion
Neil V
NV
D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002217:Carbachol / Q000494:pharmacology; D002460:Cell Line; D018678:Cholinergic Agents / Q000494:pharmacology; D005727:Ganglia, Spinal / Q000166:cytology; D006801:Humans; D051658:KCNQ2 Potassium Channel / Q000187:drug effects / Q000378:metabolism; D051659:KCNQ3 Potassium Channel / Q000187:drug effects / Q000378:metabolism; D021962:Membrane Microdomains / Q000187:drug effects* / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects; D051379:Mice; D009116:Muscarine / Q000494:pharmacology; D010095:Oxotremorine / Q000494:pharmacology; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000494:pharmacology; D026902:Potassium Channel Blockers / Q000494:pharmacology; D015221:Potassium Channels / Q000187:drug effects*; D043643:Receptor, Muscarinic M1 / Q000187:drug effects; D043587:Receptor, Muscarinic M3 / Q000187:drug effects; D011976:Receptors, Muscarinic / Q000187:drug effects
D018678:Cholinergic Agents; D051658:KCNQ2 Potassium Channel; D051659:KCNQ3 Potassium Channel; D019269:Phosphatidylinositol 4,5-Diphosphate; D026902:Potassium Channel Blockers; D015221:Potassium Channels; D043643:Receptor, Muscarinic M1; D043587:Receptor, Muscarinic M3; D011976:Receptors, Muscarinic; D010095:Oxotremorine; D009116:Muscarine; D002217:Carbachol; D002118:Calcium