Abstract
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.
Footnotes
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
doi:10.1124/mol.109.058008
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ABBREVIATIONS:
- PLC
- phospholipase C
- PIP2
- phosphatidylinositol 4,5-bisphosphate
- HEK
- human embryonic kidney
- GPCR
- G protein-coupled receptor
- HA
- hemagglutinin
- EGFP
- enhanced green fluorescent protein
- DRG
- dorsal root ganglion
- HBSS
- HEPES-buffered salt solution
- MBS
- MES-buffered saline
- MES
- N-morpholino)ethanesulfonic acid
- 77-LH-28-1
- 1-(3-(4-butyl-1-piperidinyl)propyl)-3,4-dihydro-2(1H)-quinolinone
- AD
- Alzheimer's disease
- ApoE
- apolipoprotein E.
- Received May 22, 2009.
- Accepted September 2, 2009.
- © 2009 The American Society for Pharmacology and Experimental Therapeutics
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