Receptor-Mediated Suppression of Potassium Currents Requires Colocalization within Lipid Rafts

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 ([Ca 2ϩ ] 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 HAtagged 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-␤-cyclodextrin produced a loss of localization of proteins within lipid raft membrane fractions. This pretreatment also abolished both the increase of [Ca 2ϩ ] 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-␤-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.

Functional Assembly of Kv7.1/Kv7.5 Channels With Emerging Properties on Vascular Muscle Physiology

“…22,23 For instance, displacement of heteromeric Kv7.2/ Kv7.3 M-channels from rafts prevents muscarinic-mediated suppression of the M-current in sympathetic neurons. 37 The physiological consequences of Kv7.1/Kv7.5 heteromeric channel spatial regulation require several considerations: (1) the putative association of Kv7 channels to KCNE peptides, (2) calmodulin-mediated Ca 2+ sensitivity of Kv7 channels, and (3) the physiological events initiated in cardiovascular smooth muscle rafts. First, KCNE1 and KCNE3, which associate with Kv7.1 and Kv7.5 and modulate channel trafficking and gating, are highly expressed in blood vessels.…”

Inhibition of CaV2.3 channels by NK1 receptors is sensitive to membrane cholesterol but insensitive to caveolin-1

“…Cholesterol depletion affects also the normal function of many membrane proteins, i.e., ion channels and G-protein coupled receptors [22,35,45]. We evaluated this possibility in Ca V 2.3 channels by comparing their macroscopic biophysical properties when expressed in control or MΒCD-treated HEK293 cells.…”

“…Cavin-1 is critical for caveolae formation, while the other three types of cavins regulate caveolae development, size, and stability [38]. Some functional effects of membrane rafts on ion channel physiology and on proteins related to signal transduction have been previously reported [35,50,52]. Particularly interesting is the relevance of rafts to signaling through G-protein coupled receptors [29,47].…”

Manipulation of Lipid Rafts in Neuronal Cells~!2009-09-18~!2009-12-16~!2010-03-19~!

“…M CD affects membrane raft domains and modulates the location of raft-related proteins [23,79,80]. Hence, M CD is commonly used to study lipid raft related processes in neuronal cells such as receptor mediated signaling [81][82][83] or processes related to neurodegeneration [66,[84][85][86][87][88]. Recently, the mechanism how M CD affect lipid rafts was studied in more detail: Treatment of synaptosomal plasma membranes (SPM) isolated from mouse brains with M CD significantly lowered SPM free cholesterol levels and the opposite was observed when cholesterol inclusion complexes were used [89].…”