title: Muscarinic receptor activation increases hERG channel expression through phosphorylation of ubiquitin ligase Nedd4-2.
abstract: The human ether-à-go-go-related gene (hERG) encodes the pore-forming subunit of the rapidly activating delayed rectifier potassium channel, which is important for cardiac repolarization. Reduction of hERG current due to genetic mutations or drug interferences causes long QT syndrome, leading to cardiac arrhythmias and sudden death. To date, there is no effective therapeutic method to restore or enhance hERG channel function. Using cell biology and electrophysiological methods, we found that the muscarinic receptor agonist carbachol increased the expression and function of hERG, but not ether-à-go-go or Kv1.5 channels stably expressed in human embryonic kidney cells. The carbachol-mediated increase in hERG expression was abolished by the selective M3 antagonist 4-DAMP (1,1-dimethyl-4-diphenylacetoxypiperidinium iodide) but not by the M2 antagonist AF-DX 116 (11[[2-[(diethylamino)methyl]-1-piperidinyl]-acetyl]-5,11-dihydro-6H-pyrido[2,3-b] [1,4]benzodiazepine-6-one). Treatment of cells with carbachol reduced the hERG-ubiquitin interaction and slowed the rate of hERG degradation. We previously showed that the E3 ubiquitin ligase Nedd4-2 mediates degradation of hERG channels. Here, we found that disrupting the Nedd4-2 binding domain in hERG completely eliminated the effect of carbachol on hERG channels. Carbachol treatment enhanced the phosphorylation level, but not the total level, of Nedd4-2. Blockade of the protein kinase C (PKC) pathway abolished the carbachol-induced enhancement of hERG channels. Our data suggest that muscarinic activation increases hERG channel expression by phosphorylating Nedd4-2 via the PKC pathway.
authors.affiliation: Department of Cardiovascular Medicine, First Affiliated Hospital of Xi'an Jiaotong University School of Medicine, Xi'an, China (T.W., A.M.); and Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario, Canada (T.W., A.H.-C., Y.K., Z.C., J.G., T.Y., S.M.L., W.L., J.T.F., S.Z.).
authors.forename: Tingzhong
authors.initials: T
authors.lastname: Wang
authors.forename: Andrew
authors.initials: A
authors.lastname: Hogan-Cann
authors.forename: Yudi
authors.initials: Y
authors.lastname: Kang
authors.forename: Zhi
authors.initials: Z
authors.lastname: Cui
authors.forename: Jun
authors.initials: J
authors.lastname: Guo
authors.forename: Tonghua
authors.initials: T
authors.lastname: Yang
authors.forename: Shawn M
authors.initials: SM
authors.lastname: Lamothe
authors.forename: Wentao
authors.initials: W
authors.lastname: Li
authors.forename: Aiqun
authors.initials: A
authors.lastname: Ma
authors.forename: John T
authors.initials: JT
authors.lastname: Fisher
authors.forename: Shetuan
authors.initials: S
authors.lastname: Zhang
mesh_terms: D000818:Animals; D001483:Base Sequence; D002217:Carbachol / Q000494:pharmacology; D017931:DNA Primers; D000072237:ERG1 Potassium Channel; D056827:Endosomal Sorting Complexes Required for Transport / Q000378:metabolism*; D051638:Ether-A-Go-Go Potassium Channels / Q000378:metabolism*; D005260:Female; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D008856:Microscopy, Fluorescence; D018721:Muscarinic Agonists / Q000494:pharmacology*; D000075702:Nedd4 Ubiquitin Protein Ligases; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D051381:Rats; D017207:Rats, Sprague-Dawley; D011976:Receptors, Muscarinic / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D000072237:ERG1 Potassium Channel; D056827:Endosomal Sorting Complexes Required for Transport; D051638:Ether-A-Go-Go Potassium Channels; C000606913:KCNH2 protein, human; D018721:Muscarinic Agonists; D011976:Receptors, Muscarinic; D002217:Carbachol; C000618479:NEDD4L protein, rat; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618483:Nedd4 protein, human; C000618484:Nedd4 protein, rat; C000618485:Nedd4L protein, human; D044767:Ubiquitin-Protein Ligases