M3 Muscarinic Receptor Signaling Stabilizes a Novel Mutant Human Ether-a-Go-Go-Related Gene Channel Protein via Phosphorylation of Heat Shock Factor 1 in Transfected Cells.
Long QT syndrome 2 (LQT2) is caused by mutations in the human ether-a-go-go-related gene (hERG). Most of its mutations give rise to unstable hERG proteins degraded by the proteasome. Recently, carbachol was reported to stabilize the wild-type hERG-FLAG via activation of the muscarinic type 3 receptor (M3-mAChR). Its action on mutant hERG-FLAG, however, remains uninvestigated.



A novel mutant hERG-FLAG carried 2 mutations: an amino acid substitution G572S and an in-frame insertion D1037_V1038insGD. When expressed in HEK293 cells, this mutant hERG-FLAG was degraded by the proteasome and failed to be transported to the cell surface. Carbachol restored stability of the mutant hERG-FLAG and facilitated cell-surface expression. Carbachol activated PKC, augmented phosphorylation of heat shock factor 1 (HSF1) and enhanced expression of heat shock proteins (hsps), hsp70 and hsp90. Both a M3-mAChR antagonist, 4-DAMP, and a PKC inhibitor, bisindolylmaleimide, abolished carbachol-induced stabilization of the mutant hERG-FLAG.



M3-mAChR activation leads to enhancement of hsp expression via PKC-dependent phosphorylation of HSF1, thereby stabilizing the mutant hERG-FLAG protein. Thus, M3-mAChR activators may have a therapeutic value for patients with LQT2. (Circ J 2016; 80: 2443-2452).
Division of Regenerative Medicine and Therapeutics, Department of Genetic Medicine and Regenerative Therapeutics, Institute of Regenerative Medicine and Biofunction, Tottori University Graduate School of Medical Science.
Endang
E
Mahati
Peili
P
Li
Yasutaka
Y
Kurata
Nani
N
Maharani
Nobuhito
N
Ikeda
Shinji
S
Sakata
Kazuyoshi
K
Ogura
Junichiro
J
Miake
Takeshi
T
Aiba
Wataru
W
Shimizu
Naoe
N
Nakasone
Haruaki
H
Ninomiya
Katsumi
K
Higaki
Kazuhiro
K
Yamamoto
Akira
A
Nakai
Yasuaki
Y
Shirayoshi
Ichiro
I
Hisatome
D000293:Adolescent; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D000072237:ERG1 Potassium Channel / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D000076249:Heat Shock Transcription Factors; D006801:Humans; D008133:Long QT Syndrome / Q000235:genetics / Q000378:metabolism; D008297:Male; D009154:Mutation; D010766:Phosphorylation / Q000235:genetics; D055550:Protein Stability; D043587:Receptor, Muscarinic M3 / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D004268:DNA-Binding Proteins; D000072237:ERG1 Potassium Channel; C000619706:HSF1 protein, human; D000076249:Heat Shock Transcription Factors; C000606913:KCNH2 protein, human; D043587:Receptor, Muscarinic M3; D014157:Transcription Factors