title: Functional studies on three novel HCNH2 mutations in Taiwan: identification of distinct mechanisms of channel defect and dissociation between glycosylation defect and assembly defect.
abstract: Mutations of the KCNH2 with decreased channel activity lead to congenital long QT syndrome (LQTS). We studied the electrophysiological, glycosylation, trafficking and assembly properties of three novel KCNH2 mutations identified in Taiwanese patients with LQTS (p.N633D, p.R744fs, and p.P923fs). When expressed in HEK293T cells, p.N633D and p.R744fs HERG channels displayed no HERG current while p.P923fs HERG channel showed HERG current with significantly lower (34%) current density and faster inactivation kinetics. In Western blot analysis, pR744fs was the only one with glycosylation defect, which was in consistence with the confocal microscopic findings showing that p.R744fs-GFP was the only one with trafficking defect. However, p.R744fs-GFP differed from pR744fs in being fully glycosylated while p.R744fs fusion with GFP at the N-terminus revealed glycosylation defect. To access the assembly capacity of each mutant, co-immunoprecipitation was conducted. Wild type (WT), p.N633D, and p.P923fs HERG protein showed assembly ability while p.R744fs failed to assemble with neither WT nor itself. In conclusion, we identified three novel LQTS-related KCNH2 mutations and each had a distinct mechanism of channel defect. For p.R744fs mutant, adding GFP to the C-terminus rescued the glycosylation defect but the channel was still assembly defective indicating a dissociation between glycosylation and assembly defects.
authors.affiliation: Institute of Pharmacology, School of Medicine, National Taiwan University, Taipei, Taiwan.
authors.forename: Chia-Hsiang
authors.initials: CH
authors.lastname: Hsueh
authors.forename: Wen-Pin
authors.initials: WP
authors.lastname: Chen
authors.forename: Jiunn-Lee
authors.initials: JL
authors.lastname: Lin
authors.forename: Yen-Bin
authors.initials: YB
authors.lastname: Liu
authors.forename: Ming-Jai
authors.initials: MJ
authors.lastname: Su
authors.forename: Ling-Ping
authors.initials: LP
authors.lastname: Lai
mesh_terms: D015153:Blotting, Western; D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels / Q000235:genetics* / Q000378:metabolism*; D006031:Glycosylation; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D006801:Humans; D047468:Immunoprecipitation; D007700:Kinetics; D008133:Long QT Syndrome / Q000151:congenital / Q000235:genetics* / Q000378:metabolism*; D018613:Microscopy, Confocal; D009154:Mutation; D013624:Taiwan
chemical_list: D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels; C000606913:KCNH2 protein, human; D049452:Green Fluorescent Proteins