title: Characterization of the novel mutant A78T-HERG from a long QT syndrome type 2 patient: Instability of the mutant protein and stabilization by heat shock factor 1.
abstract: BACKGROUND
  The human ether-a-go-go-related gene (HERG) encodes the α-subunit of rapidly activating delayed-rectifier potassium channels. Mutations in this gene cause long QT syndrome type 2 (LQT2). In most cases, mutations reduce the stability of the channel protein, which can be restored by heat shock (HS).
  
  
  METHODS
  We identified the novel mutant A78T-HERG in a patient with LQT2. The purpose of the current study was to characterize this mutant protein and test whether HS and heat shock factors (HSFs) could stabilize the mutant protein. A78T-HERG and wild-type HERG (WT-HERG) were expressed in HEK293 cells and analyzed by immunoblotting, immunoprecipitation, immunofluorescence, and whole-cell patch clamping.
  
  
  RESULTS
  When expressed in HEK293 cells, WT-HERG gave rise to immature and mature forms of the protein at 135 and 155 kDa, respectively. A78T-HERG gave rise only to the immature form, which was heavily ubiquitinated. The proteasome inhibitor MG132 increased the expression of immature A78T-HERG and increased both the immature and mature forms of WT-HERG. WT-HERG, but not A78T-HERG, was expressed on the plasma membrane. In whole-cell patch clamping experiments, depolarizing pulses evoked E4031-sensitive HERG channel currents in cells transfected with WT-HERG, but not in cells transfected with A78T-HERG. The A78V mutant, but not A78G mutant, remained in the immature form similarly to A78T. Maturation of the A78T-HERG protein was facilitated by HS, expression of HSF-1, or exposure to geranyl geranyl acetone.
  
  
  CONCLUSIONS
  A78T-HERG was characterized by protein instability and reduced expression on the plasma membrane. The stability of the mutant was partially restored by HSF-1, indicating that HSF-1 is a target for the treatment for LQT2 caused by the A78T mutation in HERG.
authors.affiliation: Division of Cardiovascular Medicine, Department of Molecular Medicine and Therapeutics, Faculty of Medicine, Tottori University, Japan.
authors.forename: Takehito
authors.initials: T
authors.lastname: Kondo
authors.affiliation: Division of Regenerative Medicine and Therapeutics, Tottori University Graduate School of Medical Science, Japan.
authors.forename: Ichiro
authors.initials: I
authors.lastname: Hisatome
authors.affiliation: Division of Regenerative Medicine and Therapeutics, Tottori University Graduate School of Medical Science, Japan.
authors.forename: Shouichi
authors.initials: S
authors.lastname: Yoshimura
authors.affiliation: Division of Regenerative Medicine and Therapeutics, Tottori University Graduate School of Medical Science, Japan.
authors.forename: Endang
authors.initials: E
authors.lastname: Mahati
authors.affiliation: Division of Regenerative Medicine and Therapeutics, Tottori University Graduate School of Medical Science, Japan.
authors.forename: Tomomi
authors.initials: T
authors.lastname: Notsu
authors.affiliation: Division of Regenerative Medicine and Therapeutics, Tottori University Graduate School of Medical Science, Japan.
authors.forename: Peili
authors.initials: P
authors.lastname: Li
authors.affiliation: Division of Cardiovascular Medicine, Department of Molecular Medicine and Therapeutics, Faculty of Medicine, Tottori University, Japan.
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Iitsuka
authors.affiliation: Division of Cardiovascular Medicine, Department of Molecular Medicine and Therapeutics, Faculty of Medicine, Tottori University, Japan.
authors.forename: Masaru
authors.initials: M
authors.lastname: Kato
authors.affiliation: Division of Cardiovascular Medicine, Department of Molecular Medicine and Therapeutics, Faculty of Medicine, Tottori University, Japan.
authors.forename: Kazuyoshi
authors.initials: K
authors.lastname: Ogura
authors.affiliation: Division of Cardiovascular Medicine, Department of Molecular Medicine and Therapeutics, Faculty of Medicine, Tottori University, Japan.
authors.forename: Junichiro
authors.initials: J
authors.lastname: Miake
authors.affiliation: Department of Cardiovascular Medicine, National Cerebral and Cardiovascular Center, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Aiba
authors.affiliation: Division of Cardiology and Regenerative Medicine, Nippon Medical School, Japan.
authors.forename: Wataru
authors.initials: W
authors.lastname: Shimizu
authors.affiliation: Department of Physiology II, Kanazawa Medical University, Japan.
authors.forename: Yasutaka
authors.initials: Y
authors.lastname: Kurata
authors.affiliation: Department of Pediatrics, Faculty of Medicine, Tottori University, Japan.
authors.forename: Shinji
authors.initials: S
authors.lastname: Sakata
authors.affiliation: Department of Biological Regulation, Faculty of Medicine, Tottori University, Japan.
authors.forename: Naoe
authors.initials: N
authors.lastname: Nakasone
authors.affiliation: Department of Biological Regulation, Faculty of Medicine, Tottori University, Japan.
authors.forename: Haruaki
authors.initials: H
authors.lastname: Ninomiya
authors.affiliation: Department of Biochemistry and Molecular Biology, Yamaguchi University School of Medicine, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Nakai
authors.affiliation: Division of Functional Genomics, Research Center for Bioscience and Technology, Tottori University, Japan.
authors.forename: Katsumi
authors.initials: K
authors.lastname: Higaki
authors.affiliation: Department of Chemistry and Biotechnology, Graduate School of Engineering, Tottori University, Japan.
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Kawata
authors.affiliation: Division of Regenerative Medicine and Therapeutics, Tottori University Graduate School of Medical Science, Japan.
authors.forename: Yasuaki
authors.initials: Y
authors.lastname: Shirayoshi
authors.affiliation: Division of Regenerative Medicine and Therapeutics, Tottori University Graduate School of Medical Science, Japan.
authors.forename: Akio
authors.initials: A
authors.lastname: Yoshida
authors.affiliation: Division of Cardiovascular Medicine, Department of Molecular Medicine and Therapeutics, Faculty of Medicine, Tottori University, Japan.
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Yamamoto
keywords: GGA, geranyl geranyl acetone; HERG, human-ether-a-go-go-related gene; HS, heat shock; HSF, heat shock factor; HSP, heat shock protein; Heat shock factor; Human ether-a-go-go-related gene; LQTS, long QT syndrome; Long QT syndrome