title: E3 ligase CHIP and Hsc70 regulate Kv1.5 protein expression and function in mammalian cells.
abstract: Kv1.5 confers ultra-rapid delayed-rectifier potassium channel current (IKur) which contributes to repolarization of the atrial action potential. Kv1.5 proteins, degraded via the ubiquitin-proteasome pathway, decreased in some atrial fibrillation patients. Carboxyl-terminus heat shock cognate 70-interacting protein (CHIP), an E3 ubiquitin ligase, is known to ubiquitinate short-lived proteins. Here, we investigated the roles of CHIP in Kv1.5 degradation to provide insights into the mechanisms of Kv1.5 decreases and treatments targeting Kv1.5 for atrial fibrillation. Coexpression of CHIP with Kv1.5 in HEK293 cells increased Kv1.5 protein ubiquitination and decreased the protein level. Immunofluorescence revealed decreases of Kv1.5 proteins in the endoplasmic reticulum and on the cell membrane. A siRNA against CHIP suppressed Kv1.5 protein ubiquitination and increased its protein level. CHIP mutants, lacking either the N-terminal tetratricopeptide region domain or the C-terminal U-box domain, failed to exert these effects on Kv1.5 proteins. Immunoprecipitation showed that CHIP formed complexes with Kv1.5 proteins and heat shock cognate protein 70 (Hsc70). Effects of Hsc70 on Kv1.5 were similar to CHIP by altering interaction of CHIP with Kv1.5 protein. Coexpression of CHIP and Hsc70 with Kv1.5 additionally enhanced Kv1.5 ubiquitination. Kv1.5 currents were decreased by overexpression of CHIP or Hsc70 but were increased by knockdown of CHIP or Hsc70 in HEK 293 cells stably expressing Kv1.5. These effects of CHIP and Hsc70 were also observed on endogenous Kv1.5 in HL-1 mouse cardiomyocytes, decreasing IKur and prolonging action potential duration. These results indicate that CHIP decreases the Kv1.5 protein level and functional channel by facilitating its degradation in concert with chaperone Hsc70.
authors.affiliation: Department of Genetic Medicine and Regenerative Therapeutics, Institute of Regenerative Medicine and Biofunction, Tottori University, Graduate School of Medical Science, Nishimachi 36, Yonago, Japan. Electronic address: peili-li@med.tottori-u.ac.jp.
authors.forename: Peili
authors.initials: P
authors.lastname: Li
authors.affiliation: Department of Physiology, Kanazawa Medical University, Ishikawa 920-0293, Japan.
authors.forename: Yasutaka
authors.initials: Y
authors.lastname: Kurata
authors.affiliation: Department of Genetic Medicine and Regenerative Therapeutics, Institute of Regenerative Medicine and Biofunction, Tottori University, Graduate School of Medical Science, Nishimachi 36, Yonago, Japan.
authors.forename: Nani
authors.initials: N
authors.lastname: Maharani
authors.affiliation: Department of Genetic Medicine and Regenerative Therapeutics, Institute of Regenerative Medicine and Biofunction, Tottori University, Graduate School of Medical Science, Nishimachi 36, Yonago, Japan.
authors.forename: Endang
authors.initials: E
authors.lastname: Mahati
authors.affiliation: Department of Human Genome Science, Tottori University, Faculty of Medicine, Nishichou 86, Yonago 683, Japan.
authors.forename: Katsumi
authors.initials: K
authors.lastname: Higaki
authors.affiliation: Department of Genetic Medicine and Regenerative Therapeutics, Institute of Regenerative Medicine and Biofunction, Tottori University, Graduate School of Medical Science, Nishimachi 36, Yonago, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Hasegawa
authors.affiliation: Department of Genetic Medicine and Regenerative Therapeutics, Institute of Regenerative Medicine and Biofunction, Tottori University, Graduate School of Medical Science, Nishimachi 36, Yonago, Japan.
authors.forename: Yasuaki
authors.initials: Y
authors.lastname: Shirayoshi
authors.affiliation: Department of Genetic Medicine and Regenerative Therapeutics, Institute of Regenerative Medicine and Biofunction, Tottori University, Graduate School of Medical Science, Nishimachi 36, Yonago, Japan.
authors.forename: Akio
authors.initials: A
authors.lastname: Yoshida
authors.affiliation: Department of Cardiovascular Medicine, Tottori University, Faculty of Medicine, Nishichou 86, Yonago 683, Japan.
authors.forename: Tatehito
authors.initials: T
authors.lastname: Kondo
authors.affiliation: Division of Health Administration and Promotion, Tottori University, Faculty of Medicine, Nishichou 86, Yonago 683, Japan.
authors.forename: Youichi
authors.initials: Y
authors.lastname: Kurozawa
authors.affiliation: Department of Cardiovascular Medicine, Tottori University, Faculty of Medicine, Nishichou 86, Yonago 683, Japan.
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Yamamoto
authors.affiliation: Department of Biological Regulation, Tottori University, Faculty of Medicine, Nishichou 86, Yonago 683, Japan.
authors.forename: Haruaki
authors.initials: H
authors.lastname: Ninomiya
authors.affiliation: Department of Genetic Medicine and Regenerative Therapeutics, Institute of Regenerative Medicine and Biofunction, Tottori University, Graduate School of Medical Science, Nishimachi 36, Yonago, Japan.
authors.forename: Ichiro
authors.initials: I
authors.lastname: Hisatome
mesh_terms: D000818:Animals; D001281:Atrial Fibrillation / Q000235:genetics* / Q000473:pathology; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D050883:HSC70 Heat-Shock Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D051666:Kv1.5 Potassium Channel / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D034741:RNA, Small Interfering; D015398:Signal Transduction; D044767:Ubiquitin-Protein Ligases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D054875:Ubiquitination / Q000235:genetics
chemical_list: D050883:HSC70 Heat-Shock Proteins; C494371:HSPA8 protein, human; C496981:KCNA5 protein, human; D051666:Kv1.5 Potassium Channel; D034741:RNA, Small Interfering; C492951:Stub1 protein, mouse; D044767:Ubiquitin-Protein Ligases
keywords: CHIP E3 ligase; Hsc70; Kv1.5 protein; Proteasome; Ubiquitination