title: Helix O modulates voltage dependency of CLC-1.
abstract: The chloride channel (CLC) family of proteins consists of channels and transporters that share similarities in architecture and play essential roles in physiological functions. Among the CLC family, CLC-1 channels have the representative homodimeric double-barreled structure carrying two gating processes. One is protopore gating that acts on each pore independently by glutamate residue (Eext). The other is common gating that closes both pores simultaneously in association with large conformational changes across each subunit. In skeletal muscle, CLC-1 is associated with maintaining normal sarcolemmal excitability, and a number of myotonic mutants were reported to modify the channel gating of CLC-1. In this study, we characterized highly conserved helix O as a key determinant of structural stability in CLC-1. Supporting this hypothesis, myotonic mutant (G523D) at N-terminal of helix O showed the activation at hyperpolarizing membrane potentials with a reversed voltage dependency. However, introducing glutamate at serine residue (S537) at the C-terminal of the helix O on G523D restored WT-like voltage dependency of the common gate and showed proton insensitive voltage dependency. To further validate this significant site, site-specific mutagenesis experiments was performed on V292 that is highly conserved as glutamate in antiporter and closely located to S537 and showed that this area is essential for channel function. Taken together, the results of our study suggest the importance of helix O as the main contributor for stable structure of evolutionary conserved CLC proteins and its key role in voltage dependency of the CLC-1. Furthermore, the C-terminal of the helix O can offer a clue for possible proton involvement in CLC-1 channel.
authors.affiliation: Department of Physiology, Seoul National University, College of Medicine, 28 Yeongeon-dong, Jongno-gu, Seoul, 110-799, Republic of Korea.
authors.forename: Ju Yong
authors.initials: JY
authors.lastname: Seong
authors.affiliation: Department of Physiology, Seoul National University, College of Medicine, 28 Yeongeon-dong, Jongno-gu, Seoul, 110-799, Republic of Korea.
authors.forename: Kotdaji
authors.initials: K
authors.lastname: Ha
authors.affiliation: Department of Physiology, Seoul National University, College of Medicine, 28 Yeongeon-dong, Jongno-gu, Seoul, 110-799, Republic of Korea.
authors.forename: Chansik
authors.initials: C
authors.lastname: Hong
authors.affiliation: Department of Physiology, Seoul National University, College of Medicine, 28 Yeongeon-dong, Jongno-gu, Seoul, 110-799, Republic of Korea.
authors.forename: Jongyun
authors.initials: J
authors.lastname: Myeong
authors.affiliation: Korea Brain Research Institute (KBRI), Daegu, 41068, Republic of Korea.
authors.forename: Hyun-Ho
authors.initials: HH
authors.lastname: Lim
authors.affiliation: Department of Physiology, College of Medicine, Gachon University, Incheon, 461-701, Republic of Korea.
authors.forename: Dongki
authors.initials: D
authors.lastname: Yang
authors.affiliation: Department of Physiology, Seoul National University, College of Medicine, 28 Yeongeon-dong, Jongno-gu, Seoul, 110-799, Republic of Korea. insuk@snu.ac.kr.
authors.forename: Insuk
authors.initials: I
authors.lastname: So
mesh_terms: D002460:Cell Line; D018118:Chloride Channels / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D015640:Ion Channel Gating / Q000502:physiology; D018482:Muscle, Skeletal / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D017433:Protein Structure, Secondary
chemical_list: C086321:CLC-1 channel; D018118:Chloride Channels; D018698:Glutamic Acid
keywords: CLC-1; Helix O; Proton; Voltage-gated channel