title: Extracellular disulfide bridges stabilize TRPC5 dimerization, trafficking, and activity.
abstract: Crucial cysteine residues can be involved in the modulation of protein activity via the modification of thiol (-SH) groups. Among these reactions, disulfide bonds (S-S) play a key role in the folding, stability, and activity of membrane proteins. However, the regulation of extracellular cysteines in classical transient receptor potential (TRPC) channels remains controversial. Here, we examine the functional importance of the extracellular disulfide bond in TRPC5 in modulating channel gating and trafficking. Specifically, we investigated TRPC5 activity in transiently transfected HEK293 cells with wild-type (WT) or cysteine (C553 and C558) mutants in the pore loop. Using reducing agents, we determined that a disulfide linkage mediates the tetrameric formation of the TRPC5 channel. By measuring the TRPC5 current, we observed that C553S or C558S mutants completely lose channel activity induced by lanthanides or receptor stimulation. Co-expression of TRPC5 (WT) with mutants demonstrated a dominant-negative function in mutants, which inhibited the activity of TRPC5 (WT). We generated TRPC5-TRPC5 dimers and observed reduced activity of WT-mutant (C553S or C558S) dimers compared to WT-WT dimers. When pretreated with reducing agents for 12 h, the TRPC5 current decreased due to a reduction in membrane TRPC5 distribution. In addition, we identified a reduced expression of C553S mutant in plasma membrane. We analyzed a dimeric interaction of wild-type and mutant TRPC5 using co-immunoprecipitation and FRET method, indicating a weak interaction between dimeric partners. These results indicated that the disulfide bond between conserved extracellular cysteines, especially C553, is essential for functional TRPC5 activity by channel multimerization and trafficking.
authors.affiliation: Department of Physiology and Institute of Dermatological Science, Seoul National University College of Medicine, 28 Yeongeon-dong, Jongno-gu, Seoul, 110-799, South Korea.
authors.forename: Chansik
authors.initials: C
authors.lastname: Hong
authors.forename: Misun
authors.initials: M
authors.lastname: Kwak
authors.forename: Jongyun
authors.initials: J
authors.lastname: Myeong
authors.forename: Kotdaji
authors.initials: K
authors.lastname: Ha
authors.forename: Jinhong
authors.initials: J
authors.lastname: Wie
authors.forename: Ju-Hong
authors.initials: JH
authors.lastname: Jeon
authors.forename: Insuk
authors.initials: I
authors.lastname: So
mesh_terms: D000818:Animals; D003553:Cystine / Q000737:chemistry*; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D055503:Protein Multimerization; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D050052:TRPC Cation Channels / Q000737:chemistry* / Q000378:metabolism
chemical_list: D050052:TRPC Cation Channels; C491780:TRPC5 protein, human; D003553:Cystine