title: Identification of a tetrameric assembly domain in the C terminus of heat-activated TRPV1 channels.
abstract: Transient receptor potential (TRP) channels as cellular sensors are thought to function as tetramers. Yet, the molecular determinants governing channel multimerization remain largely elusive. Here we report the identification of a segment comprising 21 amino acids (residues 752-772 of mouse TRPV1) after the known TRP-like domain in the channel C terminus that functions as a tetrameric assembly domain (TAD). Purified recombinant C-terminal proteins of TRPV1-4, but not the N terminus, mediated the protein-protein interaction in an in vitro pulldown assay. Western blot analysis combined with electrophysiology and calcium imaging demonstrated that TAD exerted a robust dominant-negative effect on wild-type TRPV1. When fused with the membrane-tethered peptide Gap43, the TAD blocked the formation of stable homomultimers. Calcium imaging and current recordings showed that deletion of the TAD in a poreless TRPV1 mutant subunit suppressed its dominant-negative phenotype, confirming the involvement of the TAD in assembly of functional channels. Our findings suggest that the C-terminal TAD in TRPV1 channels functions as a domain that is conserved among TRPV1-4 and mediates a direct subunit-subunit interaction for tetrameric assembly.
authors.affiliation: Department of Neurobiology, Neuroscience Research Institute, Peking University Health Science Center, Beijing 100191, China.
authors.forename: Feng
authors.initials: F
authors.lastname: Zhang
authors.forename: Shuang
authors.initials: S
authors.lastname: Liu
authors.forename: Fan
authors.initials: F
authors.lastname: Yang
authors.forename: Jie
authors.initials: J
authors.lastname: Zheng
authors.forename: KeWei
authors.initials: K
authors.lastname: Wang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium; D019922:GAP-43 Protein; D057809:HEK293 Cells; D006358:Hot Temperature; D006801:Humans; D008297:Male; D051379:Mice; D057054:Molecular Imaging; D008969:Molecular Sequence Data; D010446:Peptide Fragments; D011485:Protein Binding; D055503:Protein Multimerization; D011993:Recombinant Fusion Proteins; D016415:Sequence Alignment; D050916:TRPV Cation Channels / Q000737:chemistry / Q000378:metabolism*
chemical_list: D019922:GAP-43 Protein; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D050916:TRPV Cation Channels; C493921:TRPV1 protein, mouse; D002118:Calcium