Identification of a tetrameric assembly domain in the C terminus of heat-activated TRPV1 channels |
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Authors: | Zhang Feng Liu Shuang Yang Fan Zheng Jie Wang KeWei |
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Affiliation: | Department of Neurobiology, Neuroscience Research Institute, Peking University Health Science Center, Beijing 100191, China. |
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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. |
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Keywords: | Calcium Imaging Fluorescence Ion Channels Oocyte Protein Assembly Protein Domains Protein Self-assembly TRPV1 Capsaicin Menthol |
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