TY - JOUR
T1 - Identification of a tetrameric assembly domain in the C terminus of heat-activated TRPV1 channels
AU - Zhang, Feng
AU - Liu, Shuang
AU - Yang, Fan
AU - Zheng, Jie
AU - Wang, Kewei
PY - 2011/4/29
Y1 - 2011/4/29
N2 - 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 comprising21amino acids(residues752-772 of mouse TRPV1) after the known TRP-like domaininthe channel Cterminus 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.
AB - 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 comprising21amino acids(residues752-772 of mouse TRPV1) after the known TRP-like domaininthe channel Cterminus 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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U2 - 10.1074/jbc.M111.223941
DO - 10.1074/jbc.M111.223941
M3 - Article
C2 - 21357419
AN - SCOPUS:79955439687
VL - 286
SP - 15308
EP - 15316
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 17
ER -