title: Direct evidence for functional TRPV1/TRPA1 heteromers.
abstract: Transient receptor potential cation channel, subfamily V, member 1 (TRPV1) plays a key role in sensing environmental hazards and in enhanced pain sensation following inflammation. A considerable proportion of TRPV1-expressing cells also express transient receptor potential cation channel, subfamily A, member 1 (TRPA1). There is evidence for a TRPV1-TRPA1 interaction that is predominantly calcium-dependent, and it has been suggested that the two proteins might form a heteromeric channel. Here, we constructed subunit concatemers to search for direct evidence for such an interaction. We found that a TRPV1::TRPV1 concatemer and TRPV1 formed channels with similar properties. A TRPV1::TRPA1 concatemer was responsive to TRPV1 agonists capsaicin, acidic pH and ethanol, but not to TRPA1 agonists. Isolated TRPV1 and TRPV1::TRPA1 imaged by atomic force microscopy (AFM) both had molecular volumes consistent with the formation of tetrameric channels. Antibodies decorated epitope tags on TRPV1 with a four-fold symmetry, as expected for a homotetramer. In contrast, pairs of antibodies decorated tags on TRPV1::TRPA1 predominantly at 180°, indicating the formation of a channel consisting of two TRPV1::TRPA1 concatemers arranged face to face. TRPV1::TRPA1 was sensitized by PKC activation and could be inhibited by a TRPV1 antagonist. TRPV1::TRPA1 was activated by heat and displayed a threshold and temperature coefficient similar to TRPV1. However, the channel formed by TRPV1::TRPA1 has only two binding sites for capsaicin and shows less total current and a smaller capsaicin-induced shift in voltage-dependent gating than TRPV1::TRPV1 or TRPV1. We conclude that the presence of TRPA1 exerts a functional inhibition on TRPV1.
authors.affiliation: Institute of Physiology and Pathophysiology, University of Erlangen-Nuremberg, Universitätsstrasse 17, 91052, Erlangen, Germany, fischer@physiologie1.uni-erlangen.de.
authors.forename: Michael J M
authors.initials: MJ
authors.lastname: Fischer
authors.forename: Dilshan
authors.initials: D
authors.lastname: Balasuriya
authors.forename: Pia
authors.initials: P
authors.lastname: Jeggle
authors.forename: Tom A
authors.initials: TA
authors.lastname: Goetze
authors.forename: Peter A
authors.initials: PA
authors.lastname: McNaughton
authors.forename: Peter W
authors.initials: PW
authors.lastname: Reeh
authors.forename: J Michael
authors.initials: JM
authors.lastname: Edwardson
mesh_terms: D001665:Binding Sites; D015220:Calcium Channels / Q000737:chemistry / Q000378:metabolism*; D002211:Capsaicin / Q000494:pharmacology; D057809:HEK293 Cells; D006801:Humans; D015640:Ion Channel Gating; D009419:Nerve Tissue Proteins / Q000819:agonists / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D055503:Protein Multimerization; D000074025:TRPA1 Cation Channel; D050916:TRPV Cation Channels / Q000819:agonists / Q000737:chemistry / Q000378:metabolism*; D050051:Transient Receptor Potential Channels / Q000819:agonists / Q000737:chemistry / Q000378:metabolism*
chemical_list: D015220:Calcium Channels; D009419:Nerve Tissue Proteins; D000074025:TRPA1 Cation Channel; C473350:TRPA1 protein, human; D050916:TRPV Cation Channels; C494482:TRPV1 protein, human; D050051:Transient Receptor Potential Channels; D002211:Capsaicin