title: The Integrity of the TRP Domain Is Pivotal for Correct TRPV1 Channel Gating.
abstract: Transient receptor potential vanilloid subtype I (TRPV1) is a thermosensory ion channel that is also gated by chemical substances such as vanilloids. Adjacent to the channel gate, this polymodal thermoTRP channel displays a TRP domain, referred to as AD1, that plays a role in subunit association and channel gating. Previous studies have shown that swapping the AD1 in TRPV1 with the cognate from the TRPV2 channel (AD2) reduces protein expression and produces a nonfunctional chimeric channel (TRPV1-AD2). Here, we used a stepwise, sequential, cumulative site-directed mutagenesis approach, based on rebuilding the AD1 domain in the TRPV1-AD2 chimera, to unveil the minimum number of amino acids needed to restore protein expression and polymodal channel activity. Unexpectedly, we found that virtually full restitution of the AD1 sequence is required to reinstate channel expression and responses to capsaicin, temperature, and voltage. This strategy identified E692, R701, and T704 in the TRP domain as important for TRPV1 activity. Even conservative mutagenesis at these sites (E692D/R701K/T704S) impaired channel expression and abolished TRPV1 activity. However, the sole mutation of these positions in the TRPV1-AD2 chimera (D692E/K701R/S704T) was not sufficient to rescue channel gating, implying that other residues in the TRP domain are necessary to endow activity to TRPV1-AD2. A biophysical analysis of a functional chimera suggested that mutations in the TRP domain raised the energetics of channel gating by altering the coupling of stimuli sensing and pore opening. These findings indicate that inter- and/or intrasubunit interactions in the TRP domain are essential for correct TRPV1 gating.
authors.affiliation: Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, Alicante, Spain.
authors.forename: Lucia
authors.initials: L
authors.lastname: Gregorio-Teruel
authors.affiliation: Department of Experimental Medicine, Section of Physiology, University of Genova, Genova, Italy.
authors.forename: Pierluigi
authors.initials: P
authors.lastname: Valente
authors.affiliation: Department of Physiology and Biophysical Sciences, State University of New York, Buffalo, New York.
authors.forename: Beiying
authors.initials: B
authors.lastname: Liu
authors.affiliation: Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, Alicante, Spain.
authors.forename: Gregorio
authors.initials: G
authors.lastname: Fernández-Ballester
authors.affiliation: Department of Physiology and Biophysical Sciences, State University of New York, Buffalo, New York.
authors.forename: Feng
authors.initials: F
authors.lastname: Qin
authors.affiliation: Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, Alicante, Spain. Electronic address: aferrer@umh.es.
authors.forename: Antonio
authors.initials: A
authors.lastname: Ferrer-Montiel
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D057809:HEK293 Cells; D006801:Humans; D015640:Ion Channel Gating; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D051381:Rats; D050916:TRPV Cation Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D050916:TRPV Cation Channels; C493922:Trpv1 protein, rat