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.

Cryo-electron microscopy structure of the TRPV2 ion channel

“…In TRPV1, the relayed (or concerted) motion of the ARD assembly, coupling domain, TRP domain and S6 appears to be important for opening of the lower gate at S6 ( Fig. 6a,b ), a result consistent with findings from many functional studies that underscore the importance of ARD, the coupling domain and the TRP domain in heat- and ligand-dependent gating 21,22,26,39,40 . In our structure of TRPV2, the selectivity-filter gate in TRPV2 is closed, but the lack of tight interactions between the pore loop and the turret is similar to the characteristics of the fully open TRPV1 structure.…”

“…Many TRP channels possess a conserved sequence known as the ‘TRP box’, which is located at the C-terminal end of S6, a region important for channel gating and desensitization 22–24 . The TRP domain constitutes a large portion of the TRP box and is sandwiched between the VSLD and the cradle formed by the linker domain and the pre-S1 helix, in a position that allows communication to be established between the transmembrane and the intracellular regions ( Fig.…”

Cryo-EM and X-ray structures of TRPV4 reveal insight into ion permeation and gating mechanisms

“…Following the β-strands, a helix-turn-helix (HTH) motif and a pre-S1 helix snugly accommodate the C-terminal TRP helix (Supplementary Fig. 5), a conserved sequence motif among TRP channels that follows the inner helix S6 and regulates channel activity 42 . The amphipathic TRP helix runs parallel to the membrane and is in close contact with the ordered S4–S5 linker, which connects the S1–S4 and pore domains.…”

Side chain flexibility and coupling between the S4‐S5 linker and the TRP domain in thermo‐sensitive TRP channels: Insights from protein modeling

“…Finally, taking into account that different allosteric models describe the gating of thermo‐sensitive TRP channels, and considering that in all of the cases the corresponding region to the TRP domain at the C‐end of the inner gate plays a central role along with the S4‐S5L during the activation of the channels, our attention was turned to the putative interactions of S4‐S5L with the highly conserved Trp residue present in the TRP box. The results in Figure show that several potential interactions can be established between the Trp residue and the S4‐S5L.…”