Identification and characterization of hydrophobic gate residues in TRP channels

Abstract

Transient receptor potential (TRP) channels, subdivided into 6 subfamilies in mammals, have essential roles in sensory physiology. They respond to remarkably diverse stimuli, comprising thermal, chemical, and mechanical modalities, through opening or closing of channel gates. In this study, we systematically substituted the hydrophobic residues within the distal fragment of pore-lining helix S6 with hydrophilic residues and, based on Xenopus oocyte and mammalian cell electrophysiology and a hydrophobic gate theory, identified hydrophobic gates in TRPV6/V5/V4/C4/M8. We found that channel activity drastically increased when TRPV6 or TRPV5, but not any of their adjacent residues, was substituted with hydrophilic residues. Channel activity strongly correlated with the hydrophilicity of the residues at those sites, suggesting that consecutive hydrophobic residues TRPV6 and TRPV5 form a double-residue gate in each channel. By the same strategy, we identified a hydrophobic single-residue gate in TRPV4, TRPC4, and TRPM8. In support of the hydrophobic gate theory, hydrophilic substitution at the gate site, which removes the hydrophobic gate seal, substantially increased the activity of TRP channels in low-activity states but had little effect on the function of activated channels. The double-residue gate channels were more sensitive to small changes in the gate's hydrophobicity or size than single-residue gate channels. The unconventional double-reside gating mechanism in TRP channels may have been evolved to respond especially to physiologic stimuli that trigger relatively small gate conformational changes.-Zheng, W., Hu, R., Cai, R., Hofmann, L., Hu, Q., Fatehi, M., Long, W., Kong, T., Tang, J., Light, P., Flockerzi, V., Cao, Y., Chen, X.-Z. Identification and characterization of hydrophobic gate residues in TRP channels.

Direct Binding between Pre-S1 and TRP-like Domains in TRPP Channels Mediates Gating and Functional Regulation by PIP2

“…Capped RNAs of TRPP3, TRPP2, TRPM8, and TRPV1 were in vitro transcripted with mMESSAGE mMACHINE kit (Ambion, Austin, TX) and injected (25–50 ng RNA in 50 nL water per oocyte) into Xenopus oocytes prepared as previously described ( Zheng et al, 2017 ). Control oocytes were injected with equal volumes of water.…”

“…Whole-mount immunofluorescence assays using Xenopus oocytes were performed as described ( Zheng et al, 2017 ). Briefly, oocytes were washed in PBS, fixed in 4% paraformaldehyde for 15 min, washed three times in PBS plus 50 mM NH 4 Cl, and then permeabilized with 0.1% Triton X-100 for 4 min.…”

A Heuristic Derived from Analysis of the Ion Channel Structural Proteome Permits the Rapid Identification of Hydrophobic Gates

“…The concept of hydrophobic gating in members of the Cys-loop family of ligand-gated ion channels, including the nicotinic acetylcholine receptor (10), GLIC (7,11), and the 5-HT3 receptor (12)(13)(14) is now well established. However, experimental and computational evidence of hydrophobic gates and barriers within other ion channels has also emerged, including for the TWIK-1 K2P channel (15), BK channels (16), the CorA magnesium channel (17), the CRAC biorxiv_global_text_v28_figs_SI.docx channel Orai (18), and members of the transient receptor potential (TRP) channel family (19)(20)(21)(22).…”

Electric Field Induced Wetting of a Hydrophobic Gate in a Model Nanopore Based on the 5-HT3Receptor Channel

“…The gating or regulation of hydrophobic gates in ion channels is thought to involve either structural changes in pore radii, or more subtle changes in hydrophobicity through e.g. rotation of helices that contain side chains of different polarity and examples of such hydrophobic gating mechanism have now been proposed in a variety of different channels 12,[14][15][16][17][18][19][20][21][22][23][24][25] and synthetic nanopores [26][27] . However, nearly all biological membranes experience a potential difference of between 50 to 200 mV across them and molecular dynamics (MD) simulations of simple model nanopores show that application of a transmembrane electric field can hydrate an otherwise de-wetted hydrophobic constriction, which is thus rendered ion permeable [28][29] .…”