Methodologies for TRPM8 Channel Study: A Comprehensive Overview

This document details the methodologies employed for the study of the TRPM8 channel, encompassing techniques from molecular cloning to its structural and functional characterization.

Cloning, Cell Culture, and Protein Expression

N-terminally fused mEGFP constructs of TRPM8 were generated using Gibson assembly. Mutagenesis was performed via PCR or Gibson assembly, with construct sequences rigorously verified by Sanger and whole-plasmid sequencing.

Expi293F cells were cultured, and a baculovirus encoding mEGFP-Pm or Hs TRPM8 was utilized for protein overexpression. Cultures were treated with sodium butyrate to enhance expression, followed by incubation for 72 hours before cells were collected, washed, and flash-frozen.

Detergent Purification for HDX–MS and Cryo-EM Samples

TRPM8 channels were purified at 4 °C from Expi293F cell pellets. Cells were resuspended in lysis buffer, which was then adjusted with 0.5% lauryl maltose neopentyl glycol (LMNG) and 0.5% glycodiosgenin (GDN), and subsequently incubated.

Lysates were centrifuged, applied to anti-GFP-nanobody resin, washed, and digested with PreScission protease. The resulting eluate was concentrated and injected onto a Superose 6 Increase column for further purification. Variations in lysis buffer pH and detergent concentrations were specifically employed for different cryo-EM conditions.

Cell Vesicle Purification for Cryo-EM

For cryo-EM studies involving cell vesicles, Expi293F cell pellets were resuspended in lysis buffer and homogenized using a Dounce homogenizer and sonicator. The lysate was clarified by centrifugation and then filtered.

The clarified lysate was passed through an ion exchange resin, and fluorinated fos-choline 8 was added to facilitate subsequent steps.

Vesicles were bound to anti-GFP nanobody resin, washed thoroughly, and eluted by proteolysis. The eluate was concentrated, subjected to size exclusion chromatography, and immediately used for cryo-EM grid preparation.

Cryo-EM Grid Preparation, Screening, and Data Collection

Cryo-EM grids were prepared for both vesicle and detergent samples utilizing specific Quantifoil grids, which were glow discharged prior to sample application. Samples were applied to grids in a Vitrobot, blotted, and plunge-frozen in liquid ethane.

For menthol-bound datasets, menthol was added to vesicles at specific temperatures just before freezing. Grids were screened using Talos Arctica or Glacios cryo-TEMs. Data collection was performed at UCSF or Janelia facilities using Titan Krios cryo-TEMs equipped with K3 or Falcon 4i cameras, respectively. Specific defocus ranges were maintained for both vesicle and detergent datasets to optimize imaging.

Cryo-EM Data Processing and Refinement

Micrographs underwent dose-weighted motion correction using either MotionCor2 or cryoSPARC. Initial models of Pm TRPM8 were generated and subsequently refined.

Particles were identified using Topaz and template picking, followed by rounds of heterogeneous refinement and 2D classification. Consensus refinements were generated, and further 3D classification was carried out in RELION or cryoSPARC, sometimes employing masks to enhance detail. Local resolution estimation was performed and visualized within UCSF ChimeraX.

Model Building, Pore Radius, and pKa Calculations

Atomic models for TRPM8 structures were generated de novo using ModelAngelo or sourced from published structures. These models were iteratively refined using Phenix and COOT, and their quality was validated with MolProbity.

The HOLE program was specifically used to determine pore radii within the TRPM8 structures. PROPKA3 was utilized for pKa calculations on specific TRPM8 structures, providing insights into their ionization states.

Hydrogen–Deuterium Exchange (HDX)

HDX experiments were initiated by diluting GDN-solubilized TRPM8 into deuterated buffer at controlled pD and temperatures. Labeling times were carefully adjusted to account for temperature dependence.

For menthol-treated conditions, 1 mM menthol was added either throughout the purification and exchange process or solely to the D2O buffer during the exchange phase.

HDX time-points were collected in triplicate to ensure reproducibility. Quenching was performed using ice-cold quench buffer, and samples were immediately injected into a valve system. Non-deuterated, in-exchange, and maximally labeled controls were also performed for comprehensive analysis.

Liquid Chromatography–Mass Spectrometry

LC-MS analysis was conducted on different instruments at UC Berkeley and UCSF, employing the same columns but with noted differences in retention times. Proteins were digested online using a NepII/pepsin protease column, and resulting peptides were desalted.

Peptides were then separated on a C18 analytical column with a linear gradient of acetonitrile and analyzed on Thermo Q Exactive or Q Exactive Plus mass spectrometers operating in positive ion mode.

HDX–MS Data Analysis and EX2 Characterization

Peptides were identified using SearchGUI and Byonic software. HDX data was processed using HDExaminer v.3.4, with bimodal fitting applied where appropriate to accurately represent isotopic distributions.

The observed exchange kinetics were definitively identified as EX2, supported by continuous shifts in isotopic envelopes and the clear pD-dependence of exchange rates. Bimodal fits were cross-validated using HX-Express3. Isotopic distributions were meticulously inspected manually, and deuteration levels were adjusted for D-content and back-exchange to ensure accuracy.

Quantifying Thermodynamic Parameters from HDX–MS

H–D exchange was analyzed using the Linderstrøm–Lang model, where observed rates (k_obs) are described by the equilibrium constant (K_eq) under EX2 kinetics. Folding free energy (ΔG) was quantified at the peptide level using a stretched-exponential method.

Changes in folding free energy (ΔΔG) were calculated for different experimental conditions to assess stability differences. Van’t Hoff analysis was employed to determine the enthalpy change (ΔH°), entropy contribution (TΔS°), and Gibbs free energy (ΔG°) extrapolated to 25 °C. Residue-level parameters were ultimately determined from a weighted average of peptide-level parameters.

Fura-2-AM Calcium Imaging and Data Analysis

Calcium imaging experiments utilized Fura-2-AM, with continuous temperature monitoring being a critical aspect. HEK293T cells were transfected with the appropriate plasmid, replated onto Matrigel-coated coverslips, and loaded with Fura-2-AM.

Cells were imaged using an inverted microscope with 340 and 380 nm excitation wavelengths. Temperature was precisely maintained by perfusing Ringer’s solution through a Peltier device. Cold stimulation involved perfusing ice-cooled Ringer’s solution, with temperature continuously monitored and synchronized with imaging acquisition. Videos of ratio images were calculated, and peak amplitudes of calcium signals were normalized to a maximum signal obtained with ionomycin.

Sequence Alignment of TRPM8 Orthologues

Sequences of avian and mammalian TRPM8 orthologues were obtained from OrthoDB v.12.2. Multiple sequence alignments were systematically generated using Clustal Omega. The outputs from these alignments were then used to plot individual sequence logo representations of TRPM8 orthologues, specifically focusing on regions near particular tyrosine/valine residues of interest.