Journal: Theranostics

Article Title: MgSO 4 as a novel hypothermia infusion solution promotes ischemic stroke recovery through Ca 2+ regulation of neurovascular units

doi: 10.7150/thno.104879

Figure Lengend Snippet: MgSO 4 as a novel hypothermia infusion solution for promoting AIS recovery by regulating Ca 2+ in NVUs. After I/R injury, the infarction region undergoes severe neural injury, BBB breakdown and microvessel occlusion, which were triggered by Ca 2+ influx into neurons, cerebral microvessel ECs and pericytes. IA-SCSI cooled the brain and promoted partial recovery from neural injury and BBB breakdown but could not rescue microvessel occlusion. In vitro cell experiments confirmed that hypothermia could partially inhibit Ca 2+ influx into neurons and cerebral microvessel ECs but did not affect the total amount of calcium ions in pericytes because hypothermia increased Ca 2+ influx through TRPM8. Moreover, IA-SCMI demonstrated significant recovery from neural injury and BBB breakdown, and enhanced microvessel circulation. The combination of hypothermia and Mg 2+ exerted stronger inhibitory effects on Ca 2+ influx compared with hypothermia alone in NVU cells. MCA, middle cerebral artery; ICA, internal carotid artery; ECA, external carotid artery; CCA, common carotid artery; IA-SCSI, intra-artery selective cooling saline infusion; IA-SCMI, intra-artery selective cooling MgSO 4 infusion; BBB, brain-blood barrier.

Article Snippet: The primary antibodies included anti-Rat CD31 (Cat#GB120005, Servicebio, China), anti-Rat Ly6G (Cat#GTX40912, GeneTex, USA), anti-Mouse ZO-1 (Cat#33-9100, Invitrogen, USA), anti-Rat α-SMA (Cat#14395-1-AP, Proteintech, USA) and anti-Rat TRPM8 (Cat#ACC-049, Alomone Labs, Israel).

Techniques: Infusion Solution, In Vitro, Saline

Journal: bioRxiv

Article Title: Deep mutational scan of the pore of the cold-sensing TRPM8 channel

doi: 10.64898/2026.04.28.721489

Figure Lengend Snippet: (A) Structural model of TRPM8 channel (left, PDB: 9B6D ) and the transmembrane domain (right) with the region between S927 and L974 shown in dark blue. EL, extracellular pore loop; PH, pore helix. (B) Cartoon of the iCasp9 landing pad expression system , showing the genomic landing pad genes before recombination (top; star, attB sequence), the plasmid coding sequences (middle; star, attP recombination sequence; green, GCaMP6s; gray, IRES2; TRPM8, blue; red diamond, T2A; scarlet, mCherry), and the plasmid genes after Bxb1 (magenta shape)-dependent recombination (bottom). (C) Menthol concentration-response relation of TRPM8, measured at +100 (closed symbols) or -100 (open symbols) mV in whole cells. Data shown as mean ± SEM (n = 6). Curves are fits to the Hill equation (100 mV, EC 50 = 0.15 ± 0.05 mM, Hill coef. = 1.50 ± 0.18; -100 mV, EC 50 = 0.32 ± 0.06 mM, Hill coef. = 1.79 ± 0.09). (D) Current magnitude at 100 mV recorded at 10 °C (Y745) or at room temperature + 2 mM menthol (all other constructs) from individual cells expressing WT or mutant TRPM8. p-values shown are from a two-sided Student’s t-test between WT and each of the mutants. (E) Representative current traces from WT and mutant channels at ±100 mV recorded at each of the conditions denoted by the trace color. Dotted magenta lines denote the zero-current level. (F) Current voltage-relations of WT and mutants under the same conditions as in (E). Data shown as mean ± SEM (n = 4). (G) Current-temperature relations of WT and mutant TRPM8 measured at +100 (closed symbols) or -100 mV (open symbols). Data shown as mean ± SEM (n = 4). Dotted magenta lines denote the zero-current level. (H) GCaMP6s fluorescence intensity histograms of 10 x 10 3 mCherry + cells expressing WT or mutant channels measured by flow cytometry at each of the conditions denoted by the trace colors.

Article Snippet: We used the backbone of the promoter-less plasmid KAM05_attB_mCherry, which was a gift from Doug Fowler (University of Washington), to onboard a synthetic gene sequence coding for the rat TRPM8 and an upstream partial Internal Ribosome Entry Site (IRES2) sequence using Twist Bioscience gene synthesis service.

Techniques: Expressing, Sequencing, Plasmid Preparation, Concentration Assay, Construct, Mutagenesis, Fluorescence, Flow Cytometry

Journal: bioRxiv

Article Title: Deep mutational scan of the pore of the cold-sensing TRPM8 channel

doi: 10.64898/2026.04.28.721489

Figure Lengend Snippet: (A) Cartoon representation of the experimental strategy used in this study. Libraries were generated from a pooled oligo library containing fragments coding for each type of single-residue missense variant within positions 927-974 of the rat TRPM8 channel. Oligo fragments were PCR-amplified and cloned into ‘acceptor’ expression plasmids by Golden Gate cloning using BsmBI sites . (B) The plasmid library was expressed in iCasp9 cells , and cells expressing mCherry were sorted by flow cytometry into groups of low, medium, or high GCaMP6s fluorescence intensity after stimulation with cold or 2 mM menthol. The genomic DNA was extracted from the sorted cells, from which the TRPM8 coding region was amplified by PCR, cleaved by a restriction endonuclease to reduce amplicon size, and prepared for next-generation sequencing. (C) NGS read counts per variant in the plasmid library, showing homogenous variant representation. (D) mCherry fluorescence intensity distribution in the library cells that were sorted. (E) GCaMP6s fluorescence intensity distribution of the library cells, measured at room temperature (dashed black curves, not sorted), or after stimulation with cold (blue) or 2 mM menthol (green) during cell sorting. Dashed red lines denote the three cell-sorting bins. (F) Deep mutational scanning score distribution for cold and menthol experiments. (G) Correlation between individual variant scores from two biological replicates. (H) GCaMP6s fluorescence responses in four silent mutations included in the library (gray traces) relative to cells expressing WT.

Article Snippet: We used the backbone of the promoter-less plasmid KAM05_attB_mCherry, which was a gift from Doug Fowler (University of Washington), to onboard a synthetic gene sequence coding for the rat TRPM8 and an upstream partial Internal Ribosome Entry Site (IRES2) sequence using Twist Bioscience gene synthesis service.

Techniques: Generated, Residue, Variant Assay, Amplification, Clone Assay, Expressing, Cloning, Plasmid Preparation, Flow Cytometry, Fluorescence, Next-Generation Sequencing, FACS

Journal: bioRxiv

Article Title: Deep mutational scan of the pore of the cold-sensing TRPM8 channel

doi: 10.64898/2026.04.28.721489

Figure Lengend Snippet: (A) GCaMP6s fluorescence intensity histograms of mCherry + cells expressing WT or mutant channels measured by flow cytometry at room temperature (black), or after simulation with cold (blue), 0.2 mM menthol (yellow), or 2 mM menthol (green). Data shown as mean ± SEM (n = 3). (B) Atomic model of the pore region of the TRPM8 D structure (PDB: 9B6D), showing each of the mutations in (A) in magenta. (C) Comparison of the WT and mutant GCaMP6s fluorescence intensity histograms for cold and 2 mM menthol (x-axis, bottom) and the deep mutational scanning scores of those same mutants (x-axis, top; black circles). Variants are grouped according to their phenotype as indicated by the color bars on the left side.

Article Snippet: We used the backbone of the promoter-less plasmid KAM05_attB_mCherry, which was a gift from Doug Fowler (University of Washington), to onboard a synthetic gene sequence coding for the rat TRPM8 and an upstream partial Internal Ribosome Entry Site (IRES2) sequence using Twist Bioscience gene synthesis service.

Techniques: Fluorescence, Expressing, Mutagenesis, Flow Cytometry, Comparison

Journal: bioRxiv

Article Title: Deep mutational scan of the pore of the cold-sensing TRPM8 channel

doi: 10.64898/2026.04.28.721489

Figure Lengend Snippet: (A, left) Structural model of TRPM8 in the desensitized state (PDB: 9B6D) as seen from the side. Pore loop residues 927-950 are colored based on their low (dark red), intermediate (pink), or high tolerance of substitutions (gray). The pore helix and ion-selectivity filter (S.F.) of the same subunit as the pore loop are shown in green, and the S4 helix of the adjacent subunit in sand. (A, right) Same model as in the left panel seen from above, and including the electron microscopy (EM) map (EMD-44255). Dashed blue rectangles denote the pore helix and S6-proximal interaction interfaces between the pore helix and the rest of the protein. Dashed black lines in the right panel denote non-covalent interactions between the pore loop and the rest of the protein. (B) Cumulative histograms of all variant scores per sequence position, colored by their high (gray), intermediate (pink), or no tolerance (dark red) of substitutions. Shading is the average Lilace standard error per position. Histograms for C929 and C940 are shown as continuous black lines. (C) Variant classification and median score color scale as in . (D) Bubble plot of individual variant cold scores. Scores are represented both by the radius of the circles and their color as denoted by the scale on the left and as displayed in . Black squares denote WT residues. (E) Side view of the atomic model and EM map of the pore helix interface of the pore loop colored as in (A). (F) Side view of the atomic model and EM map of the S6-proximal pore loop interface with colors as in (A). Dashed black lines in (E) and (F) denote non-covalent interactions between the pore loop and the rest of the protein. (G) Violin plots of the individual variant scores at positions with intermediate and high tolerance of substitutions, relative to the individual score of the cysteine-containing variants at each position (yellow). Median scores shown in blue (cold) and green (menthol).

Article Snippet: We used the backbone of the promoter-less plasmid KAM05_attB_mCherry, which was a gift from Doug Fowler (University of Washington), to onboard a synthetic gene sequence coding for the rat TRPM8 and an upstream partial Internal Ribosome Entry Site (IRES2) sequence using Twist Bioscience gene synthesis service.

Techniques: Electron Microscopy, Variant Assay, Sequencing

Journal: bioRxiv

Article Title: Deep mutational scan of the pore of the cold-sensing TRPM8 channel

doi: 10.64898/2026.04.28.721489

Figure Lengend Snippet: (A) Structural alignment of the TRPM8 pore regions from 16 distinct structures as indicated by the colors and PDB labels. F.sw – fully swapped; s.sw – semi swapped. Only one subunit is shown. (B) Bubble plot of individual variant menthol scores for the same positions as in . Scores are represented both by the radius of the circles and their color as denoted by the scale on the left on the heatmaps on . Black squares denote WT residues. (C) Amino acid sequence alignment around the region from 927 to 974 in the rat TRPM8, including sequences from multiple TRPM8 orthologues from humans to amphibians as well as human and rat sequences for TRPM2, TRPM4, and TRPM5. Dashed lines and blue bars at the bottom denote regions where the TRPM8 sequences are not fully conserved. (D) Median deep mutational scanning scores per position, highlighting each of the regions with lower sequence conservation. The shading is the SEM of the scores across the 19 variants per position. (E) Structural model of an avian TRPM8 in the semi swapped closed state (PDB: 9ZCQ). Pore loop residues 927-951 are colored based on whether each position has a low (dark red), intermediate (pink), or high tolerance (gray) of substitutions based on the deep mutational scanning scores. The S4 helix of the same subunit as the pore loop is shown in green, and the pore helix and ion-selectivity filter (S.F.) of the adjacent subunit shown in sand. Dashed blue rectangles denote the pore helix and S6-proximal interaction interfaces between the pore helix and the rest of the protein. Dashed black lines denote non-covalent interactions between the pore loop and the rest of the protein.

Article Snippet: We used the backbone of the promoter-less plasmid KAM05_attB_mCherry, which was a gift from Doug Fowler (University of Washington), to onboard a synthetic gene sequence coding for the rat TRPM8 and an upstream partial Internal Ribosome Entry Site (IRES2) sequence using Twist Bioscience gene synthesis service.

Techniques: Variant Assay, Sequencing

Journal: bioRxiv

Article Title: Deep mutational scan of the pore of the cold-sensing TRPM8 channel

doi: 10.64898/2026.04.28.721489

Figure Lengend Snippet: (A) Map of residue-residue contacts between S6 helix residues 951-974 (x-axis) and the rest of the protein (y-axis, left) in C1 (PDB: 8E4N), O (PDB: 8E4L), and D (PDB: 9B6D) structures of TRPM8. Each color square on the map represents one contact and the color of the square denotes which states have that contact. The heatmap on the bottom insert denotes the number of total atomic contacts by residues 927-974 in each state. The median cold and menthol scores per position are superposed (y-axis, right). The shading is the SEM of the scores across the 19 variants per position. (B) Atomic models of the pore of domain of one TRPM8 subunit in C1, O, and D states, with residues 927-974 colored by helix region. (C) Cumulative histograms of all variant scores per sequence position between residues 951 and 974, colored by S6 region and tolerance to substitution, as indicated in (D). Shading is the average Lilace standard error per position. (D) Variant classification per S6 helix region and tolerance of substitutions (right-most color bars), showing the median score per position as a color scale. (E) Bubble plot of individual variant cold (left) and menthol (right) scores. Black squares denote WT residues, color bars on top denote each of the different S6 helix regions discussed in the text.

Article Snippet: We used the backbone of the promoter-less plasmid KAM05_attB_mCherry, which was a gift from Doug Fowler (University of Washington), to onboard a synthetic gene sequence coding for the rat TRPM8 and an upstream partial Internal Ribosome Entry Site (IRES2) sequence using Twist Bioscience gene synthesis service.

Techniques: Residue, Variant Assay, Sequencing

Journal: bioRxiv

Article Title: Deep mutational scan of the pore of the cold-sensing TRPM8 channel

doi: 10.64898/2026.04.28.721489

Figure Lengend Snippet: (A) Side view of the upper half of a portion of the transmembrane domain from C1 (PDB: 8E4N), O (PDB: 8E4L), and D (PDB: 9B6D) structures of TRPM8. S6 residues of one subunit are colored by their median menthol score on a scale as in , and the pore helix (P.H.) and selectivity filter of that same subunit are shown in green. S1-S4 helices of an adjacent subunit are shown in sand color. Dashed rectangles denote the approximate regions displayed in (B). (B) Magnified views of the structural models in (A), including the EM maps (C1, EMD-27893; O, EMD-27891; D, EMD-44255). Dashed black lines denote non-covalent interactions between the S6 and the rest of the protein. (C) Bubble plot of individual variant cold and menthol scores of the four N-terminal residues in the S6 as well as P958. Black squares denote WT residues. (D-G) Individual variant cold (blue) and menthol (green) scores as a function of substituting side chain volume or hydrophobicity , distinguishing between polar and charged substituting amino acids (closed symbols) or non-polar and aromatics (open symbols). Error bars are the Lilace standard error per variant. Substitutions with proline and glycine were omitted. Vertical dashed lines denote the WT-residue properties.

Article Snippet: We used the backbone of the promoter-less plasmid KAM05_attB_mCherry, which was a gift from Doug Fowler (University of Washington), to onboard a synthetic gene sequence coding for the rat TRPM8 and an upstream partial Internal Ribosome Entry Site (IRES2) sequence using Twist Bioscience gene synthesis service.

Techniques: Variant Assay, Residue

Journal: bioRxiv

Article Title: Deep mutational scan of the pore of the cold-sensing TRPM8 channel

doi: 10.64898/2026.04.28.721489

Figure Lengend Snippet: (A) Depiction of an S6 helix and adjacent pore elements viewed from above in C1 (PDB: 8E4N), O (PDB: 8E4L), and D (PDB: 9B6D) structures of TRPM8. S6 residues I955 to S966 are colored by helix region, the S5, pore helix (P.H.) and selectivity filter (S.F.) of the same subunit colored in green, and the S4 and pore helices of the adjacent subunit in sand. The transmembrane domain in the tetrameric TRPM8 complex in the O state is shown on the bottom left insert, with each subunit shown in a different color. (B) Individual variant cold (blue) and menthol (green) scores as a function of substituting side chain volume or hydrophobicity , distinguishing between polar and charged substituting amino acids (closed symbols) or non-polar and aromatics (open symbols). Error bars are the Lilace standard error per variant. Substitutions with proline and glycine were omitted. Dashed vertical lines denote the WT-residue side chain volume or hydrophobicity. (C) Structural depiction of the Y963 and M964 interaction interface in C1, O, and D structures, with colors as in (A). Dashed black lines denote non-covalent interactions between the S6 and the rest of the protein. (D) Individual variant cold (blue) and menthol (green) scores as a function of substituting side chain volume. Error bars are the Lilace standard error per variant. Substitutions with proline and glycine were omitted. Dashed vertical lines denote the WT-residue side chain volume.

Article Snippet: We used the backbone of the promoter-less plasmid KAM05_attB_mCherry, which was a gift from Doug Fowler (University of Washington), to onboard a synthetic gene sequence coding for the rat TRPM8 and an upstream partial Internal Ribosome Entry Site (IRES2) sequence using Twist Bioscience gene synthesis service.

Techniques: Variant Assay, Residue

Journal: bioRxiv

Article Title: Deep mutational scan of the pore of the cold-sensing TRPM8 channel

doi: 10.64898/2026.04.28.721489

Figure Lengend Snippet: (A) Transmembrane domain in the tetrameric TRPM8 complex in the O state with each subunit shown in a different color. (B) Depiction of an S6 helix and adjacent pore elements viewed from the membrane side in C1 (PDB: 8E4N), O (PDB: 8E4L), and D (PDB: 9B6D) structures of TRPM8. S6 residues L965-N974 are colored by helix region, the S5, pore helix (P.H.) and selectivity filter (S.F.) of the same subunit colored in green, and the S4, S4-S5 linker, S5 and pore helices of the adjacent subunit in sand. Dashed black lines denote non-covalent interactions between the S6 and the rest of the protein. (C) Individual variant cold (blue) and menthol (green) scores as a function of substituting side chain volume or hydrophobicity , distinguishing between polar and charged substituting amino acids (closed symbols) or non-polar and aromatics (open symbols). Error bars are the Lilace standard error per variant. Substitutions with proline and glycine were omitted. Vertical dashed lines denote the WT-residue properties. (D) Representative current families at ± 100 mV recorded at distinct temperatures denoted by the trace colors from whole cells expressing WT or mutant channels. Dashed lines denote the zero-current level. (E) Normalized current-temperature relations obtained from experiments as in (D) for WT and mutant TRPM8. Data shown as mean ± SEM (n = 4). (F) Normalized current-voltage relations recorded at each of the conditions denoted by symbol color for WT and mutant TRPM8. Data shown as mean ± SEM (n = 4).

Article Snippet: We used the backbone of the promoter-less plasmid KAM05_attB_mCherry, which was a gift from Doug Fowler (University of Washington), to onboard a synthetic gene sequence coding for the rat TRPM8 and an upstream partial Internal Ribosome Entry Site (IRES2) sequence using Twist Bioscience gene synthesis service.

Techniques: Membrane, Variant Assay, Residue, Expressing, Mutagenesis

Journal: bioRxiv

Article Title: Deep mutational scan of the pore of the cold-sensing TRPM8 channel

doi: 10.64898/2026.04.28.721489

Figure Lengend Snippet: (A) Transmembrane domain of TRPM8 in the fully swapped (f.sw) O (top, PDB: 8E4L) state, as well as in semi swapped (s.sw) C (middle; PDB: 9ZCQ) and O (bottom; PDB: 9PAR) states of an avian TRPM8. Subunit coloring is the same in all four complexes. (B) Depiction of an S6 helix and adjacent pore elements viewed from above in semi swapped (s.sw) C (PDB: 9ZCQ) and O (PDB: 9PAR) state structures of TRPM8 . S6 residues I955 to S966 are colored by helix region as in , the S5, pore helix (P.H.) and selectivity filter (S.F.) of the adjacent subunit colored in sand color, and the S4 and pore helices of the same subunit as the depicted S6 helix shown in green. (C, top) Structural depiction of the Y963 and M964 interaction interface in the two semi swapped structures with colors as in (A). (C, bottom) Depiction of an S6 helix and surrounding pore elements viewed from the membrane side in semi swapped structures of TRPM8. Residues and ribbons are colored as in (A). Dashed black lines denote non-covalent interactions between the S6 and the rest of the protein. (D) Map of residue-residue contacts between S6 helix residues 951-974 (x-axis) and the rest of the protein (y-axis, left) in a fully swapped open state of the human TRPM8 (top; PDB: 9PB5), a fully swapped closed state of the human TRPM8 (middle top; PDB: 9P8Y), a semi swapped open state of an avian TRPM8 (middle bottom; PDB: 9PAR), and a semi swapped closed state of an avian TRPM8 (top; PDB: 9ZCQ) . Each color square on the map represents a contact and the colors denote each of the S6 regions as discussed in the text. The heatmap on the bottom insert denotes the number of total atomic contacts by the residues in the S6. The median cold and menthol scores per position are superposed (y-axis, right). The shading is the SEM of the scores across the 19 variants per position.

Article Snippet: We used the backbone of the promoter-less plasmid KAM05_attB_mCherry, which was a gift from Doug Fowler (University of Washington), to onboard a synthetic gene sequence coding for the rat TRPM8 and an upstream partial Internal Ribosome Entry Site (IRES2) sequence using Twist Bioscience gene synthesis service.

Techniques: Membrane, Residue

Journal: bioRxiv

Article Title: Deep mutational scan of the pore of the cold-sensing TRPM8 channel

doi: 10.64898/2026.04.28.721489

Figure Lengend Snippet: (A) Structural alignment of the pore region in C1 (magenta, PDB: 8E4N) and D (gray, PDB: 9B6D) structures viewed from above. Arrows denote inferred movements upon channel opening within (1) the S6-distal interface and (2) S6-proximal interfaces of the pore loop, as well as within (3) the pore helices and (4) S4, S5, and S6 helices. (B) Cartoon of the TRPM8 pore of one subunit (green) and the S4 helices of the adjacent subunit (sand) in the closed state. Arrows denote the direction of movement of the pore helix during gating. (C, D) Structural alignment of the pore region in O (aqua, PDB: 8E4L) and D (gray, PDB: 9B6D) structures viewed from above (C) or from the side (D). Dashed arrows denote dynamics within the ion-selectivity filter that are constrained by the pore loop helix, solid arrows denote the same movements as in (A). (E) Cartoon of the TRPM8 pore of one subunit (green) and the S4 helices of the adjacent subunit (sand) in the open state. Numbers denote each of the five regions of S6 that are described in the text. (F) Cartoon illustrating how open state-dependent (green arrows) and -independent (black arrows) contacts that stabilize S6 helices in the open state of TRPM8 go from weaker (left, dashed arrows) to stronger (right, filled arrows) as the temperature decreases. (G) The S6 helices in heat-activated TRPM channels are stabilized by a larger proportion of state-independent contacts, making the helices and pore less dynamic.

Article Snippet: We used the backbone of the promoter-less plasmid KAM05_attB_mCherry, which was a gift from Doug Fowler (University of Washington), to onboard a synthetic gene sequence coding for the rat TRPM8 and an upstream partial Internal Ribosome Entry Site (IRES2) sequence using Twist Bioscience gene synthesis service.

Techniques:

Journal: bioRxiv

Article Title: Deep mutational scan of the pore of the cold-sensing TRPM8 channel

doi: 10.64898/2026.04.28.721489

Figure Lengend Snippet: (A) Map of state-dependent and independent residue-residue contacts involving the upper portion of the S6 helix and the rest of the protein, identified in closed and open-state structures of the TRPM2 (PDB: 6DRJ, 6DRK ) and TRPM4 (PDB: 9MRT, 9MTA ; 9B8Y , 6BQR ) channels. Each color square on the map represents one residue-residue contact and the color of the square denotes its state dependence. The heatmap on the bottom insert denotes the number of total atomic contacts between each residue in the S6 and other residues in the rest of the protein. The portion of S6 that was included in this analysis was determined based on the amino acid sequence alignment between TRPM8 and the other channels . (B) Median variant pathogenicity score obtained from scaled AlphaMissense predictions for all variants per position for the human TRPM2 (yellow), TRPM4 (magenta), and TRPM5 channels (purple), compared with the scaled predictions for TRPM8 (black) as well as the experimental scaled scores for cold (blue) and menthol (green). The shading is the SEM across the difference scores for the 19 variants per position.

Article Snippet: We used the backbone of the promoter-less plasmid KAM05_attB_mCherry, which was a gift from Doug Fowler (University of Washington), to onboard a synthetic gene sequence coding for the rat TRPM8 and an upstream partial Internal Ribosome Entry Site (IRES2) sequence using Twist Bioscience gene synthesis service.

Techniques: Residue, Sequencing, Variant Assay

Journal: ACS Omega

Article Title: Identification of Phialomustin‑B (PHL-B) as a Novel Natural TRPA1 Channel Inhibitor

doi: 10.1021/acsomega.5c06063

Figure Lengend Snippet: Lack of inhibitory effect of PHL-B on TRPV1, TRPV4, and TRPM8 channels. (A, C, E) Concentration-dependent relationships of PHL-B on TRPV1, TRPV4, and TRPM8 channels upon stimulation with capsaicin, HTS, and menthol, respectively. Capsazepine, HC-067047, and sesamin were used as reference inhibitors for TRPV1, TRPV4, and TRPM8 channels, respectively. (B, D, F) Plots of normalized fluorescence vs drug concentration at different time points for each channel, where the lack of effect of PHL-B can be clearly visualized. These data could not be fitted with a Hill equation; hence, the IC 50 values were not determined. Data are expressed as means ± SEM, N = 3–5 independent experiments.

Article Snippet: The rat TRPM8 plasmid was obtained from Addgene (#64879).

Techniques: Concentration Assay, Fluorescence

Journal: bioRxiv

Article Title: Reciprocal regulation among TRPV1 channels and phosphoi nos itide 3-kinase in response to nerve growth factor

doi: 10.1101/346718

Figure Lengend Snippet: Time course of NGF-induced changes in fluorescence intensity. NGF (100 ng/mL) was applied during the times indicated by the black bar/gray shading. Traces represent the mean, error bars are SEM. Control and TRPV1 data same as in with error bars removed for clarity. (A-B) Averaged normalized TIRF intensity of Akt-PH from cells transfected with TrkA/p75 NTR and Akt-PH and: (A) no channel (control; blue; n=75); TRPV1 (orange; n=122); TRPV2 (black; n=61); TRPV4 (yellow; n=29) (B) no channel (control; blue; n=75); TRPM4 (n=63); TRPM8 (n=73); TRPA1 (n=59). (C-D) Averaged normalized TIRF intensity of individual TRP channels. Color scheme as in (A-B) with the cell numbers as follows: (C) TRPV1 (n=94); TRPV2 (n=62); TRPV4 (n=48); (D) TRPM4 (n=65); TRPM8 (n=67); TRPA1 (n=52).

Article Snippet: TRPV4-EGFP (human) in pEGFP was obtained from Tim Plant (Charite-Universitatsmedizine, Berlin) ( ). pEGFP-TRPM8 (rat) in pEGFP was obtained from Addgene (#64879, Addgene, Cambridge, MA).

Techniques: Fluorescence, Transfection

Journal: Cephalalgia

Article Title: Functional interactions between transient receptor potential M8 and transient receptor potential V1 in the trigeminal system: Relevance to migraine pathophysiology

doi: 10.1177/0333102417712719

Figure Lengend Snippet: Temporal profiles of the heat pain threshold temperatures after the induction of IS-induced meningeal inflammation. (a) IS-induced meningeal inflammation lowered the heat pain threshold temperature at six hours and Days 1 and 2 in vehicle (DMSO)-treated wild-type mice (filled triangle, N = 30 from six independent experiments). In contrast, there was no change in threshold temperature when compared to that of pre-IS Baseline in icilin-pretreated WT mice (filled circle, N = 30 from six independent experiments). * p < 0.001, ANOVA, Bonferroni’s post hoc test, compared to baseline. $p < 0.001, ANOVA, Bonferroni’s post hoc test, between-group difference at the same time point. (b) In sham-operated wild-type mice, there were no temporal changes in the heat pain threshold temperature. Icilin did not affect the heat pain threshold temperature. (N = 30 from six independent experiments in each group). (c) In TRPM8 KO mice, there were no antinociceptive effects of icilin on IS-induced threshold temperature changes (N = 30 from six independent experiments in each group). (d) In sham-operated TRPM8 KO mice, the heat pain threshold temperature did not change throughout the experimental period. There was no significant effect of icilin on the heat pain threshold temperature. (N = 30 from six independent experiments in each group).

Article Snippet: The EmGFP-rat full-length TRPM8-V5 expression vector was transfected into PC12 cells using Lipofectamine 2000 (Life Technologies).

Techniques:

Journal: Cephalalgia

Article Title: Functional interactions between transient receptor potential M8 and transient receptor potential V1 in the trigeminal system: Relevance to migraine pathophysiology

doi: 10.1177/0333102417712719

Figure Lengend Snippet: Quantitative data of thermal pain threshold changes.

Article Snippet: The EmGFP-rat full-length TRPM8-V5 expression vector was transfected into PC12 cells using Lipofectamine 2000 (Life Technologies).

Techniques:

Journal: Cephalalgia

Article Title: Functional interactions between transient receptor potential M8 and transient receptor potential V1 in the trigeminal system: Relevance to migraine pathophysiology

doi: 10.1177/0333102417712719

Figure Lengend Snippet: Changes in TRPM8 and TRPV1 expression in TG neurons during IS-induced meningeal inflammation. (a) Photomicrographs showing TRPM8-positive and TRPV1-positive TG neurons. TG neurons double positive for TRPM8 and TRPV1 are indicated by arrowheads. Bar = 15 µm. (b) Left: The ratios of TRPM8-positive (white) and TRPV1-positive (black) neurons to total neurons are shown in the bar graph. ** p < 0.001, compared to control, ANOVA, Bonferroni post hoc test. Right: The colocalization ratio of TRPM8 and TRPV1 in TG neurons is shown in the bar graph. ** p < 0.001, compared to control, ANOVA, Bonferroni post hoc test. (c) There was no significant immunoreactivity in TRPM8 KO mouse TG tissue with the same TRPM8 antibody. Bar = 20 µm. (d) TG neurons positive for TRPM8 transcripts visualized by in situ hybridization. For simplicity, only data for Sham (sham-operated animals) and Day 2 and Day 6 post-IS are displayed. Bar = 30 µm.

Article Snippet: The EmGFP-rat full-length TRPM8-V5 expression vector was transfected into PC12 cells using Lipofectamine 2000 (Life Technologies).

Techniques: Expressing, In Situ Hybridization

Journal: Cephalalgia

Article Title: Functional interactions between transient receptor potential M8 and transient receptor potential V1 in the trigeminal system: Relevance to migraine pathophysiology

doi: 10.1177/0333102417712719

Figure Lengend Snippet: Functional interactions between TRPM8 and TRPV1 in PC12 cells. (a) The expression construct for the EmGFP-rat full-length TRPM8-V5 epitope fusion protein. Expression was driven by the CMV promoter. (b) Calcium imaging with Rhod-2 AM revealed that the intracellular calcium increase induced by icilin was dependent on the TRPM8 expression level, such that a higher calcium rise was observed at lower icilin concentrations in high TRPM8-expressing cells. The regions of calcium concentration measurement are encircled. Low, Intermediate, and High indicate the expression levels of TRPM8 (EmGFP). Bar: 10 µm. (c) Upper row: Dose and time-dependent increases in JNK phosphorylation evoked by capsaicin (Caps) in rat full-length TRPV1-stably expressing PC12 cells. Lower row: In the absence of TRPM8 expression (left), icilin treatment did not significantly change the magnitude of TRPV1-induced JNK phosphorylation. In the presence of TRPM8 expression, icilin treatment attenuated TRPV1-induced JNK phosphorylation. A densitometric analysis of western blots is presented in the bar graph. Ratios of capsaicin-induced JNK phosphorylation in icilin-treated cells to that in vehicle-treated cells are compared between TRPM8-null and TRPM8-expressing cells. ** p = 0.004, unpaired t-test. Cells were treated with 50 µM capsaicin for 60 min. Where noted, cells were pretreated 30 min prior with 100 nM icilin.

Article Snippet: The EmGFP-rat full-length TRPM8-V5 expression vector was transfected into PC12 cells using Lipofectamine 2000 (Life Technologies).

Techniques: Functional Assay, Expressing, Construct, Imaging, Concentration Assay, Stable Transfection, Western Blot

Journal: Cephalalgia

Article Title: Functional interactions between transient receptor potential M8 and transient receptor potential V1 in the trigeminal system: Relevance to migraine pathophysiology

doi: 10.1177/0333102417712719

Figure Lengend Snippet: Proposed mechanism by which facial TRPM8 activation alleviates meningeal inflammation-mediated thermal allodynia. (a) In the resting state, there are few TG neurons that express both TRPV1 and TRPM8. Some of the dural afferent TG neurons send collaterals to the face as well. (b) Meningeal inflammation can activate TRPV1-positive TG neurons, which causes headache and facial thermal allodynia. (c) After a while, TRPM8 expression is enhanced by transcriptional upregulation. As a consequence, the number of TRPM8/TRPV1-positive TG neurons increases. Such TRPM8 upregulation in TRPV1-positive cells also occurs in TG neurons innervating both the dura and face. (d) In this condition, facial TRPM8 activation can alleviate TRPV1-mediated thermal allodynia and, possibly, headache. In this paradigm, opposing actions derived from the intracranial (dura) and extracranial (facial tissue) tissue can interact with each other in a cell-autonomous fashion. TNC: trigeminal nucleus caudalis.

Article Snippet: The EmGFP-rat full-length TRPM8-V5 expression vector was transfected into PC12 cells using Lipofectamine 2000 (Life Technologies).

Techniques: Activation Assay, Expressing, Derivative Assay

Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

Article Title: Neurturin Overexpression in Skin Enhances Expression of TRPM8 in Cutaneous Sensory Neurons and Leads to Behavioral Sensitivity to Cold and Menthol

doi: 10.1523/JNEUROSCI.4012-12.2013

Figure Lengend Snippet: Sequences of primers used for real-time RT-PCR assays.

Article Snippet: 30 μg of protein was boiled in 5×SDS loading buffer for 10 min, separated on an 8% SDS-PAGE gel, transferred to PVDF membranes, blocked, and incubated with primary antibodies at 4°C overnight (rat anti-TRPM8, 1:1000 and rabbit-anti-GAPDH, 1:5000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

Techniques: Quantitative RT-PCR

Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

Article Title: Neurturin Overexpression in Skin Enhances Expression of TRPM8 in Cutaneous Sensory Neurons and Leads to Behavioral Sensitivity to Cold and Menthol

doi: 10.1523/JNEUROSCI.4012-12.2013

Figure Lengend Snippet: Change in gene expression in lumbar DRG and TG of NRTN-OE mice. ASIC2a, GFRα2, P2X 3 ,Ret, Runx1 and TRPM8 were increased in ganglia of NRTN-OE mice relative to WT mice.

Article Snippet: 30 μg of protein was boiled in 5×SDS loading buffer for 10 min, separated on an 8% SDS-PAGE gel, transferred to PVDF membranes, blocked, and incubated with primary antibodies at 4°C overnight (rat anti-TRPM8, 1:1000 and rabbit-anti-GAPDH, 1:5000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

Techniques: Expressing

Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

Article Title: Neurturin Overexpression in Skin Enhances Expression of TRPM8 in Cutaneous Sensory Neurons and Leads to Behavioral Sensitivity to Cold and Menthol

doi: 10.1523/JNEUROSCI.4012-12.2013

Figure Lengend Snippet: In WT DRG (A-C), TRPM8-staining was not detectable in IB4-binding neurons, whereas in NRTN-OE DRG (D-F), most IB4-binding neurons express TRPM8 (indicated by arrows). Immunoblot analysis of pooled L2-L4 DRG from WT (n=3) and NRTN-OE (n=3) mice (G). TRPM8 protein level was greater in DRG from NRTN-OE mice. TRPM8 protein was undetectable in WT ganglia under the conditions used. Calibration bar=20 μm.

Article Snippet: 30 μg of protein was boiled in 5×SDS loading buffer for 10 min, separated on an 8% SDS-PAGE gel, transferred to PVDF membranes, blocked, and incubated with primary antibodies at 4°C overnight (rat anti-TRPM8, 1:1000 and rabbit-anti-GAPDH, 1:5000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

Techniques: Staining, Binding Assay, Western Blot