Fig 2: Icilin activation of TRPM8 in CHO cells and rat DRG neurons.Antagonism of icilin induced activation of human TRPM8 recombinantly expressed by CHO cells (A) and rat DRG neurons (B) by additional poly- and monoclonal antibodies generated against the third extracellular pore loop. a. Alomone ACC-049. b. MyBiosource MBS609041. c. Creative Diagnostics CABT37242RH. d. Thermo Scientific OST00133W. e. Antibodies Online ABIN351226. f. Lifespan Biosciences LS-B6668. g. Enzo Lifesciences BML-SA664. h. M8-B. i. 1 µM icilin. j. 1 µM icilin + peptide (SDVD GTTYDFAHC). k. Buffer. A. Note the complete block of TRPM8 channel activation by ACC-049 (a), MyBiosource (b) and Enzo Lifesciences (g) antibodies at the single concentration tested. Small molecule positive control M8-B also completely blocked TRPM8 channel activation (h). B. Five out of seven antibodies tested (a, b, e, f, g) block icilin activation of rat DRG neurons by 70–80%, two antibodies (c, d) are ineffective. Values are means of triplicate measures in a single experiment and expressed as percent of control (POC). 45Ca2+ uptake of CHO-TRPM8 cells activated with 1 µM icilin and antigen peptide (j) was considered as 100 percent and wells with only assay buffer plus 45Ca2+ were set as zero percent.

Fig 3: ACC-049 is a selective inhibitor of TRPM8.Specificity of ACC-049 (2.5 µM) for blocking human TRPM8 activation induced by the specific natural agonist cold (A) or synthetic agonist icilin (D). No effect of ACC-049 on noxious cold induced human TRPA1 (B) or heat induced TRPV1 activation (C). Small molecule antagonists AMG9090 and AMG6541 are the positive control for TRPA1 (B) or TRPV1 (C) blockage, respectively. Note the near complete blockade of TRPM8 activation by ACC-049 at 2.5 µM, similar to that by the positive small molecule antagonist control M8-B (A). Neither control IgG, nor peptide-absorbed ACC-049, or peptide alone blocked activation of any of the channels tested (A–D). Values are means of triplicate measures in a single experiment and expressed as percent of control (POC). Agonist induced 45Ca2+ uptake in the absence of antibodies (no Ab) was considered as 100 percent and wells with small molecule antagonists plus 45Ca2+ were set as zero percent.

Fig 4: Cold activation of TRPM8.Concentration dependent antagonism of cold activation (10°C) of the human (A), rat (B), or mouse (C) TRPM8 channels by ACC-049, control IgG and M8-B measured by 45calcium uptake. Note the right shifted concentration response of ACC-049 on human TRPM8 (A) compared to rat (B) or mouse (C) TRPM8, while the small molecule antagonist positive control M8-B exhibited comparable responses on TRPM8 channels of all species tested (A–C). Values are means of triplicate measures in a single experiment and expressed as percent of control (POC). Cold induced 45Ca2+ uptake was considered as 100 percent and wells with M8-B at 1 µM plus 45Ca2+ were set as zero percent.

Fig 5: Menthol activation of TRPM8.Concentration dependent antagonism of menthol induced activation of the human (A), rat (B) or mouse (C) TRPM8 channels by ACC-049, control IgG and M8-B measured by 45calcium uptake. Human TRPM8 channel activation was blocked by ACC-049 in a concentration dependent manner (A), but there was no antagonistic effect of ACC-049 on either rat (B) or mouse (C) TRPM8 channels activated by menthol. The small molecule antagonist positive control M8-B exhibited comparable responses on TRPM8 channels of all species tested (A–C). Values are means of triplicate measures and expressed as percent of control (POC). Menthol induced 45Ca2+ uptake was considered as 100 percent and wells with only assay buffer plus 45Ca2+ were set as zero percent.