Journal: Bioscience Reports

Article Title: Human TRPM8 and TRPA1 pain channels, including a gene variant with increased sensitivity to agonists (TRPA1 R797T), exhibit differential regulation by SRC-tyrosine kinase inhibitor

doi: 10.1042/BSR20140061

Figure Lengend Snippet: Short list of functionally informative mutations in mammalianTRPM8 and TRPA1 (excluding N-terminal domain)

Article Snippet: The potent TRPM8 agonist WS 12 [(1R,2S)- N -(4-methoxyphenyl)-5-methyl-2-(1-methylethyl)cyclohexanecarbo-xamide], the TRPM8 antagonist AMTB [ N -(3-Aminopropyl)-2-[(3-methylphenyl)methoxy]- N -(2-thienylmethyl)benzamide] hydrochloride and TRPA1 antagonists HC 030031 [2-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-7H-purin-7-yl)- N -(4-isopropylphenyl)acetamide] and A967079 were from Tocris bioscience.

Techniques: Binding Assay, Glycoproteomics, Activity Assay

Journal: Bioscience Reports

Article Title: Human TRPM8 and TRPA1 pain channels, including a gene variant with increased sensitivity to agonists (TRPA1 R797T), exhibit differential regulation by SRC-tyrosine kinase inhibitor

doi: 10.1042/BSR20140061

Figure Lengend Snippet: Relative fluorescence intensity units (RFU) are plotted versus time (s). ( a ) HEK-293 cells expressing human TRPM8 prepared in the presence of increasing concentrations of probenecid (A: 0.13 mM, B: 1.3 mM, C: 2.5 mM) treated with 40 nM WS 12 (injected at first arrow), then 2 μM A23187 (arrow with asterisk, indicated for first trace). Exposure to probenecid suppressed the response to WS 12. ( b ) Example Ca 2+ transient traces showing dose-dependent WS 12 activation of TRPM8 SV 762,763 EL double residue mutant expressed in HEK-293 cells (A: 1 μM, B: 100 nM, C: 30 nM WS 12). ( c ) The Ca 2+ transient response to 125 nM WS 12 in SH-SY5Y cells transfected with TRPM8 was similar in magnitude to the response to 2 nM bradykinin-mediated activation of endogenous bradykinin receptors, ( d ) ( e ) Dose–response curves derived from measurements of peak intracellular Ca 2+ fluorescence in HEK-293 and SH-SY5Y cell clones expressing TRPM8 constructs. Estimation of EC 50 for WS 12 (mean±S.E.M., n =3 independent experiments each performed in triplicate). Mean EC 50 values for reference sequence and mutant TRPM8 were not statistically significant in either cell line. ( f ) Estimated IC 50 values for TRPM8 antagonist AMTB determined using a constant dose of 150 nM WS 12, (mean±S.E.M., n =3). Mean IC 50 values for wild-type and mutant TRPM8 were not statistically significant in either cell type.

Article Snippet: The potent TRPM8 agonist WS 12 [(1R,2S)- N -(4-methoxyphenyl)-5-methyl-2-(1-methylethyl)cyclohexanecarbo-xamide], the TRPM8 antagonist AMTB [ N -(3-Aminopropyl)-2-[(3-methylphenyl)methoxy]- N -(2-thienylmethyl)benzamide] hydrochloride and TRPA1 antagonists HC 030031 [2-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-7H-purin-7-yl)- N -(4-isopropylphenyl)acetamide] and A967079 were from Tocris bioscience.

Techniques: Fluorescence, Expressing, Injection, Activation Assay, Residue, Mutagenesis, Transfection, Derivative Assay, Clone Assay, Construct, Sequencing

Journal: Bioscience Reports

Article Title: Human TRPM8 and TRPA1 pain channels, including a gene variant with increased sensitivity to agonists (TRPA1 R797T), exhibit differential regulation by SRC-tyrosine kinase inhibitor

doi: 10.1042/BSR20140061

Figure Lengend Snippet: ( a–e ) Pre-treatment with PP2 caused a dose-dependent inhibition of agonist -induced elevation in intracellular Ca 2+ (150 nM WS 12). PP3 did not inhibit responses to agonist, ( e ) The IC 50 for inhibition of TRPM8 response was approximately 24 μM PP2, ( f ) Dose–response experiments were repeated on more than three separate occasions.

Article Snippet: The potent TRPM8 agonist WS 12 [(1R,2S)- N -(4-methoxyphenyl)-5-methyl-2-(1-methylethyl)cyclohexanecarbo-xamide], the TRPM8 antagonist AMTB [ N -(3-Aminopropyl)-2-[(3-methylphenyl)methoxy]- N -(2-thienylmethyl)benzamide] hydrochloride and TRPA1 antagonists HC 030031 [2-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-7H-purin-7-yl)- N -(4-isopropylphenyl)acetamide] and A967079 were from Tocris bioscience.

Techniques: Inhibition