Morgan Kevin, Sadofsky Laura R, Crow Christopher, Morice Alyn H
*Respiratory Medicine, Centre for Cardiovascular and Metabolic Research, Daisy Building, University of Hull and Hull York Medical School, Castle Hill Hospital, Cottingham HU16 5JQ, East Yorkshire, U.K.
Biosci Rep. 2014 Aug 6;34(4):e00131. doi: 10.1042/BSR20140061.
TRPM8 (transient receptor potential M8) and TRPA1 (transient receptor potential A1) are cold-temperature-sensitive nociceptors expressed in sensory neurons but their behaviour in neuronal cells is poorly understood. Therefore DNA expression constructs containing human TRPM8 or TRPA1 cDNAs were transfected into HEK (human embryonic kidney cells)-293 or SH-SY5Y neuroblastoma cells and G418 resistant clones analysed for effects of agonists and antagonists on intracellular Ca2+ levels. Approximately 51% of HEK-293 and 12% of SH-SY5Y cell clones expressed the transfected TRP channel. TRPM8 and TRPA1 assays were inhibited by probenecid, indicating the need to avoid this agent in TRP channel studies. A double-residue mutation in ICL-1 (intracellular loop-1) of TRPM8 (SV762,763EL, mimicking serine phosphorylation) or one in the C-terminal tail region (FK1045,1046AG, a lysine knockout) retained sensitivity to agonists (WS 12, menthol) and antagonist {AMTB [N-(3-Aminopropyl)-2-[(3-methylphenyl)methoxy]-N-(2-thienylmethyl)benzamide]}. SNP (single nucleotide polymorphism) variants in TRPA1 ICL-1 (R797T, S804N) and TRPA1 fusion protein containing C-terminal (His)10 retained sensitivity to agonists (cinnamaldehyde, allyl-isothiocyanate, carvacrol, eugenol) and antagonists (HC-030031, A967079). One SNP variant, 797T, possessed increased sensitivity to agonists. TRPA1 became repressed in SH-SY5Y clones but was rapidly rescued by Src-family inhibitor PP2 [4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine]. Conversely, TRPM8 in SH-SY5Y cells was inhibited by PP2. Further studies utilizing SH-SY5Y may identify structural features of TRPA1 and TRPM8 involved in conferring differential post-translational regulation.
瞬时受体电位M8（TRPM8）和瞬时受体电位A1（TRPA1）是在感觉神经元中表达的冷敏伤害感受器，但它们在神经元细胞中的行为尚不清楚。因此，将含有人类TRPM8或TRPA1 cDNA的DNA表达构建体转染到HEK（人胚肾细胞）-293或SH-SY5Y神经母细胞瘤细胞中，并分析G418抗性克隆中激动剂和拮抗剂对细胞内Ca2+水平的影响。约51%的HEK-293细胞克隆和12%的SH-SY5Y细胞克隆表达了转染的TRP通道。丙磺舒可抑制TRPM8和TRPA1检测，这表明在TRP通道研究中需要避免使用该药物。TRPM8的胞内环-1（ICL-1）中的双残基突变（SV762,763EL，模拟丝氨酸磷酸化）或C末端尾部区域中的一个突变（FK1045,1046AG，赖氨酸敲除）对激动剂（WS 12、薄荷醇）和拮抗剂{AMTB [N-(3-氨丙基)-2-[(3-甲基苯基)甲氧基]-N-(2-噻吩基甲基)苯甲酰胺]}仍保持敏感性。TRPA1的ICL-1中的单核苷酸多态性（SNP）变体（R797T、S804N）和含有C末端（His）10的TRPA1融合蛋白对激动剂（肉桂醛、烯丙基异硫氰酸酯、香芹酚、丁香酚）和拮抗剂（HC-030031、A967079）仍保持敏感性。一种SNP变体797T对激动剂的敏感性增加。TRPA1在SH-SY5Y克隆中受到抑制，但Src家族抑制剂PP2 [4-氨基-5-(4-氯苯基)-7-(叔丁基)吡唑并[3,4-d]嘧啶]可迅速使其恢复。相反，PP2可抑制SH-SY5Y细胞中的TRPM8。利用SH-SY5Y进行的进一步研究可能会确定TRPA1和TRPM8中参与赋予翻译后差异调控的结构特征。
