Morgan Kevin, Sadofsky Laura Rachel, Morice Alyn Hugh
Centre for Cardiovascular and Metabolic Research, Hull-York Medical School, University of Hull, Daisy Building, Castle Hill Hospital, Cottingham, Hull HU16 5JQ, U.K.
Biosci Rep. 2015 Sep 1;35(5):e00255. doi: 10.1042/BSR20150108.
Multiple mis-sense variants of TRPA1 (transient receptor potential A1) and TRPM8 (transient receptor potential M8) are recorded in the human genome single nt polymorphism (SNP) database, but their potential impact on channel signalling in patho-physiology is not fully explored. Variants, mostly quite rare in the general human population, alter sites in different structural domains of these homo-tetrameric ion channel proteins. The effects of individual SNPs affecting the large cytoplasmic N-terminal domain have not been completely documented for TRPM8 or TRPA1. We examined the Ca(2+) signalling properties of a short-list of eight variants affecting the N-terminal domain by individual expression in human embryonic kidney HEK293 or neuroblastoma (SH-SY5Y) cell lines (four SNP variants for TRPM8: G150R, K423N, R475C, R485W and four for TRPA1: Y69C, A366D, E477K, D573A). These were compared with TRPA1 SNP variants affecting intracellular loops located beyond the N-terminal domain and associated with gain of function (such as increased sensitivity to agonists: TRPA1 R797T and N855S). A substitution in TRPA1 (Y69C) exhibited high expression/sensitivity to agonists (high iCa(2+)max (maximum level of intracellular calcium ion), similar to R797T, but less sensitive than N855S), whereas each of the other non-conservative substitutions exhibited poor signalling response (low iCa(2+)max). Responses from these poorly expressed variants could be salvaged, to different extents, by pre-treating cells with the Src (Src protein) family inhibitor protein kinase inhibitor PP2 (PP2: 4-Amino-3-(4-chlorophenyl)-1-(t-butyl)-1H-pyrazolo[3,4-d]pyrimidine, 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), or with micromolar Zn(2+). The TRPA1 variants and several experimental mutants (TRPA1 Y97F, Y226F and YY654-655FF) expressed poorly in SH-SY5Y compared with HEK293 cells. More in-depth studies are needed to identify SNP variants eliciting gain of function in these TRP (transient receptor potential) channels and to assess their roles in medical conditions.
在人类基因组单核苷酸多态性（SNP）数据库中记录了瞬时受体电位A1（TRPA1）和瞬时受体电位M8（TRPM8）的多个错义变体，但它们在病理生理学中对通道信号传导的潜在影响尚未得到充分研究。这些变体在普通人群中大多相当罕见，它们改变了这些同四聚体离子通道蛋白不同结构域中的位点。对于TRPM8或TRPA1，影响大的胞质N末端结构域的单个SNP的作用尚未完全记录。我们通过在人胚肾HEK293或神经母细胞瘤（SH-SY5Y）细胞系中单独表达，研究了影响N末端结构域的八个变体的短名单的Ca(2+)信号特性（TRPM8的四个SNP变体：G150R、K423N、R475C、R485W和TRPA1的四个：Y69C、A366D、E477K、D573A）。将这些与影响位于N末端结构域之外的细胞内环并与功能增强相关的TRPA1 SNP变体（如对激动剂的敏感性增加：TRPA1 R797T和N855S）进行比较。TRPA1中的一个替代（Y69C）表现出对激动剂的高表达/敏感性（高iCa(2+)max（细胞内钙离子的最大水平），类似于R797T，但比N855S敏感性低），而其他非保守替代中的每一个都表现出差的信号反应（低iCa(2+)max）。通过用Src（Src蛋白）家族抑制剂蛋白激酶抑制剂PP2（PP2：4-氨基-3-(4-氯苯基)-1-(叔丁基)-1H-吡唑并[3,4-d]嘧啶，4-氨基-5-(4-氯苯基)-7-(叔丁基)吡唑并[3,4-d]嘧啶）或微摩尔Zn(2+)预处理细胞，可以在不同程度上挽救这些低表达变体的反应。与HEK293细胞相比，TRPA1变体和几个实验突变体（TRPA1 Y97F、Y226F和YY654-655FF）在SH-SY5Y中的表达较差。需要更深入的研究来鉴定在这些瞬时受体电位（TRP）通道中引发功能增强的SNP变体，并评估它们在医学病症中的作用。
