*Department of Pharmacology, Infectious Disease Medical Research Center, College of Medicine, Hallym University, Chuncheon 200-702, South Korea.
Biochem J. 2013 Nov 1;455(3):319-27. doi: 10.1042/BJ20130303.
TRPM2 (transient receptor potential melastatin 2) is a non-selective Ca2+-permeable cation channel activated by ADPR (adenosine diphosphoribose) and H2O2. It is widely expressed in mammalian cells and plays an important role in the regulation of various cell functions. However, the mechanisms of TRPM2 channel activation are not fully understood. Previously, we reported that TRPM2 channel activation is induced by high intracellular Cl- concentration. In the present study, we investigated the functional role of Lys1110 in the membrane-proximal C-terminal region by site-directed mutagenesis. Replacement of the positively charged amino acid lysine (Lys1110) with the neutrally charged amino acid asparagine (K1110N) or the negatively charged amino acid glutamic acid (K1110E) generated mutants that failed to induce an increase in free cytosolic calcium concentration ([Ca2+]i) not only by intracellular injection of Cl-, but also by H2O2 or ADPR. However, a mutant generated by replacing the lysine residue with a positively charged amino acid arginine (K1110R) displayed channel activity similar to wild-type TRPM2. Interestingly, in the K1107N/K1110N double-point mutant, the impaired function of the K1110N mutant in response to ADPR and H2O2, but not to Cl-, was recovered. There were no changes in protein expression, membrane trafficking and oligomerization of the mutant channels. The extent of [Ca2+]i increase by H2O2 in HEK (human embryonic kidney)-293 cells expressing TRPM2 mutants was well correlated with the degree of susceptibility to H2O2-induced cell death. These results display the crucial role of a positively charged amino acid residue at position 1110 for TRPM2 channel activity.
瞬时受体电位 melastatin 2 型（TRPM2）是一种非选择性 Ca2+通透阳离子通道，可被 ADPR（二磷酸腺苷核糖）和 H2O2 激活。它广泛表达于哺乳动物细胞，在调节各种细胞功能方面发挥着重要作用。然而，TRPM2 通道激活的机制尚未完全阐明。先前我们报道过，高细胞内 Cl-浓度可诱导 TRPM2 通道激活。在本研究中，我们通过定点突变研究了膜近端 C 末端区域赖氨酸 1110 （Lys1110）的功能作用。用不带电荷的氨基酸天冬酰胺（K1110N）或带负电荷的氨基酸谷氨酸（K1110E）替换带正电荷的氨基酸赖氨酸（Lys1110），生成的突变体不仅不能通过细胞内 Cl-注入，也不能通过 H2O2 或 ADPR 诱导游离细胞内钙浓度（[Ca2+]i）增加。然而，用带正电荷的氨基酸精氨酸（K1110R）替换赖氨酸残基的突变体显示出与野生型 TRPM2 相似的通道活性。有趣的是，在 K1107N/K1110N 双点突变体中，对 ADPR 和 H2O2 反应的 K1110N 突变体的功能障碍（但对 Cl-无反应）得到了恢复。突变通道的蛋白表达、膜转运和寡聚化没有变化。表达 TRPM2 突变体的 HEK（人胚肾）-293 细胞中 H2O2 引起的 [Ca2+]i 增加程度与对 H2O2 诱导细胞死亡的敏感性程度密切相关。这些结果表明，第 1110 位带正电荷的氨基酸残基对 TRPM2 通道活性起着至关重要的作用。
