为确定H9N2猪流感病毒(H9N2-SIV)通过瞬时受体电位通道M2(TRPM2)介导肺微血管上皮细胞(PMVEC)铁死亡的分子机制,使用H9N2-SIV接种PMVEC,构建TRPM2-siRNA质粒并转染细胞。用透射电镜观察细胞超微结构,用荧光探针法检测活性氧(ROS)、Ca^(...为确定H9N2猪流感病毒(H9N2-SIV)通过瞬时受体电位通道M2(TRPM2)介导肺微血管上皮细胞(PMVEC)铁死亡的分子机制,使用H9N2-SIV接种PMVEC,构建TRPM2-siRNA质粒并转染细胞。用透射电镜观察细胞超微结构,用荧光探针法检测活性氧(ROS)、Ca^(2+)和Fe^(2+);用生化试剂盒检测丙二醛(MDA)和谷胱甘肽(GSH)含量,并通过荧光定量PCR和Western-blot检测葡萄糖调节蛋白78(GRP78)、TRPM2、蛋白激酶R样内质网激酶(PERK)、活化转录因子4(ATF4)、阳离子转运调控样蛋白1(CHAC1)、谷胱甘肽过氧化物酶4(GPX4)的m RNA和蛋白表达水平。结果显示,H9N2-SIV感染可诱导细胞铁死亡,敲低TRPM2可以减少细胞内ROS水平,降低Ca^(2+)、Fe^(2+)及MDA含量,GSH水平明显增加;此外,GRP78、PERK、ATF4、CHAC1 m RNA和蛋白表达水平下调,GPX4的m RNA和蛋白表达水平上调。结果表明,H9N2-SIV感染可诱导细胞铁死亡,其可通过激活TRPM2使Ca^(2+)内流增多,进而激活PERK/ATF4/CHAC1信号通路,加速GSH耗竭,抑制GPX4的活性,促进细胞铁死亡。展开更多
Transient receptor potential melastatin 2(TRPM2) is an important ion channel that represents a potential target for treating injury caused by cerebral ischemia. However, it is unclear whether reducing TRPM2 expression...Transient receptor potential melastatin 2(TRPM2) is an important ion channel that represents a potential target for treating injury caused by cerebral ischemia. However, it is unclear whether reducing TRPM2 expression can help repair cerebral injury, and if so what the mechanism underlying this process involves. This study investigated the protective effect of reducing TRPM2 expression on pheochromocytoma(PC12) cells injured by oxygen-glucose deprivation(OGD). PC12 cells were transfected with plasmid encoding TRPM2 shRNAS, then subjected to OGD by incubation in glucose-free medium under hypoxic conditions for 8 hours, after which the cells were allowed to reoxygenate for 24 hours. Apoptotic cells, mitochondrial membrane potentials, reactive oxygen species levels, and cellular calcium levels were detected using flow cytometry. The relative expression of C-X-C motif chemokine ligand 2(CXCL2), NACHT, LRR, and PYD domain–containing protein 3(NALP3), and caspase-1 were detected using fluorescence-based quantitative reverse transcription-polymerase chain reaction and western blotting. The rates of apoptosis, mitochondrial membrane potentials, reactive oxygen species levels, and cellular calcium levels in the TRPM2-shRNA + OGD group were lower than those observed in the OGD group. Taken together, these results suggest that TRPM2 knockdown reduces OGD-induced neuronal injury, potentially by inhibiting apoptosis and reducing oxidative stress levels, mitochondrial membrane potentials, intracellular calcium concentrations, and NLRP3 inflammasome activation.展开更多
As a crucial signaling molecule, calcium plays a critical role in many physiological and pathological processes by regulating ion channel activity. Recently, one study resolved the structure of the transient receptor ...As a crucial signaling molecule, calcium plays a critical role in many physiological and pathological processes by regulating ion channel activity. Recently, one study resolved the structure of the transient receptor potential melastatin 2(TRPM2) channel from Nematostella vectensis(nvTRPM2). This identified a calcium-binding site in the S2–S3 loop, while its effect on channel gating remains unclear. Here, we investigated the role of this calcium-binding site in both nvTRPM2 and human TRPM2(hTRPM2) by mutagenesis and patch-clamp recording. Unlike hTRPM2, nvT RPM2 cannot be activated by calcium alone. Moreover, the inactivation rate of nvTRPM2 was decreased as intracellular calcium concentration was increased. In addition, our results showed that the four key residues in the calcium-binding site of S2–S3 loop have similar effects on the gating processes of nvTRPM2 and hTRPM2. Among them, the mutations at negatively charged residues(glutamate and aspartate) substantially decreased the currents of nvT RPM2 and hTRPM2. This suggests that these sites are essential for calcium-dependent channel gating. For the charge-neutralizing residues(glutamine and asparagine) in the calcium-binding site, our data showed that glutamine mutating to alanine or glutamate did not affect the channel activity, but glutamine mutating to lysine caused loss of function. Asparagine mutating to aspartate still remained functional, while asparagine mutating to alanine or lysine led to little channel activity. These results suggest that the side chain of glutamine has a less contribution to channel gating than does asparagine. However, our data indicated that both glutamine mutating to alanine or glutamate and asparagine mutating to aspartate accelerated the channel inactivation rate, suggesting that the calcium-binding site in the S2–S3 loop is important for calcium-dependent channel inactivation. Taken together, our results uncovered the effect of four key residues in the S2–S3 loop of TRPM2 on the TRPM2 gating process.展开更多
文摘为确定H9N2猪流感病毒(H9N2-SIV)通过瞬时受体电位通道M2(TRPM2)介导肺微血管上皮细胞(PMVEC)铁死亡的分子机制,使用H9N2-SIV接种PMVEC,构建TRPM2-siRNA质粒并转染细胞。用透射电镜观察细胞超微结构,用荧光探针法检测活性氧(ROS)、Ca^(2+)和Fe^(2+);用生化试剂盒检测丙二醛(MDA)和谷胱甘肽(GSH)含量,并通过荧光定量PCR和Western-blot检测葡萄糖调节蛋白78(GRP78)、TRPM2、蛋白激酶R样内质网激酶(PERK)、活化转录因子4(ATF4)、阳离子转运调控样蛋白1(CHAC1)、谷胱甘肽过氧化物酶4(GPX4)的m RNA和蛋白表达水平。结果显示,H9N2-SIV感染可诱导细胞铁死亡,敲低TRPM2可以减少细胞内ROS水平,降低Ca^(2+)、Fe^(2+)及MDA含量,GSH水平明显增加;此外,GRP78、PERK、ATF4、CHAC1 m RNA和蛋白表达水平下调,GPX4的m RNA和蛋白表达水平上调。结果表明,H9N2-SIV感染可诱导细胞铁死亡,其可通过激活TRPM2使Ca^(2+)内流增多,进而激活PERK/ATF4/CHAC1信号通路,加速GSH耗竭,抑制GPX4的活性,促进细胞铁死亡。
基金supported by grants from the Hong Kong Research Grant Committee Theme-based Research Scheme(TBRS)(T13-706/11,AoE/M-05/12,CUHK2/CRF/11G,CUHK478011,CUHK478413)the National Natural Science Foundation of China(No.31171100)
基金supported by the National Natural Science Foundation of China,Nos.81671532,81771625(to XF)the Jiangsu Provincial Key Medical Discipline of China,No.ZDXKA2016013(to XF)+3 种基金the Jiangsu Provincial Medical Youth Talent of China,No.QNRC2016758(to XF)the Jiangsu Province Women and Children Health Research Project of China,No.F201750(to XF)the Public Health Technology Project of Suzhou City of China,No.SYS201765(to XF)a grant from the Department of Pediatrics Clinical Center of Suzhou City of China,No.Szzx201504(to XF)。
文摘Transient receptor potential melastatin 2(TRPM2) is an important ion channel that represents a potential target for treating injury caused by cerebral ischemia. However, it is unclear whether reducing TRPM2 expression can help repair cerebral injury, and if so what the mechanism underlying this process involves. This study investigated the protective effect of reducing TRPM2 expression on pheochromocytoma(PC12) cells injured by oxygen-glucose deprivation(OGD). PC12 cells were transfected with plasmid encoding TRPM2 shRNAS, then subjected to OGD by incubation in glucose-free medium under hypoxic conditions for 8 hours, after which the cells were allowed to reoxygenate for 24 hours. Apoptotic cells, mitochondrial membrane potentials, reactive oxygen species levels, and cellular calcium levels were detected using flow cytometry. The relative expression of C-X-C motif chemokine ligand 2(CXCL2), NACHT, LRR, and PYD domain–containing protein 3(NALP3), and caspase-1 were detected using fluorescence-based quantitative reverse transcription-polymerase chain reaction and western blotting. The rates of apoptosis, mitochondrial membrane potentials, reactive oxygen species levels, and cellular calcium levels in the TRPM2-shRNA + OGD group were lower than those observed in the OGD group. Taken together, these results suggest that TRPM2 knockdown reduces OGD-induced neuronal injury, potentially by inhibiting apoptosis and reducing oxidative stress levels, mitochondrial membrane potentials, intracellular calcium concentrations, and NLRP3 inflammasome activation.
基金Project supported by the National Natural Science Foundation oX f China(Nos.81371302,81571127,and 31872796)the National Basic Research Program(973)of China(No.2014CB910300)+1 种基金the National Major New Drugs Innovation and Development(No.2018ZX X09711001-004-005)the Zhejiang Provincial Natural Science Foundation of China(Nos.LR16H090001 and LY19B020013)
文摘As a crucial signaling molecule, calcium plays a critical role in many physiological and pathological processes by regulating ion channel activity. Recently, one study resolved the structure of the transient receptor potential melastatin 2(TRPM2) channel from Nematostella vectensis(nvTRPM2). This identified a calcium-binding site in the S2–S3 loop, while its effect on channel gating remains unclear. Here, we investigated the role of this calcium-binding site in both nvTRPM2 and human TRPM2(hTRPM2) by mutagenesis and patch-clamp recording. Unlike hTRPM2, nvT RPM2 cannot be activated by calcium alone. Moreover, the inactivation rate of nvTRPM2 was decreased as intracellular calcium concentration was increased. In addition, our results showed that the four key residues in the calcium-binding site of S2–S3 loop have similar effects on the gating processes of nvTRPM2 and hTRPM2. Among them, the mutations at negatively charged residues(glutamate and aspartate) substantially decreased the currents of nvT RPM2 and hTRPM2. This suggests that these sites are essential for calcium-dependent channel gating. For the charge-neutralizing residues(glutamine and asparagine) in the calcium-binding site, our data showed that glutamine mutating to alanine or glutamate did not affect the channel activity, but glutamine mutating to lysine caused loss of function. Asparagine mutating to aspartate still remained functional, while asparagine mutating to alanine or lysine led to little channel activity. These results suggest that the side chain of glutamine has a less contribution to channel gating than does asparagine. However, our data indicated that both glutamine mutating to alanine or glutamate and asparagine mutating to aspartate accelerated the channel inactivation rate, suggesting that the calcium-binding site in the S2–S3 loop is important for calcium-dependent channel inactivation. Taken together, our results uncovered the effect of four key residues in the S2–S3 loop of TRPM2 on the TRPM2 gating process.
