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首页> 外文期刊>The Biochemical Journal >Expression of gp91(phox)/Nox2 in COS-7 cells: cellular localization of the protein and the detection of outward proton currents
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Expression of gp91(phox)/Nox2 in COS-7 cells: cellular localization of the protein and the detection of outward proton currents

机译：gp91（phox）/ Nox2在COS-7细胞中的表达：蛋白质的细胞定位和外向质子电流的检测

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We have reported previously that gp91(phox), expressed in CHO (Chinese hamster ovary) cells, functions as a voltage-dependent proton channel. However, others have reported that COS-7 cells expressing gp91(phox) failed to exhibit outward proton currents, and concluded that gp91(phox) does not function as a proton channel. To investigate this clear difference in findings, we have examined the expression and cellular localization of the fusion protein EGFP-C-91, in which gp91(phox) is fused to the C-terminus of enhanced green fluorescent protein. EGFP-C-91 was observed in the plasma membrane and intracellular membranes of 30% of the transected COS-7 cells. In the remaining COS-7 cells, EGFP-C-91 was detected in the intracellular membranes only. In CHO cells EGFP-C-91 was present in both the plasma membrane and the intracellular membranes of all transfected cells. Under the whole-ccll configuration. outward currents were recorded from COS-7 cells expressing gp91(phox). These increased in magnitude and lost their 'droop' overtime as the pipette solution equilibrated with the cell cytoplasm (50 min). The threshold activation voltage for the currents was shifted by similar to60 mV for a I unit difference in bath pH. Zn2+ inhibited the outward currents observed in COS-7 cells expressing gp91(phox). The tail current reversal potential was -64 mV at a PHo (external PH) of 8.0, -40 mV at PHo 7.4 and -8 mV at PHo 7.0, indicating that the current arises from the movement of protons. Outward currents were exhibited by 37.5% of the COS-7 cells expressing gp91(phox). Proton currents were recorded following the excision of inside-out patches from cells transfected with gp91(phox). The presence of outward proton currents in COS-7 cells expressing gp91(phox) provides further support for our proposed role for gp91(phox) as the NADPH oxidase-associated proton channel.
机译：我们以前已经报道过，在CHO（中国仓鼠卵巢）细胞中表达的gp91（phox）可以作为电压依赖性质子通道。然而，其他人已经报道表达gp91（phox）的COS-7细胞不能表现出向外的质子电流，并得出结论说gp91（phox）不能作为质子通道。为了调查这一发现的明显差异，我们检查了融合蛋白EGFP-C-91的表达和细胞定位，其中gp91（phox）与增强型绿色荧光蛋白的C末端融合。在被切除的COS-7细胞的30​​％的质膜和细胞内膜中观察到EGFP-C-91。在其余的COS-7细胞中，仅在细胞内膜中检测到EGFP-C-91。在CHO细胞中，所有转染细胞的质膜和细胞内膜均存在EGFP-C-91。在整个ccll配置下。记录从表达gp91（phox）的COS-7细胞流出的电流。随着移液溶液与细胞质平衡（50分钟），这些溶液的量级增加并失去了其“下降”时间。对于电流pH的I单位差异，电流的阈值激活电压偏移了约60 mV。 Zn2 +抑制表达gp91（phox）的COS-7细胞中的外向电流。在8.0的PHo（外部PH）下，尾电流反向电位为-64 mV，在PHo 7.4时为-40 mV，在PHo 7.0时为-8 mV，表明电流是由质子的运动引起的。 37.5％的表达gp91（phox）的COS-7细胞表现出外向电流。在从用gp91（phox）转染的细胞中切除内向外的斑块后，记录质子电流。表达gp91（phox）的COS-7细胞中外向质子电流的存在为我们提议的gp91（phox）作为NADPH氧化酶相关质子通道的作用提供了进一步的支持。

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