Modulation of the voltage-dependent anion channel (VDAC) by glutamate
Modulation of the voltage-dependent anion channel (VDAC) by glutamate
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DOI：
10.1023/a:1005670527340
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发表时间：
2000-12-01
影响因子：
3
通讯作者：
Shoshan-Barmatz, V
中科院分区：
文献类型：
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作者：
Gincel, D;Silberberg, SD;Shoshan-Barmatz, V
电压依赖性阴离子通道（VDAC），也称为线粒体孔蛋白，是可渗透阴离子、阳离子、ATP 和其他代谢物的大通道。除了羟基磷灰石柱之外，还使用反应性红色琼脂糖柱从羊脑突触体或大鼠肝线粒体中纯化 VDAC。红色琼脂糖柱允许进一步纯化（98%），蛋白质浓度超过十倍，降低Triton X-100浓度，和/或用其他去污剂（例如Nonidet P-40或辛基葡萄糖苷）替代Triton X-100。这种纯化的 VDAC 重组为平面脂质双层，在 1 M 氯化钠中、10 mV 下具有 3.7 +/- 0.1 nS 的单一最大电导，并且可渗透大阳离子和阴离子。在最大传导状态下，Na+、乙酰胆碱(+)、多巴胺(+)和谷氨酸(-)相对于Cl-的渗透率估计分别为0.73、0.6、0.44和0.4。相反，在亚导状态下，谷氨酸盐是不可渗透的，而对乙酰胆碱(+)的相对渗透性增加，而对多巴胺(+)的相对渗透性保持不变。在渗透性实验中使用的高浓度（0.1-0.5 M）下，谷氨酸消除了 VDAC 通道电导电压依赖性的钟形形状。研究发现，在 1 M 氯化钠存在的情况下，浓度为 1 至 20 mM 的谷氨酸可调节 VDAC 通道活性。在单通道实验中，在低电压（+/-10 mV）下，谷氨酸会引起通道在完全打开状态和长期低传导状态或短期关闭状态之间快速波动。在低电压下，通道活性的谷氨酸修饰取决于电压，需要通道短时间（20-60 秒）暴露于高膜电位。谷氨酸的作用是特定的，因为它是在 1 M 氯化钠存在的情况下观察到的，而用天冬氨酸或 GABA 则无法获得。这些结果表明 VDAC 具有调节其活性的特定谷氨酸结合位点。
The voltage-dependent anion channel (VDAC), also known as mitochondrial porin, is a large channel permeable to anions, cations, ATP, and other metabolites. VDAC was purified from sheep brain synaptosomes or rat liver mitochondria using a reactive red-agarose column, in addition to the hydroxyapatitate column. The red-agarose column allowed further purification lover 98%), concentration of the protein over ten-fold, decreasing Triton X-100 concentration, and/or replacing Triton X-100 with other detergents, such as Nonidet P-40 or octylglucoside. This purified VDAC reconstituted into planar-lipid bilayer, had a unitary maximal conductance of 3.7 +/- 0.1 nS in 1 M NaCl, at 10 mV and was permeable to both large cations and anions. In the maximal conducting state, the permeability ratios for Na+, acetylcholine(+), dopamine(+) and glutamate(-), relative to Cl-, were estimated to be 0.73, 0.6, 0.44, and 0.4, respectively. In contrast, in the subconducting state, glutamate- was impermeable, while the relative permeability to acetylcholine(+) increased and to dopamine(+) remained unchanged. At the high concentrations (0.1-0.5 M) used in the permeability experiments, glutamate eliminated the bell shape of the voltage dependence of VDAC channel conductance. Glutamate at concentrations of 1 to 20 mM, in the presence of 1 M NaCl, was found to modulate the VDAC channel activity. In single-channel experiments, at low voltages (+/-10 mV), glutamate induced rapid fluctuations of the channel between the fully open state and long-lived low-conducting states or short-lived closed state. Glutamate modification of the channel activity, at low voltages, is dependent on voltage, requiring short-time (20-60 sec) exposure of the channel to high membrane potentials. The effect of glutamate is specific, since it was observed in the presence of 1 M NaCl and it was not obtained with aspartate or GABA. These results suggest that VDAC possesses a specific glutamate-binding site that modulates its activity.