Pentameric assembly of a neuronal glutamate transporter
Pentameric assembly of a neuronal glutamate transporter
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DOI：
10.1073/pnas.97.15.8641
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发表时间：
2000-07-18
影响因子：
11.1
通讯作者：
Zampighi, GA
中科院分区：
文献类型：
--
作者：
Eskandari, S;Kreman, M;Zampighi, GA
冷冻断裂电子显微镜用于研究人神经元谷氨酸转运体（EAAT 3）的结构。EAAT 3在非洲爪蟾卵母细胞中表达，其功能与同一细胞质膜上转运蛋白的总数相关。当最大电荷响应于一系列跨膜电压脉冲而移动时，测定功能。从一个独特的10 nm的冷冻断裂颗粒的密度，这只出现在EAAT 3表达后的原生质面中确定质膜中的转运蛋白的数量。EAAT 3最大载体介导的电荷和10 nm颗粒的总数之间的线性相关性表明，该颗粒代表质膜中的功能性EAAT 3。EAAT 3在质膜中的横截面积（48 +/- 5 nm（2））预测转运蛋白复合物中有35 +/- 3个跨膜α-螺旋。该信息沿着二级结构模型（6-10个跨膜α-螺旋）表明EAAT 3的寡聚状态，EAAT 3颗粒的形状为五边形，其中可以鉴定出五个结构域。它们表现出五重对称，因为它们看起来像等边五边形，顶点的角度是110度。每个结构域似乎有助于细胞外质量，该质量向细胞外空间中投射约3 nm。所有五个区域的投影都朝着一个穿过五边形中心的轴逐渐变细，使运输机复合体看起来像一个基于五边形的金字塔。EAAT 3的五聚体结构提供了对其作为谷氨酸转运蛋白和谷氨酸门控氯离子通道的功能的新见解。
Freeze-fracture electron microscopy was used to study the structure of a human neuronal glutamate transporter (EAAT3). EAAT3 was expressed in Xenopus laevis oocytes, and its function was correlated with the total number of transporters in the plasma membrane of the same cells. Function was assayed as the maximum charge moved in response to a series of transmembrane voltage pulses. The number of transporters in the plasma membrane was determined from the density of a distinct 10-nm freeze-fracture particle, which appeared in the protoplasmic face only after EAAT3 expression. The linear correlation between EAAT3 maximum carrier-mediated charge and the total number of the 10-nm particles suggested that this particle represented functional EAAT3 in the plasma membrane. The cross-sectional area of EAAT3 in the plasma membrane (48 +/- 5 nm(2)) predicted 35 +/- 3 transmembrane alpha-helices in the transporter complex. This information along with secondary structure models (6-10 transmembrane alpha-helices) suggested an oligomeric state for EAAT3, EAAT3 particles were pentagonal in shape in which five domains could be identified. They exhibited fivefold symmetry because they appeared as equilateral pentagons and the angle at the vertices was 110 degrees. Each domain appeared to contribute to an extracellular mass that projects approximate to 3 nm into the extracellular space. Projections from all five domains taper toward an axis passing through the center of the pentagon, giving the transporter complex the appearance of a penton-based pyramid. The pentameric structure of EAAT3 offers new insights into its function as both a glutamate transporter and a glutamate-gated chloride channel.