Dynamics of membrane channels as revealed by freeze-fracture replica labeling

谷氨酸在体内和体外都能引起星形胶质细胞肿胀。虽然星形胶质细胞上表达了几种不同类型的离子型和代谢型谷氨酸受体，但它们在谷氨酸诱导的细胞肿胀中的功能作用尚不清楚。利用冷冻断裂复制免疫电子显微镜，我们发现代谢性谷氨酸受体3(MGluR3)和水通道水通道蛋白4(AQP4)在星形胶质细胞膜的一个特殊的膜结构域上有独特的共定位，称为颗粒正交阵列(OAPs)。将mGluR3和AQP4的cDNA导入中国仓鼠卵巢细胞，观察mGluR3和AQP4是否组装成OAP。复制免疫电子显微镜显示，两种分子都高度集中在类OAPs结构中。MGluR3、谷氨酸和DCG-IV激动剂破坏mGluR3和AQP4的共定位，并降低这些细胞内OAPs样结构的密度。激动剂在原位星形胶质细胞中也观察到了类似的作用。对转基因细胞的功能分析表明，激动剂增加了细胞膜的透水性，导致细胞肿胀，可能是以cAMP依赖的方式。这些发现表明，mGluR3和AQP4组装形成OAP，结构和功能状态的变化受mGluR3的活性控制。因此，我们提出了星形胶质细胞渗透容量调节的mGluR3-AQP4偶联途径。

Glutamate is known to cause swelling of astrocytes both in situ and in vitro. Although several different types of ionotropic and metabotropic glutamate receptors are expressed on astrocytes, their functional role in the glutamate-induced cell swelling is poorly understood. Using freeze-fracture replica immunoelectron microscopy, we have found a distinctive co-localization of metabotropic glutamate receptor 3 (mGluR3) and water channel aquaporn 4 (AQP4) on a specialized membrane domain, called orthogonal arrays of particles (OAPs), of astrocyte membranes in situ. To examine whether mGluR3 and AQP4 assemble to form OAPs, their cDNAs were introduced into Chinese hamster ovary cells. Replica immunoelectron microscopy revealed that both molecules were highly concentrated in OAPs-like structure. Agonists of mGluR3, glutamate and DCG-IV, disrupted co-localization of mGluR3 and AQP4, and decreased the density of OAPs-like structure in these cells. Similar effects of the agonists were also observed in astrocytes in situ. Functional analysis using the transfected cells showed that the agonists increased water permeability across cell membranes leading to cell swelling, probably in a cAMP-dependent manner. These findings suggest that mGluR3 and AQP4 assemble to form OAPs, and changes in the structural and functional states are governed by the activity of mGluR3. Therefore, we propose a pathway, mGluR3-AQP4 coupling, of osmotic cell volume regulation, of astrocytes.