van Hoek A N, Yang B, Kirmiz S, Brown D
Renal Unit and Department of Pathology, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA.
J Membr Biol. 1998 Oct 1;165(3):243-54. doi: 10.1007/s002329900438.
Several studies suggest that aquaporin water channels can be identified in membranes by freeze-fracture electron microscopy. For this report, Chinese Hamster ovary cells were stably transfected with cDNAs encoding aquaporins 1-5. Measurement of the osmotic water permeability of the cells confirmed that functional protein was expressed and delivered to the plasma membrane. By freeze-fracture electron microscopy, a 20% increase in intramembrane particle (IMP) density was found in plasma membranes of cells expressing AQP2, 3 and 5, and a 100% increase was measured in AQP1-expressing cells, when compared to mock-transfected cells. On membranes of cells expressing AQP4, large aggregates of IMPs were organized into orthogonal arrays, which occupied 10-20% of the membrane surface. IMP aggregates were never seen in AQP2-transfected cells. Hexagonally packed IMP clusters were detected in approximately 5% of the membranes from AQP3-expressing cells. Particle size-distribution analysis of rotary shadowed IMPs showed a significant shift from 13. 5 (control cells) to 8.5 nm or less in AQP-expressing cells; size distribution analysis of unidirectionally shadowed IMPs also showed a significant change when compared to control. Some IMPs in AQP expressing cells had features consistent with the idea that aquaporins are assembled as tetramers. The results demonstrate that in transfected CHO cells, AQP transfection modifies the general appearance and number of IMPs on the plasma membrane, and show that only AQP4 assembles into well-defined IMP arrays.
多项研究表明，水通道蛋白水通道可通过冷冻蚀刻电子显微镜在膜中得以识别。在本报告中，用编码水通道蛋白1 - 5的cDNA稳定转染了中国仓鼠卵巢细胞。对细胞的渗透水通透性进行测量，证实表达了功能性蛋白并转运至质膜。通过冷冻蚀刻电子显微镜观察发现，与mock转染细胞相比，表达AQP2、3和5的细胞的质膜中膜内颗粒（IMP）密度增加了20%，而表达AQP1的细胞中IMP密度增加了100%。在表达AQP4的细胞的膜上，IMP的大聚集体排列成正交阵列，占据膜表面的10 - 20%。在AQP2转染的细胞中从未见过IMP聚集体。在约5%的表达AQP3的细胞的膜中检测到六边形排列的IMP簇。对旋转阴影处理的IMP进行粒度分布分析显示，与对照细胞（13.5nm）相比，表达AQP的细胞中显著转变为8.5nm或更小；与对照相比，单向阴影处理的IMP的粒度分布分析也显示出显著变化。表达AQP的细胞中的一些IMP具有与水通道蛋白组装成四聚体这一观点相符的特征。结果表明，在转染的CHO细胞中，AQP转染改变了质膜上IMP的总体外观和数量，并表明只有AQP4组装成明确的IMP阵列。
