Sorbo Jan Gunnar, Moe Svein Erik, Ottersen Ole Petter, Holen Torgeir
Center for Molecular Biology and Neuroscience (CMBN), and Nordic Center of Excellence for Research in Water Imbalance Related Disorders (WIRED), University of Oslo, Oslo 0317, Norway.
Biochemistry. 2008 Feb 26;47(8):2631-7. doi: 10.1021/bi702146k. Epub 2008 Feb 2.
Square arrays are prominent structures in plasma membranes of brain, muscle, and kidneys with an unknown function. So far, the analysis of these arrays has been restricted to freeze fracture preparations, which have shown square arrays to contain the water channel Aquaporin-4 (AQP4). Using Blue-Native PAGE immunoblots, we provide evidence that higher-order AQP4 complexes correspond to square arrays, with the AQP4 isoform M23 playing a dominant role. Our data are consistent with the idea that square arrays consist of aggregates of AQP4 tetramers complexed with multiples of dimers. By comparison, Aquaporin-1 and Aquaporin-9 form tetramers, but not higher-order complexes. AQP4 square arrays are stable under several biochemical purification steps. Analyzing the internal composition of the higher-order complexes by 2D gels, we demonstrate that the square arrays in addition to M23 also invariably contain AQP4, M1, and a novel AQP4 isoform that we call Mz. The visualization AQP4 square arrays by a rapid, biochemical assay provides new insight in the molecular organization of square arrays and gives further proof of the heterogeneity of AQP4 square arrays in vivo.
方形阵列是大脑、肌肉和肾脏质膜中的显著结构，其功能未知。到目前为止，对这些阵列的分析仅限于冷冻断裂标本，这些标本显示方形阵列包含水通道水通道蛋白4（AQP4）。使用蓝色非变性聚丙烯酰胺凝胶电泳免疫印迹，我们提供证据表明高阶AQP4复合物对应于方形阵列，其中AQP4亚型M23起主导作用。我们的数据与方形阵列由与多个二聚体复合的AQP4四聚体聚集体组成的观点一致。相比之下，水通道蛋白1和水通道蛋白9形成四聚体，但不形成高阶复合物。AQP4方形阵列在几个生化纯化步骤中是稳定的。通过二维凝胶分析高阶复合物的内部组成，我们证明方形阵列除了M23外，还总是包含AQP4、M1和一种我们称为Mz的新型AQP4亚型。通过快速生化分析可视化AQP4方形阵列，为方形阵列的分子组织提供了新的见解，并进一步证明了体内AQP4方形阵列的异质性。
