The molecular composition of square arrays.
ABSTRACT 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.