Maintenance of cell volume against osmotic change is crucial for proper cell functions. Leucine-rich repeat-containing 8 proteins are anion-selective channels that extrude anions to decrease the cell volume on cellular swelling. Here, we present the structure of human leucine-rich repeat-containing 8A, determined by single-particle cryo-electron microscopy. The structure shows a hexameric assembly, and the transmembrane region features a topology similar to gap junction channels. The LRR region, with 15 leucine-rich repeats, forms a long, twisted arc. The channel pore is located along the central axis and constricted on the extracellular side, where highly conserved polar and charged residues at the tip of the extracellular helix contribute to permeability to anions and other osmolytes. Two structural populations were identified, corresponding to compact and relaxed conformations. Comparing the two conformations suggests that the LRR region is flexible and mobile, with rigid-body motions, which might be implicated in structural transitions on pore opening.
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We thank the members of the Nureki laboratory, the Kikkawa laboratory, and the RIKEN Center for Biosystems Dynamics Research, especially M. Takemoto and R. Taniguchi (Nureki laboratory) for assistance with the manuscript preparation and H. Shigematsu (RIKEN) for microscope maintenance. We also thank Y. Araiso (Kyoto Sangyo University) for instructions regarding the digitonin preparation; K. Touhara (The Rockefeller University) for advice about the anti-GFP nanobody preparation; and K. Iwasaki, N. Miyazaki, and A. Kawamoto (Osaka University) for optimization of the cryo-EM experiment. This work was supported by a grant from the National Key R&D Program of China (2016YFA0502800); by the RIKEN Pioneering Project ‘Dynamic Structural Biology’; by the Platform for Drug Discovery, Informatics and Structural Life Science from the Japan Agency for Medical Research and Development (AMED) to O.N.; by AMED (grant no. JP17gm5010001) to H.I.; by a MEXT Grant-in-Aid for Specially Promoted Research (grant no. 16H06294) to O.N.; by the National Natural Science Foundation of China (grant no. 31571083) to Z.Y.; by the Program for Professor of Special Appointment (Eastern Scholar of Shanghai, TP2014008) to Z.Y.; by the Shanghai Rising-Star Program (4QA1400800) to Z.Y.; by the Young 1000 Talent Program of China to Z.Y.; and by JSPS KAKENHI (grant nos. 17J06101 to G.K.; 17K15086 to K.W.; 25221302 to H.I.; 17H03640 to R.I.; 24227004 to O.N.). This research was also supported by the Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) from AMED (grant nos. JP18am0101072, JP18am0101082 and JP18am0101115).