14. Bergmeyer, H. U. Methods of Enzymatic Analysis (Verlag Chemie, Weinheim/Bergstr., 1974). 15. Kushnir, M. M., Komaromy-Hiller, G., Shushan, B.,Urry, F. M. & Roberts, W. L. Analysis of dicarboxylic acids by tandem mass spectrometry. High-throughput quantitative measurement of methylmalonic acid in serum, plasma, and urine. Clin. Chem. 47, 1993–2002 (2001). 16. Martin, M., Ferrier, B. & Baverel, G. Transport and utilization of alpha-ketoglutarate by the rat kidney in vivo. Pflugers Arch. 413, 217–224 (1989). 17. Gullans, S. R., Kone, B. C., Avison, M. J. & Giebisch, G. Succinate alters respiration, membrane potential, and intracellular K þ in proximal tubule. Am. J. Physiol. 255, F1170–F1177 (1988). 18. Gullans, S. R., Brazy, P. C., Dennis, V. W. & Mandel,L. J. Interactions between gluconeogenesisand sodium transport in rabbit proximal tubule. Am. J. Physiol. 246, F859–F869 (1984). 19. Krebs, H. A. Rate control of the tricarboxylic acid cycle. Adv. Enzyme Regul. 8, 335–353 (1970). 20. Hems, D. A. & Brosnan, J. T. Effects of ischaemia on content of metabolites in rat liver and kidney in vivo. Biochem. J. 120, 105–111 (1970). 21. Pan, L. et al. Critical roles of a cyclic AMP responsive element and an E-box in regulation of mouse renin gene expression. J. Biol. Chem. 276, 45530–45538 (2001). 22. Hackenthal, E., Paul, M., Ganten, D. & Taugner, R. Morphology, physiology, and molecular biology of renin secretion. Physiol. Rev. 70, 1067–1116 (1990). 23. An, S. et al. Identification and characterization of a melanin-concentrating hormone receptor. Proc. Natl Acad. Sci. USA 98, 7576–7581 (2001). 24. Brandish, P. E., Hill, L. A., Zheng, W. & Scolnick, E. M. Scintillation proximity assayof inositol phosphates in cell extracts: high-throughput measurement of G-protein-coupled receptor activation. Anal. Biochem. 313, 311–318 (2003). 25. Chuang, P. T., Kawcak, T. & McMahon, A. P. Feedback control of mammalian Hedgehog signaling by the Hedgehog-binding protein, Hip1, modulates Fgf signaling during branching morphogenesis of the lung. Genes Dev. 17, 342–347 (2003). 26. Palczewski, K. et al. Crystal structure of rhodopsin: A G protein-coupled receptor. Science 289, 739–745 (2000). 27. Krege, J. H., Hodgin, J. B., Hagaman, J. R. & Smithies, O. A noninvasive computerized tail-cuff system for measuring blood pressure in mice. Hypertension 25, 1111–1115 (1995). 28. Sugiyama, F. et al. QTL associated with blood pressure, heart rate, and heart weight in CBA/CaJ and BALB/cJ mice. Physiol. Genomics 10, 5–12 (2002). 29. Silva, A. P. et al. Bilateral nephrectomy delays gastric emptying of a liquid meal in awake rats. Ren. Fail. 24, 275–284 (2002). 30. Woronicz,J. D., Gao, X., Cao, Z., Rothe,M. & Goeddel, D. V. IkB kinase-b: NF-kB activation and complex formation with IkB kinase-a and NIK. Science 278, 866–869 (1997). Supplementary Information accompanies the paper on www.nature.com/nature. Acknowledgements We thank J. Reagan for critical comments during ligand identification; L. Yang for sharing reagents for in situ hybridization; G. Cutler, J. Knop, H. Baribault, J. Ma, S.-C. Miao, W. Inman, C. Ogden, S. Shuttleworth and M. Rich for providing support and discussions; and D. Goeddel, B. Lemmon and T. Hoey for critical reading of the manuscript. Competing interests statement The authors declare that they have no competing financial interests. Correspondence and requests for materials should be addressed to L.L. (ling@tularik.com). .............................................................. Aquaporin-0 membrane junctions reveal the structure of a closed water pore Tamir Gonen 1 , Piotr Sliz 2 , Joerg Kistler 3 , Yifan Cheng 1 & Thomas Walz 1 1 Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA 2 Howard Hughes Medical Institute and Children’s Hospital Laboratory of Molecular Medicine, 320 Longwood Avenue, and Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA 3 School of Biological Sciences, University of Auckland, Auckland PO Box 92019, New Zealand ............................................................................................................................................................................. The lens-specific water pore aquaporin-0 (AQP0) is the only aquaporin known to form membrane junctions in vivo 1 . We show here that AQP0 from the lens core, containing some carboxy- terminally cleaved AQP0 2,3 , forms double-layered crystals that recapitulate in vivo junctions. We present the structure of the AQP0 membrane junction as determined by electron crystal- lography. The junction is formed by three localized interactions between AQP0 molecules in adjoining membranes, mainly mediated by proline residues conserved in AQP0s from different species but not present in most other aquaporins. Whereas all previously determined aquaporin structures show the pore in an open conformation 4–9 , the water pore is closed in AQP0 junc- tions. The water pathway in AQP0 also contains an additional pore constriction, not seen in other known aquaporin struc- tures 4–9 , which may be responsible for pore gating. AQP0 is a member of the aquaporin family, members of which form pores that are either highly selective for water (aquaporins) or also permeable to other small neutral solutes such as glycerol (aquaglyceroporins) (reviewed in ref. 10). To date, the atomic structures of three aquaporins have been determined (AQP1 4–6 , GlpF 7,8 and AQPZ 9 ). Sequence alignment shows AQP0 to be closely related to the pure water channel AQP1 (43.6% identity, 62.6% similarity). The presence of His 172, a residue conserved only in aquaporins but substituted in aquaglyceroporins, also suggests that AQP0 forms a pure water pore. AQP0 water permeability at neutral pH is approximately 40 times lower than that of AQP1 11 , but AQP0 water conductance doubles under mildly acidic conditions 12 . In the case of aquaporins in plant roots, a pH-dependent closure of the water pores has been reported 13 . Thus, evidence suggests that certain aquaporin pores are gated. AQP0 water pores are considered essential for the lens micro- circulation system, proposed to supply deeper-lying fibre cells with nutrients and to clear waste products 14,15 . Unlike all other aqua- porins, AQP0 is also present in membrane junctions. It is particu- larly enriched in the 11–13 nm thin junctions between lens fibre cells, that feature square AQP0 arrays 1 . Atomic force microscopy analysis of in vitro reconstituted AQP0 two-dimensional crystals demonstrated these crystals to be double-layered 16 . Using AQP0 from the core of sheep lenses, where some of the AQP0 is proteolytically cleaved near the C terminus at various sites in an age-dependent manner 2,3 , we reproduced the double-layered two-dimensional crystals 16,17 . When core AQP0 was reconstituted at a lipid-to-protein ratio of 0.25 (w/w), large membrane sheets formed (.6 mm) that in some cases showed two parallel edges, revealing them to be double-layered (Fig. 1a). The crystals showing p422 symmetry had lattice constants of a ¼ b ¼ 65.5 A ˚ and a thickness of 11 nm (Fig. 2a), the same dimensions as thin junctions in the lens 1 . Double-layered AQP0 two-dimensional crystals are therefore likely to recapitulate thin lens fibre cell junctions. Electron diffraction analysis of AQP0 crystals (tilted to an angle of up to 708) produced strong diffraction spots to 3 A ˚ resolution in all directions (Fig. 1b, c; the electron crystallographic data are summarized in Table 1). As the crystal structure of the homologous bovine AQP1 was available 5 , we determined the structure of the AQP0 membrane junction by molecular replacement, thus avoiding the cumbersome and time-consuming process of collecting high- resolution images of tilted specimens. Sequencing of cloned sheep AQP0 showed an identical amino acid sequence to bovine AQP0 18 , with the exception of three conservative (S20T, M90V and S240T) and one non-conservative substitutions (C14F). Our model (Fig. 2a) shows unique features that enable AQP0 to form membrane junction interactions. These differ from those previously suggested based on atomic force microscopy data 16 . The extracellular surface of AQP0 is rather flat and the interactions are mediated by direct contacts of the corresponding loops in the opposing AQP0 molecules (Fig. 2a). Loop C, connecting a-helices three and four, is significantly shorter than in AQP1 and GlpF. The shortened loop C (also seen in AQP2, AQP5, AQP6 and AQP8) is crucial for the formation of the very tight AQP0 junction, as it allows three specific interactions to be formed that are mediated almost exclusively by proline residues. The most striking interaction involves Pro 38, in extracellular loop A. The proline residues (Pro 38) from eight symmetry-related AQP0 molecules in the stacked tetramers come together to form a letters to nature NATURE | VOL 429 | 13 MAY 2004 | www.nature.com/nature 193 ©2004 Nature Publishing Group