Two mechanisms have been proposed to account for solute permeation of lipid bilayers. Partitioning into the hydrophobic phase of the bilayer, followed by diffusion, is accepted by many for the permeation of water and other small neutral solutes, but transient pores have also been proposed to account for both water and ionic solute permeation. These two mechanisms make distinctively different predictions about the permeability coefficient as a function of bilayer thickness. Whereas the solubility-diffusion mechanism predicts only a modest variation related to bilayer thickness, the pore model predicts an exponential relationship. To test these models, we measured the permeability of phospholipid bilayers to protons, potassium ions, water, urea, and glycerol. Bilayers were prepared as liposomes, and thickness was varied systematically by using unsaturated lipids with chain lengths ranging from 14 to 24 carbon atoms. The permeability coefficient of water and neutral polar solutes displayed a modest dependence on bilayer thickness, with an approximately linear fivefold decrease as the carbon number varied from 14 to 24 atoms. In contrast, the permeability to protons and potassium ions decreased sharply by two orders of magnitude between 14 and 18 carbon atoms, and leveled off, when the chain length was further extended to 24 carbon atoms. The results for water and the neutral permeating solutes are best explained by the solubility-diffusion mechanism. The results for protons and potassium ions in shorter-chain lipids are consistent with the transient pore model, but better fit the theoretical line predicted by the solubility-diffusion model at longer chain lengths.

In all living cells, coordination of solute and water movement across cell membranes is of critical importance for osmotic balance. The current concept is that these processes are of distinct biophysical nature. Here we report the expression cloning of a liver cDNA encoding a unique promiscuous solute channel (AQP9) that confers high permeability for both solutes and water. AQP9 mediates passage of a wide variety of non-charged solutes including carbamides, polyols, purines, and pyrimidines in a phloretin-and mercury-sensitive manner, whereas amino acids, cyclic sugars, Na ؉ , K ؉ , Cl ؊ , and deprotonated monocarboxylates are excluded. The properties of AQP9 define a new evolutionary branch of the major intrinsic protein family of aquaporin proteins and describe a previously unknown mechanism by which a large variety of solutes and water can pass through a single pore, enabling rapid cellular uptake or exit of metabolites with minimal osmotic perturbation.Transport of solutes such as ions, nutrients, neurotransmitters, and metabolic waste products across cell membranes is of fundamental importance to all mammalian cells. Despite the identification of many selective solute transporters and water channels (1-4), it has remained unclear how transport of large amounts of solutes is coordinated with water movement in metabolically highly active cells such as hepatocytes, spermatocytes, neurons, and glia. The liver is a major site of production and elimination of metabolites such as urea, nucleotides, and ketone bodies, and substantial amounts of these solutes must rapidly cross the hepatocyte plasma membrane with minimal osmotic perturbation (5). In testis, a solute transport mechanism is presumably required to supply nutrients to rapidly growing spermatocytes and to provide an exit pathway for metabolites. In brain, regulation of solute transport is critical because osmolality changes in extracellular fluids can affect neuronal cell function (6).Among metabolically active tissues, liver was selected as a target for expression cloning of a new solute-transporting protein because a phloretin-sensitive urea exit mechanism had been described (7-9). MATERIALS AND METHODSExpression Cloning-Total RNA was extracted from rats fed a high protein diet (50%, w/w) for 2 weeks. Poly(A) ϩ RNA purified by oligo(dT) chromatography was size-fractionated by preparative agarose gel electrophoresis (30). Specific fractions were screened for 1 mM [14 C]urea uptake activity in RNA-injected Xenopus oocytes (4, 30). A directional cDNA library was constructed from the positive fraction by using the SuperScript Plasmid System (Life Technologies, Inc.), and cDNA clones were screened for urea uptake (4). Northern Analysis and in Situ Hybridization-Poly(A)ϩ RNA (3 g) from rat tissues was electrophoresed in a formaldehyde-agarose gel and transferred to a nylon membrane. The filter was probed with 32 Plabeled full-length AQP9 1 cDNA, hybridized at 42°C, and washed with 0.1% SDS, 0.1ϫ SSC, at 65°C. Autoradiography was performed at Ϫ80°C for 5 days...

Aquaporins (AQP) are members of the major intrinsic protein (MIP) superfamily of integral membrane proteins and facilitate water transport in various eukaryotes and prokaryotes. The archetypal aquaporin AQP1 is a partly glycosylated water-selective channel that is widely expressed in the plasma membranes of several water-permeable epithelial and endothelial cells. Here we report the three-dimensional structure of deglycosylated, human erythrocyte AQP1, determined at 7 A resolution in the membrane plane by electron crystallography of frozen-hydrated two-dimensional crystals. The structure has an inplane, intramolecular 2-fold axis of symmetry located in the hydrophobic core of the bilayer. The AQP1 monomer is composed of six membrane-spanning, tilted alpha-helices. These helices form a barrel that encloses a vestibular region leading to the water-selective channel, which is outlined by densities attributed to the functionally important NPA boxes and their bridges to the surrounding helices. The intramolecular symmetry within the AQP1 molecule represents a new motif for the topology and design of membrane protein channels, and is a simple and elegant solution to the problem of bidirectional transport across the bilayer.