Peter Agre

Background information. Aquaporins (AQPs) are a family of channels permeable to water and some small solutes. In mammals, 13 members (AQP0–AQP12) have been found. AQP8 is widely distributed in many tissues and organs. Previous studies in frog oocytes suggested that AQP8 was permeable to water, urea and ammonium, but no direct characterization had yet been reported.Results. We expressed recombinant rAQP8, hAQP8 and mAQP8 (rat, human and mouse AQP8 respectively) in yeast, purified the proteins to homogeneity and reconstituted them into proteoliposomes. Although showing high sequence similarity, AQP8 proteins from the three species had to be purified with different detergents prior to reconstitution. In stopped‐flow studies, all three AQP8 proteoliposomes showed water permeability, which was inhibited by mercuric chloride and rescued by 2‐mercaptoethanol. rAQP8 and hAQP8 proteoliposomes did not transport glycerol or urea but were permeable to formamide, which was also inhibited by mercuric chloride. In the oocyte transport assay, hAQP8‐injected oocytes showed significantly higher [¹⁴C]methylammonium uptake than water‐injected oocytes.Conclusions. In the present study, we successfully purified rAQP8, hAQP8 and mAQP8 proteins and characterized their biochemical and biophysical properties. All three AQP8 proteins transport water. rAQP8 and hAQP8 are not permeable to urea or glycerol. Moreover, hAQP8 is permeable to ammonium analogues (formamide and methylammonium). Our results suggest that AQP8 may transport ammonium in vivo and physiologically contribute to the acid—base equilibrium.

The aquaporins are a family of integral membrane proteins composed of two subfamilies: the orthodox aquaporins, which transport only water, and the aquaglyceroporins, which transport glycerol, urea, or other small solutes. Two recently described aquaporins, numbers 11 and 12, appear to be more distantly related to the other mammalian aquaporins and aquaglyceroporins. We report on the characterization of Aquaporin-11 (AQP11). AQP11 RNA and protein is found in multiple rat tissues, including kidney, liver, testes and brain. AQP11 has a unique distribution in brain, appearing in Purkinje cell dendrites, hippocampal neurons of CA1 and CA2, and cerebral cortical neurons. Immunofluorescent staining of Purkinje cells indicates that AQP11 is intracellular. Unlike other aquaporins, Xenopus oocytes expressing AQP11 in the plasma membrane failed to transport water, glycerol, urea, or ions. AQP11 is functionally distinct from other proteins of the aquaporin superfamily and could represent a new aquaporin subfamily. Further studies are necessary to elucidate the role of AQP11 in the brain.

Both prokaryotic and eukaryotic cells need to be able to transport ammonia gas. In their Perspective, Knepper and Agre discuss an exciting study ( Khademi et al.) that reports resolution of the crystallographic structure of a bacterial ammonia transport channel, AmtB, to an astonishing 1.35 angstroms, an amazing feat for an integral membrane protein. The structure reveals how ammonia is transported in bacteria and sheds light on how related ammonia transport proteins work in eukaryotic cells.

Although water is the major component of all biological fluids, the molecular pathways for water transport across cell membranes eluded identification until the discovery of the aquaporin family of water channels. The atomic structure of mammalian AQP1 illustrates how this family of proteins is freely permeated by water but not protons (hydronium ions, H₃O⁺). Definition of the subcellular sites of expression predicted their physiological functions and potential clinical disorders. Analysis of several human disease states has confirmed that aquaporins are involved in multiple different illnesses including abnormalities of kidney function, loss of vision, onset of brain edema, starvation, and arsenic toxicity.

Aquaglyceroporins form the subset of the aquaporin water channel family that is permeable to glycerol and certain small, uncharged solutes. AQP9 has unusually broad solute permeability and is expressed in hepatocyte plasma membranes. Proteoliposomes reconstituted with expressed, purified rat AQP9 protein were compared with simple liposomes for solute permeability. At pH 7.5, AQP9 proteoliposomes exhibited Hg ²⁺ -inhibitible glycerol and urea permeabilities that were increased 63-fold and 90-fold over background. β-Hydroxybutyrate permeability was not increased above background, and osmotic water permeability was only minimally elevated. During starvation, the liver takes up glycerol for gluconeogenesis. Expression of AQP9 in liver was induced up to 20-fold in rats fasted for 24–96 h, and the AQP9 level gradually declined after refeeding. No changes in liver AQP9 levels were observed in rats fed ketogenic diets or high-protein diets, but AQP9 levels were elevated in livers of rats made diabetic by streptozotocin injection. When blood glucose levels of the diabetic rats were restored to normal by insulin treatments, the AQP9 levels returned to baseline. Confocal immunofluorescence revealed AQP9 immunostaining on the sinusoidal surfaces of hepatocyte plates throughout the livers of control rats. Denser immunostaining was observed in the same distribution in livers of fasted and streptozotocin-treated rats. We conclude that AQP9 serves as membrane channel in hepatocytes for glycerol and urea at physiological pH, but not for β-hydroxybutyrate. In addition, levels of AQP9 expression fluctuate depending on the nutritional status of the subject and the circulating insulin levels.

The water permeability of biological membranes has been a longstanding problem in physiology, but the proteins responsible for this remained unknown until discovery of the aquaporin 1 (AQP1) water channel protein. AQP1 is selectively permeated by water driven by osmotic gradients. The atomic structure of human AQP1 has recently been defined. Each subunit of the tetramer contains an individual aqueous pore that permits single‐file passage of water molecules but interrupts the hydrogen bonding needed for passage of protons. At least 10 mammalian aquaporins have been identified, and these are selectively permeated by water (aquaporins) or water plus glycerol (aquaglyceroporins). The sites of expression coincide closely with the clinical phenotypes ‐ ranging from congenital cataracts to nephrogenic diabetes insipidus. More than 200 members of the aquaporin family have been found in plants, microbials, invertebrates and vertebrates, and their importance to the physiology of these organisms is being uncovered.