pH replica-exchange method based on discrete protonation states

We propose a new algorithm for obtaining proton titration curves of ionizable residues. The algorithm is a pH replica-exchange method (PHREM), which is based on the constant pH algorithm of Mongan et al. (J Comput Chem 2004;25:2038-2048). In the original replica-exchange method, simulations of different replicas are performed at different temperatures, and the temperatures are exchanged between the replicas. In our PHREM, simulations of different replicas are performed at different pH values, and the pHs are exchanged between the replicas. The PHREM was applied to a blocked amino acid and to two protein systems (snake cardiotoxin and turkey ovomucoid third domain), in conjunction with a generalized Born implicit solvent. The performance and accuracy of this algorithm and the original constant pH method (PHMD) were compared. For a single set of simulations at different pHs, the use of PHREM yields more accurate Hill coefficients of titratable residues. By performing multiple sets of constant pH simulations started with different initial states, the accuracy of predicted pK(a) values and Hill coefficients obtained with PHREM and PHMD methods becomes comparable. However, the PHREM algorithm exhibits better samplings of the protonation states of titratable residues and less scatter of the titration points and thus better precision of measured pK(a) values and Hill coefficients. In addition, PHREM exhibits faster convergence of individual simulations than the original constant pH algorithm.     

pH and calcium regulate the water permeability of aquaporin 0

Aquaporins increase the water permeability in many cell types across many species. We investigated the effects of external pH and Ca(2+) on water permeability of Xenopus oocytes injected with aquaporin cRNA by measuring the rate of swelling in hypotonic solutions. Lowering pH to 6.5 increased the water permeability of aquaporin (AQP0) 3.4 +/- 0.4-fold. Diethylpyrocarbonate pretreatment increased water permeability 4.2 +/- 0.5-fold and abolished pH sensitivity, suggesting that the pH regulation is mediated by an external histidine. Lowering Ca(2+) increased water permeability 4.1 +/- 0. 4-fold. The effects of Ca(2+) and pH each required the presence of histidine 40, indicating a critical role of this amino acid in facilitating the modulation of water permeability. Clamping intracellular Ca(2+) at high or low values abolished sensitivity to external Ca(2+), suggesting that Ca(2+) acts at an internal site. Three different calmodulin inhibitors each increased AQP0 water permeability, suggesting that Ca(2+) may act through calmodulin. None of the above altered the water permeability induced by AQP1 or AQP4. Because the greatest change in AQP0 water permeability is in the normal pH range found in the lens (7.2-6.5), this paper provides evidence for regulation of an aquaporin by pH under physiological conditions.     

Acid-base equilibria in rhodopsin: dependence of the protonation state of glu134 on its environment

Glutamic acid E134 in rhodopsin is part of a highly conserved triad, D(E)RY, located near the cytoplasmic lipid/water interface in transmembrane helix 3 of G protein-coupled receptors (GPCRs). A large body of experimental evidence suggests that the protonation of E134 plays a role in the mechanism of activation of rhodopsin and other GPCRs as well. For E134 to change its protonation state, its pK(a) value must shift from values below physiological pH to higher values. Because of the proximity of the triad to the lipid/water interface, it was hypothesized that a change in solvent around E134 from water to lipid could induce such a shift in pK(a). To test this hypothesis, the pK(a) values of the titratable amino acid residues in rhodopsin have been calculated and the change in solvent around E134 was modeled by shifting the position of the lipid/water interface. The approach used to carry out the pK(a) calculations takes into account the partial immersion of transmembrane proteins in lipid. Qualitative experimental evidence is available for several residues regarding their likely protonation state in rhodopsin at or near physiological pH. Comparison of the calculated pK(a) values with these experimental findings shows good agreement between the two. Notably, glutamic acids E122 and E181 were found to be protonated. The pK(a) values were then calculated for a range of lipid/water interface positions. Although the surrounding solvent of several titratable residues changed from water to lipid in this range, leading to pK(a) shifts in most cases, only for E134 would the shift lead to a change in protonation state at physiological pH. Thus, our results show that the protonation state of E134 is particularly sensitive to its environment. This sensitivity together with the location of E134 near the actual position of the lipid/water interface could be a strategic element in the mechanism of activation of rhodopsin.     

Zinc modulation of water permeability reveals that aquaporin 0 functions as a cooperative tetramer

We previously showed that the water permeability of AQP0, the water channel of the lens, increases with acid pH and that His40 is required (Németh-Cahalan, K.L., and J.E. Hall. 2000. J. Biol. Chem. 275:6777-6782; Németh-Cahalan, K.L., K. Kalman, and J.E. Hall. 2004. J. Gen. Physiol. 123:573-580). We have now investigated the effect of zinc (and other transition metals) on the water permeability of AQP0 expressed in Xenopus oocytes and determined the amino acid residues that facilitate zinc modulation. Zinc (1 mM) increased AQP0 water permeability by a factor of two and prevented any additional increase induced by acid pH. Zinc had no effect on water permeability of AQP1, AQP4 or MIPfun (AQP0 from killifish), or on mutants of AQP1 and MIPfun with added external histidines. Nickel, but not copper, had the same effect on AQP0 water permeability as zinc. A fit of the concentration dependence of the zinc effect to the Hill equation gives a coefficient greater than three, suggesting that binding of more than one zinc ion is necessary to enhance water permeability. His40 and His122 are necessary for zinc modulation of AQP0 water permeability, implying structural constraints for zinc binding and functional modulation. The change in water permeability was highly sensitive to a coinjected zinc-insensitive mutant and a single insensitive monomer completely abolished zinc modulation. Our results suggest a model in which positive cooperativity among subunits of the AQP0 tetramer is required for zinc modulation, implying that the tetramer is the functional unit. The results also offer the possibility of a pharmacological approach to manipulate the water permeability and transparency of the lens.     

The pH dependence of red cell membrane transport of titratable anions studied by NMR spectroscopy

The effects of varying extracellular pH on the rates of uptake of titratable anions by human erythrocytes under conditions of constant intracellular pH have been determined for a series of highly related anions, the phosphate "analogs." These compounds are simply substituted phosphorus oxyacids, differing in the number and acidity of titratable protons: phosphate (HPO4(2-), pKa 6.8); phosphite (HPO3(2-), pKa 6.4); hypophosphite (H2PO2-); methylphosphonate ((CH3)PO3(2-), pKa 7.4); dimethylphosphinate ((CH3)2PO2-); fluorophosphate [PO3F2-, pKa 4.7); and thiophosphate (HSPO3(2-), pKa 5.5). Suspensions of intact, Cl(-)-loaded erythrocytes (intracellular pH, 7.2) were incubated at 37 degrees C in isotonic buffers (pH 4-8) containing 60 mM phosphate analog for specified time intervals, whereupon influx was halted by the addition of 1 mM 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS), an inhibitor of anion exchange. The intracellular anion concentrations were determined from 31P or 19F nuclear magnetic resonance spectra from the erythrocyte suspensions. The influx rates for the titratable phosphate analogs exhibited bimodal pH dependence, reaching maximal levels at pH values that increased with increasing anion pK. This pH-dependent behavior is consistent with a transport channel that contains a titratable regulatory site which interacts with the translocated anion. Based upon the Henderson-Hasselbalch equation, the probability that a titratable anion will have an electric charge of equal magnitude to that of the titratable carrier is highest at a pH value exactly midway between the pK of the regulatory site and that of the anion. The pH maxima observed for the phosphate analogs indicate a pK for this site of 5.5 at 37 degrees C. Intracellular pH changes associated with influx indicated that transport of the "fast" anion phosphite is largely in monoionized form. Intracellular pH changes associated with transport of slow anions were predominantly determined by partial ionic equilibrium effects and did not indicate the ionization state of the transported anion.     

On the Hill plot of NMR data for titration of protein residues

The Hill plots of NMR titration data for protein residues disclose more clearly than the usual titration curves the presence of multiple weak perturbations originating from other titratable groups, and should be used whenever the conventional curve fitting is poor. For a quantitative interpretation, we derive here expressions for the Hill equation and the Hill coefficient when the titration of the observed group is perturbed by more than one titratable group. When the generalized Hill equation is fitted to the data, values of the interaction parameters between the observed group and the others are extracted provided that there are no mutual interactions between the latter groups. The method is applied to the titration data of two histidyl residues of l-arginine phosphotransferase (E.C. 2.7.3.3.) in the transition state analogue complex (enzyme-Mg²⁺-ADP-NOsk3/–l-Arg). From the Hill plots, interactions with three titratable groups are disclosed for both residues, and the fitting with the Hill equation reveals that they experience perturbations from the same three groups. Microscopic pK values are obtained for all the involved groups, indicating large changes (up to 3 pH units) upon protonation of the interacting groups. As compared to the conventional fitting procedure, the use and fitting of Hill plots yields from NMR data more information on the neighbourhood of enzyme residues and on the changes intervening therein through the steps involved in the catalysis.     

Kinetic studies with myo-inositol monophosphatase from bovine brain

The kinetic properties of myo-inositol monophosphatase with different substrates were examined with respect to inhibition by fluoride, activation or inhibition by metal ions, pH profiles, and solvent isotope effects. F- is a competitive inhibitor versus 2'-AMP and glycerol 2-phosphate, but noncompetitive (Kis = Kii) versus DL-inositol 1-phosphate, all with Ki values of approximately 45 microM. Activation by Mg2+ follows sigmoid kinetics with Hill constants around 1.9, and random binding of substrate and metal ion. At high concentrations, Mg2+ acts as an uncompetitive inhibitor (Ki = 4.0 mM with DL-inositol 1-phosphate at pH 8.0 and 37 degrees C). Activation and inhibition constants, and consequently the optimal concentration of Mg2+, vary considerably with substrate structure and pH. Uncompetitive inhibition by Li+ and Mg2+ is mutually exclusive, suggesting a common binding site. Lithium binding decreases at low pH with a pK value of 6.4, and at high pH with a pK of 8.9, whereas magnesium inhibition depends on deprotonation with a pK of 8.3. The pH dependence of V suggests that two groups with pK values around 6.5 have to be deprotonated for catalysis. Solvent isotope effects on V and V/Km are greater than 2 and 1, respectively, regardless of the substrate, and proton inventories are linear. These results are consistent with a model where low concentrations of Mg2+ activate the enzyme by stabilizing the pentacoordinate phosphate intermediate. Li+ as well as Mg2+ at inhibiting concentrations bind to an additional site in the enzyme-substrate complex. Hydrolysis of the phosphate ester is rate limiting and facilitated by acid-base catalysis.     

A water channel of the nematode C.elegans and its implications for channel selectivity of MIP proteins

A genome project focusingon the nematode Caenorhabditis elegans has demonstrated thepresence of eight cDNAs belonging to the major intrinsic proteinsuperfamily. We functionally characterized one of these cDNAs namedC01G6.1. Injection of C01G6.1 cRNA increased the osmotic waterpermeability (P_f) of Xenopusoocytes 11-fold and the urea permeability 4.5-fold but failed toincrease the glycerol permeability. It has been speculated that the MIPfamily may be separated into two large subfamilies based on thepresence or absence of two segments of extra amino acid residues (~15amino acids) at the second and third extracellular loops. BecauseC01G6.1 (designated AQP-CE1), AQP3, and glycerol facilitator (GlpF) all have these two segments, we replaced the segments of AQP-CE1 with thoseof AQP3 and GlpF to identify their roles. The functional characteristics of these mutants were principally similar to that ofwild-type AQP-CE1, although the values of P_f andurea permeability were decreased by 39-74% and 28-65%,respectively. These results suggest that the two segments of extraamino acid residues may not contribute to channel selectivity orformation of the route for small solutes.

     

Copper inhibits the water and glycerol permeability of aquaporin-3

Aquaporin-3 (AQP3) is an aquaglyceroporin expressed in erythrocytes and several other tissues. Erythrocytes are, together with kidney and liver, the main targets for copper toxicity. Here we report that both water and glycerol permeability of human AQP3 is inhibited by copper. Inhibition is fast, dose-dependent, and reversible. If copper is dissolved in carbonic acid-bicarbonate buffer, the natural buffer system in our body, doses in the range of those observed in Wilson disease and in copper poisoning caused significant inhibition. AQP7, another aquaglyceroporin, was insensitive to copper. Three extracellular amino acid residues, Trp128, Ser152, and His241, were identified as responsible for the effect of copper on AQP3. We have previously shown that Ser152 is involved in regulation of AQP3 by pH. The fact that Ser152 mediates regulation of AQP3 by copper may explain the phenomenon of exquisite sensitivity of human erythrocytes to copper at acidic pH. When AQP3 was co-expressed with another AQP, only glycerol but not water permeability was inhibited by copper. Our results provide a better understanding of processes that occur in severe copper metabolism defects such as Wilson disease and in copper poisoning.     

Cloning, heterologous expression, and characterization of three aquaglyceroporins from Trypanosoma brucei

Trypanosoma brucei, causative for African sleeping sickness, relies exclusively on glycolysis for ATP production. Under anaerobic conditions, glucose is converted to equimolar amounts of glycerol and pyruvate, which are both secreted from the parasite. As we have shown previously, glycerol transport in T. brucei occurs via specific membrane proteins (Wille, U., Schade, B., and Duszenko, M. (1998) Eur. J. Biochem. 256, 245-250). Here, we describe cloning and biochemical characterization of the three trypanosomal aquaglyceroporins (AQP; TbAQP1-3), which show a 40-45% identity to mammalian AQP3 and -9. AQPs belong to the major intrinsic protein family and represent channels for small non-ionic molecules. Both TbAQP1 and TbAQP3 contain two highly conserved NPA motifs within the pore-forming region, whereas TbAQP2 contains NSA and NPS motifs instead, which are only occasionally found in AQPs. For functional characterization, all three proteins were heterologously expressed in yeast and Xenopus oocytes. In the yeast fps1Delta mutant, TbAQPs suppressed hypoosmosensitivity and rendered cells to a hyper-osmosensitive phenotype, as expected for unregulated glycerol channels. Under iso- and hyperosmotic conditions, these cells constitutively released glycerol, consistent with a glycerol efflux function of TbAQP proteins. TbAQP expression in Xenopus oocytes increased permeability for water, glycerol and, interestingly, dihydroxyacetone. Except for urea, TbAQPs were virtually impermeable for other polyols; only TbAQP3 transported erythritol and ribitol. Thus, TbAQPs represent mainly water/glycerol/dihydroxyacetone channels involved in osmoregulation and glycerol metabolism in T. brucei. This function and especially the so far not investigated transport of dihydroxyacetone may be pivotal for the survival of the parasite survival under non-aerobic or osmotic stress conditions.     

Erythrocyte water permeability and renal function in double knockout mice lacking aquaporin-1 and aquaporin-3

Aquaporin (AQP) water channel AQP3 has been proposed to be the major glycerol and non-AQP1 water transporter in erythrocytes. AQP1 and AQP3 are also expressed in the kidney where their deletion in mice produces distinct forms of nephrogenic diabetes insipidus. Here AQP1/AQP3 double knockout mice were generated and analyzed to investigate the functional role of AQP3 in erythrocytes and kidneys. 53 double knockout mice were born out of 756 pups from breeding double heterozygous mice. The double knockout mice had reduced survival and impaired growth compared with the single knockout mice. Erythrocyte water permeability was 7-fold reduced by AQP1 deletion but not further reduced in AQP1/AQP3 null mice. AQP3 deletion did not affect erythrocyte glycerol permeability or its inhibition by phloretin. Daily urine output in AQP1/AQP3 double knockout mice (15 ml) was 9-fold greater than in wild-type mice, and urine osmolality (194 mosm) was 8.4-fold reduced. The mice remained polyuric after DDAVP administration or water deprivation. The renal medulla in most AQP1/AQP3 null mice by age 4 weeks was atrophic and fluid-filled due to the severe polyuria and hydronephrosis. Our data provide direct evidence that AQP3 is not functionally important in erythrocyte water or glycerol permeability. The renal function studies indicate independent roles of AQP1 and AQP3 in countercurrent exchange and collecting duct osmotic equilibration, respectively.     

Nickel and extracellular acidification inhibit the water permeability of human aquaporin-3 in lung epithelial cells

Nickel is a common cause of pneumoconiosis. Here, we show that nickel inactivates aquaporin (AQP)-3, the water channel expressed apically in epithelial cells of human terminal airways. Human AQP3 was transiently transfected into human lung cells, and water permeability was measured in transfected and neighboring untransfected cells. Incubation with NiCl2 rapidly, dose-dependently, and reversibly decreased water permeability in AQP3-expressing cells. Acidification of the extracellular medium also caused rapid, dose-dependent, and reversible inhibition of AQP3. Sensitivity of AQP3 to nickel was lower at alkaline pH than at neutral and acidic pH. Cells transfected with human AQP4 and AQP5, which are also expressed in airway epithelia, were insensitive to nickel and extracellular acidification. Zinc and cadmium, other common causes of pneumoconiosis, had no effect on the water permeability of AQP3. Three extracellular residues, Trp128, Ser152, and His241, were responsible for the blocking effect of nickel on human AQP3. Ser152 was identified as a common site for nickel and pH sensitivity. His53, Tyr124, and His154 were also involved in regulation of AQP3 by extracellular pH. In addition, the aromatic side chain of His154 was shown to be important for the water permeability of AQP3. Our results imply that nickel and extracellular pH may modulate lung water clearance and that defective water clearance may be an early component of nickel-induced lung disease.     

Single-channel permeability and glycerol affinity of human aquaglyceroporin AQP3

For its fundamental relevance, transport of water and glycerol across the erythrocyte membrane has long been investigated before and after the discovery of aquaporins (AQPs), the membrane proteins responsible for water and glycerol transport. AQP1 is abundantly expressed in the human erythrocyte for maintaining its hydrohomeostasis where AQP3 is also expressed (at a level ~30-folds lower than AQP1) facilitating glycerol transport. This research is focused on two of the remaining questions: How permeable is AQP3 to water? What is the glycerol-AQP3 affinity under near-physiological conditions? Through atomistic modelling and large-scale simulations, we found that AQP3 is two to three times more permeable to water than AQP1 and that the glycerol-AQP3 affinity is approximately 500/M. Using these computed values along with the data from the latest literature on AQP1 and on erythrocyte proteomics, we estimated the water and glycerol transport rates across the membrane of an entire erythrocyte. We used these rates to predict the time courses of erythrocyte swelling-shrinking in response to inward and outward osmotic gradients. Experimentally, we monitored the time course of human erythrocytes when subject to an osmotic or glycerol gradient with light scattering in a stopped-flow spectrometer. We observed close agreement between the experimentally measured and the computationally predicted time courses of erythrocytes, which corroborated our computational conclusions on the AQP3 water-permeability and the glycerol-AQP3 affinity.     

Assessing the quality of the homology-modeled 3D structures from electrostatic standpoint: test on bacterial nucleoside monophosphate kinase families

In this study, we address the issue of performing meaningful pK(a) calculations using homology modeled three-dimensional (3D) structures and analyze the possibility of using the calculated pK(a) values to detect structural defects in the models. For this purpose, the 3D structure of each member of five large protein families of a bacterial nucleoside monophosphate kinases (NMPK) have been modeled by means of homology-based approach. Further, we performed pK(a) calculations for the each model and for the template X-ray structures. Each bacterial NMPK family used in the study comprised on average 100 members providing a pool of sequences and 3D models large enough for reliable statistical analysis. It was shown that pK(a) values of titratable groups, which are highly conserved within a family, tend to be conserved among the models too. We demonstrated that homology modeled structures with sequence identity larger than 35% and gap percentile smaller than 10% can be used for meaningful pK(a) calculations. In addition, it was found that some highly conserved titratable groups either exhibit large pK(a) fluctuations among the models or have pK(a) values shifted by several pH units with respect to the pK(a) calculated for the X-ray structure. We demonstrated that such case usually indicates structural errors associated with the model. Thus, we argue that pK(a) calculations can be used for assessing the quality of the 3D models by monitoring fluctuations of the pK(a) values for highly conserved titratable residues within large sets of homologous proteins.     

Excretion and conservation of glycerol, and expression of aquaporins and glyceroporins, during cold acclimation in Cope's gray tree frog Hyla chrysoscelis

Cope's gray tree frog Hyla chrysoscelis accumulates glycerol during cold acclimation. We hypothesized that, during this process, gray tree frogs adjust renal filtration and/or reabsorption rates to retain accumulated glycerol. During cold acclimation, plasma concentrations of glycerol rose >200-fold, to 51 mmol/l. Although fractional water reabsorption decreased, glomerular filtration rate (GFR) and, consequently, urine flow were <5% of warm levels, and fractional glycerol reabsorption increased. In contrast, dehydrated frogs increased fractional water reabsorption, decreased GFR, and did not accumulate glycerol. We hypothesized that expression of proteins from the aquaporin (AQP)/glyceroporin (GLP) family was associated with changing patterns of water and glycerol movement. We cloned the cDNA for three such proteins, quantified mRNA expression in nine tissues using real-time quantitative PCR, and functionally characterized them using a Xenopus oocyte expression system. HC-1, an AQP1-like water channel conferring low glycerol permeability, is expressed ubiquitously in warm- and cold-acclimated tissues. HC-2, a water channel most similar to AQP2, is primarily expressed in organs of osmoregulation. HC-3, which is most similar to AQP3, is functionally characterized as a GLP, with low permeability to water but high permeability to glycerol. Aspects of expression levels and functional characteristics varied between cold and warm conditions for each of the three AQPs, suggesting a complex pattern of involvement in osmoregulation related to thermal acclimation.     

Progressive adipocyte hypertrophy in aquaporin-7-deficient mice: adipocyte glycerol permeability as a novel regulator of fat accumulation

Aquaporin-7 (AQP7) is a water/glycerol transporting protein expressed in adipocyte plasma membranes. We report here remarkable age-dependent hypertrophy in adipocytes in AQP7-deficient mice. Wild type and AQP7 null mice had similar growth at 0-16 weeks as assessed by body weight; however, by 16 weeks AQP7 null mice had 3.7-fold increased body fat mass. Adipocytes from AQP7 null mice of age 16 weeks were greatly enlarged (diameter 118 mum) compared with wild type mice (39 mum). Adipocytes from AQP7 null mice also accumulated excess glycerol (251 versus 86 nmol/mg of protein) and triglycerides (3.4 versus 1.7 mumol/mg of protein). In contrast, at age 4 weeks, adipocyte volume and body fat mass were comparable in wild type and AQP7 null mice. To investigate the mechanism(s) responsible for the progressive adipocyte hypertrophy, glycerol permeability and fat metabolism were studied in adipocytes isolated from the younger mice. Plasma membrane glycerol permeability measured by [(14)C]glycerol uptake was 3-fold reduced in AQP7-deficient adipocytes. However, adipocyte lipolysis, measured by free fatty acid release and hormone-sensitive lipase activity, and lipogenesis, measured by [(14)C]glucose incorporation into triglycerides, were not affected by AQP7 deletion. These data suggest that adipocyte hypertrophy in AQP7 deficiency results from defective glycerol exit and consequent accumulation of glycerol and triglycerides. Increasing AQP7 expression/function in adipocytes may reduce adipocyte volume and fat mass in obesity.     

Calculation of pK(a) in proteins with the microenvironment modulated-screened coulomb potential

The MM-SCP has been applied to predict pK(a) values of titratable residues in wild type and mutants of staphylococcal nuclease (SNase). The calculations were based on crystal structures made available by the Garcia-Moreno Laboratory. In the mutants, mostly deeply buried hydrophobic residues were replaced with ionizable residues, and thus their pK(a) values could be measured and calculated using various methods. The data set used here consisted of a set of WT SNase for which His pK(a) at several ionic strengths had been measured, a set of mutants for which measured pK(a) were available and a set of 11 mutants for which the measured pK(a) were not known at the time of calculation. For this latter set, blind predictions were submitted to the protein pK(a) cooperative, 2009 workshop at Telluride, where the results of the blind predictions were discussed (the RMSD of the submitted set was 1.10 pH units). The calculations on the structures with known pK(a) indicated that in addition to weaknesses of the method, structural issues were observed that led to larger errors (>1) in pK(a) predictions. For example, different crystallographic conditions or steric clashes can lead to differences in the local environment around the titratable residue, which can produce large differences in the calculated pK(a) . To gain further insight into the reliability of the MM-SCP, pK(a) of an extended set of 54 proteins belonging to several structural classes were carried out. Here some initial results from this study are reported to help place the SNase results in the appropriate context.     

Cloning of a new aquaporin (AQP10) abundantly expressed in duodenum and jejunum

A new aquaporin (AQP10) was identified in human small intestine. This gene encoded a 264-amino-acid protein with high sequence identity with AQP3 (53%), 9 (52%), and 7 (43%). These AQPs constitute one subfamily of AQP family that is differentiated from the other subfamily of AQP (AQP0, 1, 2, 4, 5, 6, and 8) by sequence homology. Ribonuclease protection assay and Northern blotting demonstrated almost exclusive expression of AQP10 mRNA in the duodenum and jejunum. In situ hybridization localized it in absorptive jejunal epithelial cells. Xenopus oocytes expressing AQP10 exhibited an increased osmotic water permeability in a mercury-sensitive manner. Although AQP10 belongs to the AQP subfamily, which has been characterized by permeability to water and neutral solutes such as urea and glycerol, it was not permeable to urea nor glycerol. The specific expression of AQP10 suggests its contribution to the water transport in the upper portion of small intestine.