Purification and functional characterization of aquaporin-8

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.     

Arsenic trioxide uptake by hexose permeases in Saccharomyces cerevisiae

Arsenic trioxide is a toxic metalloid and carcinogen that is also used as an anticancer drug, and for this reason it is important to identify the routes of arsenite uptake by cells. In this study the ability of hexose transporters to facilitate arsenic trioxide uptake in Saccharomyces cerevisiae was examined. In the absence of glucose, strains with disruption of the arsenite efflux gene ACR3 accumulated high levels of (73)As(OH)(3). The addition of glucose inhibited uptake by approximately 80%. Disruption of FPS1, the aquaglyceroporin gene, reduced glucose-independent uptake by only about 25%, and the residual uptake was nearly completely inhibited by hexoses, including glucose, galactose, mannose, and fructose but not pentoses or disaccharides. A strain lacking FPS1, ACR3, and all genes for hexose permeases except for HXT3, HXT6, HXT7, and GAL2 exhibited hexose-inhibitable (73)As(OH)(3) uptake, whereas a strain lacking all 18 hexose transport-related genes (HXT1 to HXT17 and GAL2), FPS1 and ACR3, exhibited <10% of wild type (73)As(OH)(3) transport. When HXT1, HXT3, HXT4, HXT5, HXT7, or HXT9 was individually expressed in that strain, hexose-inhibitable (73)As(OH)(3) uptake was restored. In addition, the transport of [(14)C]glucose was inhibited by As(OH)(3). These results clearly demonstrate that hexose permeases catalyze the majority of the transport of the trivalent metalloid arsenic trioxide.     

Mammalian glucose permease GLUT1 facilitates transport of arsenic trioxide and methylarsonous acid

Arsenic exposure is associated with hypertension, diabetes, and cancer. Some mammals methylate arsenic. Saccharomyces cerevisiae hexose permeases catalyze As(OH)(3) uptake. Here, we report that mammalian glucose transporter GLUT1 catalyzes As(OH)(3) and CH(3)As(OH)(2) uptake in yeast or in Xenopus laevis oocytes. Expression of GLUT1 in a yeast lacking other glucose transporters allows for growth on glucose. Yeast expressing yeast HXT1 or rat GLUT1 transport As(OH)(3) and CH(3)As(OH)(2). The K(m) of GLUT1 is to 1.2mM for CH(3)As(OH)(2), compared to a K(m) of 3mM for glucose. Inhibition between glucose and CH(3)As(OH)(2) is noncompetitive, suggesting differences between the translocation pathways of hexoses and arsenicals. Both human and rat GLUT1 catalyze uptake of both As(OH)(3) and CH(3)As(OH)(2) in oocytes. Thus GLUT1 may be a major pathway uptake of both inorganic and methylated arsenicals in erythrocytes or the epithelial cells of the blood-brain barrier, contributing to arsenic-related cardiovascular problems and neurotoxicity.     

Pentavalent methylated arsenicals are substrates of human AQP9

Liver aquaglyceroporin AQP9 facilitates movement of trivalent inorganic arsenite (As^III) and organic monomethylarsonous acid (MAs^III). However, the transport pathway for the two major pentavalent arsenic cellular metabolites, MAs^V and DMAs^V, remains unknown in mammals. These products of arsenic metabolism, in particular DMAs^V, are the major arsenicals excreted in the urine of mammals. In this study, we examined the uptake of the two pentavalent organic arsenicals by human AQP9 in Xenopus laevis oocytes. Xenopus laevis oocytes microinjected with AQP9 cRNA exhibited uptake of both MAs^V and DMAs^V in a pH-dependent manner. The rate of transport was much higher at acidic pH (pH5.5) than at neutral pH. Hg(II), an aquaporin inhibitor, inhibited transport of As^III, MAs^III, MAs^V and DMAs^V via AQP9. However, phloretin, which inhibits water and glycerol permeation via AQP9, can only inhibit transport of pentavalent MAs^V and DMAs^V but not trivalent As^III and MAs^III, indicating the translocation mechanisms of these arsenic species are not exactly the same. Reagents such as FCCP, valinomycin and nigericin that dissipate transmembrane proton potential or change the transmemebrane pH gradient did not significantly inhibit all arsenic transport via AQP9, suggesting the transport of pentavalent arsenic is not proton coupled. The results suggest that in addition to the initial uptake of trivalent inorganic As^III inside cells, AQP9 plays a dual role in the detoxification of arsenic metabolites by facilitating efflux from cells.     

Equilibrium characterization of the As(III)-cysteine and the As(III)-glutathione systems in aqueous solution

Some arsenic compounds were the first antimicrobial agents specifically synthesized for the treatment of infectious diseases such as syphilis and trypanosomiasis. More recently, arsenic trioxide has been shown to be efficient in the treatment of acute promyelocytic leukemia. The exact mechanism of action has not been elucidated yet, but it seems to be related to arsenic binding to vicinal thiol groups of regulatory proteins. Glutathione is the major intracellular thiol and plays important roles in the cellular defense and metabolism. This paper reports on a study of the interactions between arsenic(III) and either cysteine or glutathione in aqueous solution. The behavior observed for the As(III)-glutathione system is very similar to that of As(III)-cysteine. In both cases, the formation of two complexes in aqueous solution was evidenced by NMR and electronic spectroscopies and by potentiometry. The formation constants of the cysteine complexes [As(H(-1)Cys)(3)], log K = 29.84(6), and [As(H(-2)Cys)(OH)(2)](-), log K = 12.01(9), and of the glutathione complexes [As(H(-2)GS)(3)](3-), log K = 32.0(6), and [As(H(-3)GS)(OH)(2)](2-), log K = 10(3) were calculated from potentiometric and spectroscopic data. In both cases, the [As(HL)(3)] species, in which the amine groups are protonated, predominate from acidic to neutral media, and the [As(L)(OH)(2)] species appear in basic medium (the charges were omitted for the sake of simplicity). Spectroscopic data clearly show that the arsenite-binding site in both complexes is the sulfur atom of cysteine. In the [As(L)(OH)(2)] species, the coordination sphere is completed by two hydroxyl groups. In both cases, arsenic probably adopts a trigonal pyramidal geometry. Above pH 10, the formation of [As(OH)(2)O](-) excludes the thiolates from arsenic coordination sites. At physiological pH, almost 80% of the ligand is present as [As(HL)(3)].     

ATP binding at human P2X1 receptors. Contribution of aromatic and basic amino acids revealed using mutagenesis and partial agonists

P2X receptors comprise a family of ATP-gated ion channels with the basic amino acids Lys-68, Arg-292, and Lys-309 (P2X(1) receptor numbering) contributing to agonist potency. In many ATP-binding proteins aromatic amino acids coordinate the binding of the adenine group. There are 20 conserved aromatic amino acids in the extracellular ligand binding loop of at least 6 of the 7 P2X receptors. We used alanine replacement mutagenesis to determine the effects of individual conserved aromatic residues on the properties of human P2X(1) receptors expressed in Xenopus oocytes. ATP evoked concentration-dependent (EC(50) approximately 1 microm) desensitizing currents at wild-type receptors and for the majority of mutants there was no change (10 residues) or a <6-fold decrease in ATP potency (6 mutants). Mutants F195A and W259A failed to form detectable channels at the cell surface. F185A and F291A produced 10- and 160-fold decreases in ATP potency. The partial agonists 2',3'-O-(4-benzoyl)-ATP (BzATP) and P(1),P(5)-di(adenosine 5')-pentaphosphate (Ap(5)A) were tested on a range of mutants that decreased ATP potency to determine whether this resulted predominantly from changes in agonist binding or gating of the channel. At K68A and K309A receptors BzATP and Ap(5)A had essentially no agonist activity but antagonized, or for R292A potentiated, ATP responses. At F185A receptors BzATP was an antagonist but Ap(5)A no longer showed affinity for the receptor. These results suggest that residues Lys-68, Phe-185, Phe-291, Arg-292, and Lys-309 contribute to ligand binding at P2X(1) receptors, with Phe-185 and Phe-291 coordinating the binding of the adenine ring of ATP.     

Role of AQP9 in transport of monomethyselenic acid and selenite

AQP9 is an aquaglyceroporin with a very broad substrate spectrum. In addition to its orthodox nutrient substrates, AQP9 also transports multiple neutral and ionic arsenic species including arsenic trioxide, monomethylarsenous acid (MAs^III) and dimethylarsenic acid (DMA^V). Here we discovered a new group of AQP9 substrates which includes two clinical relevant selenium species. We showed that AQP9 efficiently transports monomethylselenic acid (MSeA) with a preference for acidic pH, which has been demonstrated in Xenopus laevis oocyte following the overexpression of human AQP9. Specific inhibitors that dissipate transmembrane proton potential or change the transmembrane pH gradient, such as FCCP, valinomycin and nigericin did not significantly inhibit MSeA uptake, suggesting MSeA transport is not proton coupled. AQP9 was also found to transport ionic selenite and lactate, with much less efficiency compared with MSeA uptake. Selenite and lactate uptake via AQP9 is pH dependent and inhibited by FCCP and nigericin, but not valinomycin. The selenite and lactate uptake via AQP9 can be inhibited by different lactate analogs, indicating that their translocation share similar mechanisms. AQP9 transport of MSeA, selenite and lactate is all inhibited by a previously identified AQP9 inhibitor, phloretin, and the AQP9 substrate arsenite (As^III). These newly identified AQP9 selenium substrates imply that AQP9 play a significant role in MSeA uptake and possibly selenite uptake involved in cancer therapy under specific microenvironments.     

Contributions of conserved residues at the gating interface of glycine receptors

Glycine receptors (GlyRs) are chloride channels that mediate fast inhibitory neurotransmission and are members of the pentameric ligand-gated ion channel (pLGIC) family. The interface between the ligand binding domain and the transmembrane domain of pLGICs has been proposed to be crucial for channel gating and is lined by a number of charged and aromatic side chains that are highly conserved among different pLGICs. However, little is known about specific interactions between these residues that are likely to be important for gating in α1 GlyRs. Here we use the introduction of cysteine pairs and the in vivo nonsense suppression method to incorporate unnatural amino acids to probe the electrostatic and hydrophobic contributions of five highly conserved side chains near the interface, Glu-53, Phe-145, Asp-148, Phe-187, and Arg-218. Our results suggest a salt bridge between Asp-148 in loop 7 and Arg-218 in the pre-M1 domain that is crucial for channel gating. We further propose that Phe-145 and Phe-187 play important roles in stabilizing this interaction by providing a hydrophobic environment. In contrast to the equivalent residues in loop 2 of other pLGICs, the negative charge at Glu-53 α1 GlyRs is not crucial for normal channel function. These findings help decipher the GlyR gating pathway and show that distinct residue interaction patterns exist in different pLGICs. Furthermore, a salt bridge between Asp-148 and Arg-218 would provide a possible mechanistic explanation for the pathophysiologically relevant hyperekplexia, or startle disease, mutant Arg-218 → Gln.     

Aquaglyceroporins serve as metabolic gateways in adiposity and insulin resistance control

Aquaglyceroporins (AQP3, AQP7, AQP9 and AQP10) encompass a subfamily of aquaporins that allow the movement of water and other small solutes, especially glycerol, through cell membranes. Adipose tissue constitutes a major source of glycerol via AQP7. We have recently reported that, in addition to the well-known expression of AQP7 in adipose tissue, AQP3 and AQP9 are also expressed in omental and subcutaneous fat depots. Moreover, insulin and leptin act as regulators of aquaglyceroporins through the PI3K/Akt/mTOR pathway. AQP3 and AQP7 appear to facilitate glycerol efflux from adipose tissue while reducing the glycerol influx into hepatocytes via AQP9 to prevent the excessive lipid accumulation and the subsequent aggravation of hyperglycemia in human obesity. This Extra View focuses on the control of glycerol release by aquaglyceroporins in the adipose tissue and briefly discusses the importance of glycerol as a substrate for hepatic gluconeogenesis, pancreatic insulin secretion and cardiac ATP production.     

A mutational analysis of active site residues in trans-3-chloroacrylic acid dehalogenase

trans-3-Chloroacrylic acid dehalogenase (CaaD) catalyzes the hydrolytic dehalogenation of trans-3-haloacrylates to yield malonate semialdehyde by a mechanism utilizing βPro-1, αArg-8, αArg-11, and αGlu-52. These residues are implicated in a promiscuous hydratase activity where 2-oxo-3-pentynoate is processed to acetopyruvate. The roles of three nearby residues (βAsn-39, αPhe-39, and αPhe-50) are unexplored. Mutants were constructed at these positions (βN39A, αF39A, αF39T, αF50A and αF50Y) and kinetic parameters determined along with those of the αR8K and αR11K mutants. Analysis indicates that αArg-8, αArg-11, and βAsn-39 are critical for dehalogenase activity whereas αArg-11 and αPhe-50 are critical for hydratase activity. Docking studies suggest structural bases for these observations.     

Aquaglyceroporins serve as metabolic gateways in adiposity and insulin resistance control

Aquaglyceroporins (AQP3, AQP7, AQP9 and AQP10) encompass a subfamily of aquaporins that allow the movement of water and other small solutes, especially glycerol, through cell membranes. Adipose tissue constitutes a major source of glycerol via AQP7. We have recently reported that, in addition to the well-known expression of AQP7 in adipose tissue, AQP3 and AQP9 are also expressed in omental and subcutaneous fat depots. Moreover, insulin and leptin act as regulators of aquaglyceroporins through the PI3K/Akt/mTOR pathway. AQP3 and AQP7 appear to facilitate glycerol efflux from adipose tissue while reducing the glycerol influx into hepatocytes via AQP9 to prevent the excessive lipid accumulation and the subsequent aggravation of hyperglycemia in human obesity. This Extra View focuses on the control of glycerol release by aquaglyceroporins in the adipose tissue and briefly discusses the importance of glycerol as a substrate for hepatic gluconeogenesis, pancreatic insulin secretion and cardiac ATP production.Key words: glycerol, aquaporin, fat accumulation, glucose homeostasis, insulin secretion, ATP production     

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.     

Mercury chloride decreases the water permeability of aquaporin-4-reconstituted proteoliposomes.

BACKGROUND INFORMATION: Mercurials inhibit AQPs (aquaporins), and site-directed mutagenesis has identified Cys(189) as a site of the mercurial inhibition of AQP1. On the other hand, AQP4 has been considered to be a mercury-insensitive water channel because it does not have the reactive cysteine residue corresponding to Cys(189) of AQP1. Indeed, the osmotic water permeability (P(f)) of AQP4 expressed in various types of cells, including Xenopus oocytes, is not inhibited by HgCl2. To examine the direct effects of mercurials on AQP4 in a proteoliposome reconstitution system, His-tagged rAQP4 [corrected] (rat AQP4) M23 was expressed in Saccharomyces cerevisiae, purified with an Ni2+-nitrilotriacetate affinity column, and reconstituted into liposomes with the dilution method. RESULTS: The water permeability of AQP4 proteoliposomes with or without HgCl2 was measured with a stopped-flow apparatus. Surprisingly, the P(f) of AQP4 proteoliposomes was significantly decreased by 5 microM HgCl2 within 30 s, and this effect was completely reversed by 2-mercaptoethanol. The dose- and time-dependent inhibitory effects of Hg2+ suggest that the sensitivity to mercury of AQP4 is different from that of AQP1. Site-directed mutagenesis of six cysteine residues of AQP4 demonstrated that Cys(178), which is located at loop D facing the intracellular side, is a target responding to Hg2+. We confirmed that AQP4 is reconstituted into liposome in a bidirectional orientation. CONCLUSIONS: Our results suggest that mercury inhibits the P(f) of AQP4 by mechanisms different from those for AQP1 and that AQP4 may be gated by modification of a cysteine residue in cytoplasmic loop D.     

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.     

Cloning and identification of a new member of water channel (AQP10) as an aquaglyceroporin

Recently, a new member of aquaporins was reported as AQP10 [Biochem. Biophys. Res. Commun. 287 (2001) 814], which is incompletely spliced to lose the sixth transmembrane domain and has poor water and no glycerol/urea permeabilities. Independently, we identified a similar clone in human. Our AQP10 consists of 301 amino acids with a highly conserved sixth transmembrane domain. AQP10 has higher identity with aquaglyceroporins (50% with AQP9, 48% with AQP3, 42% with AQP7) and lower identity with other aquaporins (32% with AQP1 and AQP8). AQP10 is expressed only in the small intestine with (approximately 2 kb). RNase protection assay revealed the absence of the unspliced form, supporting the authenticity of our clone. When expressed in Xenopus oocytes, AQP10 stimulated osmotic water permeability sixfold in a mercury-sensitive manner. Glycerol and urea uptakes were also stimulated, while adenine uptake was not. The genome structure of AQP10 is similar to those of other aquaglyceroporins (AQP3, AQP7, AQP9) with six exons. We conclude that AQP10 represents a new member of aquaglyceroporins functionally as well as structurally.     

Water transport in human aquaporin-4: molecular dynamics (MD) simulations

Aquaporin-4 (AQP4) is the predominant water channel in the central nervous system, where it has been reported to be involved in many pathophysiological roles including water transport. In this paper, the AQP4 tetramer was modeled from its PDB structure file, embedded in a palmitoyl-oleoyl-phosphatidyl-choline (POPC) lipid bilayer, solvated in water, then minimized and equilibrated by means of molecular dynamics simulations. Analysis of the equilibrated structure showed that the central pore along the fourfold axis of the tetramers is formed with hydrophobic amino acid residues. In particular, Phe-195, Leu-191 and Leu-75, form the narrowest part of the pore. Therefore water molecules are not expected to transport through the central pore, which was confirmed by MD simulations. Each monomer of the AQP4 tetramers forms a channel whose walls consist mostly of hydrophilic residues. There are eight water molecules in single file observed in each of the four channels, transporting through the selectivity filter containing Arg-216, His-201, Phe-77, Ala-210, and the two conserved Asn-Pro-Ala (NPA) motifs containing Asn-213 and Asn-97. By using Brownian dynamics fluctuation–dissipation-theorem (BD-FDT), the overall free-energy profile was obtained for water transporting through AQP4 for the first time, which gives a complete map of the entire channel of water permeation.