Aquaglyceroporins Research Articles

Structural features of the aromatic/arginine constriction in the aquaglyceroporin GintAQPF2 are responsible for glycerol impermeability in arbuscular mycorrhizal symbiosis

Carbon transport in arbuscular mycorrhizal (AM) symbiosis is of fundamental importance. However, the role of glycerol transport in AM symbiosis has not yet been resolved. Glycerol transport across the cell membrane is mediated by aquaglyceroporins (AQGPs), whereas our previous study revealed that it was disfavoured by GintAQPF2, an AQGP from AM fungi (AMF). Here, we analysed the function of two amino acid residues in the aromatic/arginine (ar/R) constriction known as the major selectivity filter in AQGPs. Replacement of phenylalanine-94 (Phe-94) by alanine (Ala) enlarged the diameter of the ar/R constriction and resulted in an increased intracellular glycerol accumulation and thus survival rate of yeast cells at high glycerol levels, while individual or joint replacement of Phe-94 and Ala-234 by tryptophan and glycine induced a closed state of GintAQPF2, suggesting that the potential double gates (Phe94-Phe243 and arginine-249) of the ar/R constriction also likely determined solute permeability. To figure out whether GintAQPF2 functions were relevant to the establishment of AM symbiosis, genomic analyses of four representative fungi with different lifestyles were performed. We found that glycerol facilitators existed in the facultative fungi (the ectomycorrhizal fungus Laccaria bicolor and hemibiotrophic pathogen Magnaporthe oryzae), but not in the obligatory fungi (the AMF Rhizophagus irregularis and necrotrophic pathogen Fusarium verticillioides), revealing a conserved pattern of glycerol transport in symbionts and pathogens. Our results suggested that glycerol blocks due to the special structural features of the ar/R constriction in the only AMF AQGP could potentially play a role in the establishment of AM symbiosis.

Identification of key residues involved in Si transport by the aquaglyceroporins

We recently demonstrated that the aquaglyceroporins (AQGPs) could act as potent transporters for orthosilicic acid (H4SiO4). Although interesting, this finding raised the question of whether water and H4SiO4, the transportable form of Si, permeate AQGPs by interacting with the same region of the pore, especially in view of the difference in molecular radius between the two substrates. Here, our goal was to identify residues that endow the AQGPs with the ability to facilitate Si diffusion by examining the transport characteristics of mutants in which residues were interchanged between a water-permeable but Si-impermeable channel (aquaporin 1 [AQP1]) and a Si-permeable but water-impermeable channel (AQP10). Our results indicate that the composition of the arginine filter (XX/R), known to include three residues that play an important role in water transport, may also be involved in Si selectivity. Interchanging the identities of the nonarginine residues within this filter causes Si transport to increase by approximately sevenfold in AQP1 and to decrease by approximately threefold in AQP10, whereas water transport and channel expression remain unaffected. Our results further indicate that two additional residues in the AQP arginine filter may be involved in substrate selectivity: replacing one of the residues has a profound effect on water permeability, and replacing the other has a profound effect on Si permeability. This study has thus led to the identification of residues that could play a key role in Si transport by the AQGPs and shown that substrate selectivity is likely ensured by more than one checkpoint within or near the pore.

Expression and clinical significance of aquaglyceroporins in human hepatocellular carcinoma.

Aquaglyceroporins (AQPs) are a subset of the aquaporin family, and are permeable to water and glycerol. The aim of the present study was to determine the expression and clinical significance of three AQPs, AQP3,7and9in hepatocellular carcinoma (HCC). Fresh HCC and adjacent non‑tumorous liver tissues were collected from68patients diagnosed with HCC. The expression levels of AQP3,7and9were detected by reverse transcription‑quantitative polymerase chain reaction, western blotting and immunohistochemical analysis. The association between the expression of AQPs and clinicopathological parameters of HCC were investigated. Compared with non‑tumorous liver tissue, HCC tissues exhibited a significant (P<0.05) increase in the expression of AQP3and a concomitant reduction in the expression levels of AQP7and AQP9, at both the mRNA and protein levels. Immunohistochemistry revealed that AQP9was dominantly localized on the plasma membrane of hepatocytes, while AQP3and AQP7exhibited a predominantly cytoplasmic and nuclear distribution. High expression of AQP3was significantly (P<0.05) associated with low expression levels of AQP7and AQP9. High expression of AQP3was correlated with tumor grade (P=0.017), tumor stage (P=0.010) and lymphatic metastasis (P=0.031). Low expression of AQP7was correlated with tumor grade (P=0.043). AQP3was upregulated, and AQP7and AQP9 were downregulated in HCC. A high expression of AQP3and low expression of AQP7was significantly associated with the aggressive features of HCC.

1,3-propanediol binds deep inside the channel to inhibit water permeation through aquaporins.

Aquaporins and aquaglyceroporins (AQPs) are membrane channel proteins responsible for transport of water and for transport of glycerol in addition to water across the cell membrane, respectively. They are expressed throughout the human body and also in other forms of life. Inhibitors of human AQPs have been sought for therapeutic treatment for various medical conditions including hypertension, refractory edema, neurotoxic brain edema, and so forth. Conducting all-atom molecular dynamics simulations, we computed the binding affinity of acetazolamide to human AQP4 that agrees closely with in vitro experiments. Using this validated computational method, we found that 1,3-propanediol (PDO) binds deep inside the AQP4 channel to inhibit that particular aquaporin efficaciously. Furthermore, we used the same method to compute the affinities of PDO binding to four other AQPs and one aquaglyceroporin whose atomic coordinates are available from the protein data bank (PDB). For bovine AQP1, human AQP2, AQP4, AQP5, and Plasmodium falciparum PfAQP whose structures were resolved with high resolution, we obtained definitive predictions on the PDO dissociation constant. For human AQP1 whose PDB coordinates are less accurate, we estimated the dissociation constant with a rather large error bar. Taking into account the fact that PDO is generally recognized as safe by the US FDA, we predict that PDO can be an effective diuretic which directly modulates water flow through the protein channels. It should be free from the serious side effects associated with other diuretics that change the hydro-homeostasis indirectly by altering the osmotic gradients.

Sexual Dimorphism of Adipose and Hepatic Aquaglyceroporins in Health and Metabolic Disorders.

Gender differences in the relative risk of developing metabolic complications, such as insulin resistance or non-alcoholic fatty liver disease (NAFLD), have been reported. The deregulation of glycerol metabolism partly contributes to the onset of these metabolic diseases, since glycerol constitutes a key substrate for the synthesis of triacylglycerols (TAGs) as well as for hepatic gluconeogenesis. The present mini-review covers the sex-­related differences in glycerol metabolism and aquaglyceroporins (AQPs) and its impact in the control of adipose and hepatic fat accumulation as well as in whole-body glucose homeostasis. Plasma glycerol concentrations are increased in women compared to men probably due to the higher lipolytic rate and larger AQP7 amounts in visceral fat as well as the well-known sexual dimorphism in fat mass with women showing higher adiposity. AQP9 represents the primary route for glycerol uptake in hepatocytes, where glycerol is converted by the glycerol-kinase enzyme into glycerol-3-phosphate, a key substrate for de novo synthesis of glucose and TAG. In spite of showing similar hepatic AQP9 protein, women exhibit lower hepatocyte glycerol permeability than men, which might contribute to their lower prevalence of insulin resistance and NAFLD.

Intra-helical salt-bridge and helix destabilizing residues within the same helical turn: Role of functionally important loop E half-helix in channel regulation of major intrinsic proteins

The superfamily of major intrinsic proteins (MIPs) includes aquaporin (AQP) and aquaglyceroporin (AQGP) and it is involved in the transport of water and neutral solutes across the membrane. Diverse MIP sequences adopt a unique hour-glass fold with six transmembrane helices (TM1 to TM6) and two half-helices (LB and LE). Loop E contains one of the two conserved NPA motifs and contributes two residues to the aromatic/arginine selectivity filter. Function and regulation of majority of MIP channels are not yet characterized. We have analyzed the loop E region of 1468 MIP sequences and their structural models from six different organism groups. They can be phylogenetically clustered into AQGPs, AQPs, plant MIPs and other MIPs. The LE half-helix in all AQGPs contains an intra-helical salt-bridge and helix-breaking residues Gly/Pro within the same helical turn. All non-AQGPs lack this salt-bridge but have the helix destabilizing Gly and/or Pro in the same positions. However, the segment connecting LE half-helix and TM6 is longer by 10–15 residues in AQGPs compared to all non-AQGPs. We speculate that this longer loop in AQGPs and the LE half-helix of non-AQGPs will be relatively more flexible and this could be functionally important. Molecular dynamics simulations on glycerol-specific GlpF, water-transporting AQP1, its mutant and a fungal AQP channel confirm these predictions. Thus two distinct regions of loop E, one in AQGPs and the other in non-AQGPs, seem to be capable of modulating the transport. These regions can also act in conjunction with other extracellular residues/segments to regulate MIP channel transport.

A novel variant of aquaporin 3 is expressed in killifish (Fundulus heteroclitus) intestine

Killifish (Fundulus heteroclitus) are euryhaline teleosts that are widely used in environmental and toxicological studies, and they are tolerant to arsenic, in part due to very low assimilation of arsenic from the environment. The mechanism of arsenic uptake by the intestine, a major route of arsenic uptake in humans is unknown. Thus, the goal of this study was to determine if aquaglyceroporins (AQPs), which transport water and other small molecules including arsenite across cell membranes, are expressed in the killifish intestine, and whether AQP expression is affected by osmotic stress. Through RT-PCR and sequence analysis of PCR amplicons, we demonstrated that the intestine expresses kfAQP3a and kfAQP3b, two previously identified variants, and also identified a novel variant of killifish AQP3 (kfAQP3c) in the intestine. The variants likely represent alternate splice forms. A BLAST search of the F. heteroclitus reference genome revealed that the AQP3 gene resides on a single locus, while an alignment of the AQP3 sequence among 384 individuals from eight population ranging from Rhode Island to North Carolina revealed that its coding sequence was remarkably conserved with no fixed polymorphism residing in the region that distinguishes these variants. We further demonstrate that the novel variant transports arsenite into HEK293T cells. Whereas kfAQP3a, which does not transport arsenite, was expressed in both freshwater (FW) and saltwater (SW) acclimated fish, kfAQP3b, an arsenic transporter, was expressed only in FW acclimated fish, and kfAQP3c was expressed only in SW acclimated fish. Thus, we have identified a novel, putative splice variant of kfAQP3, kfAQP3c, which transports arsenic and is expressed only in SW acclimated fish.

Melarsoprol sensitivity profile of Trypanosoma brucei gambiense isolates from cured and relapsed sleeping sickness patients from the Democratic Republic of the Congo.

BackgroundSleeping sickness caused by Trypanosoma brucei (T.b.) gambiense constitutes a serious health problem in sub-Sahara Africa. In some foci, alarmingly high relapse rates were observed in patients treated with melarsoprol, which used to be the first line treatment for patients in the neurological disease stage. Particularly problematic was the situation in Mbuji-Mayi, East Kasai Province in the Democratic Republic of the Congo with a 57% relapse rate compared to a 5% relapse rate in Masi-Manimba, Bandundu Province. The present study aimed at investigating the mechanisms underlying the high relapse rate in Mbuji-Mayi using an extended collection of recently isolated T.b. gambiense strains from Mbuji-Mayi and from Masi-Manimba.Methodology/Principal FindingsForty five T.b. gambiense strains were used. Forty one were isolated from patients that were cured or relapsed after melarsoprol treatment in Mbuji-Mayi. In vivo drug sensitivity tests provide evidence of reduced melarsoprol sensitivity in these strains. This reduced melarsoprol sensitivity was not attributable to mutations in TbAT1. However, in all these strains, irrespective of the patient treatment outcome, the two aquaglyceroporin (AQP) 2 and 3 genes are replaced by chimeric AQP2/3 genes that may be associated with resistance to pentamidine and melarsoprol. The 4 T.b. gambiense strains isolated in Masi-Manimba contain both wild-type AQP2 and a different chimeric AQP2/3. These findings suggest that the reduced in vivo melarsoprol sensitivity of the Mbuji-Mayi strains and the high relapse rates in that sleeping sickness focus are caused by mutations in the AQP2/AQP3 locus and not by mutations in TbAT1.Conclusions/SignificanceWe conclude that mutations in the TbAQP2/3 locus of the local T.b. gambiense strains may explain the high melarsoprol relapse rates in the Mbuji-Mayi focus but other factors must also be involved in the treatment outcome of individual patients.

New subfamilies of major intrinsic proteins in fungi suggest novel transport properties in fungal channels: implications for the host-fungal interactions.

BackgroundAquaporins (AQPs) and aquaglyceroporins (AQGPs) belong to the superfamily of Major Intrinsic Proteins (MIPs) and are involved in the transport of water and neutral solutes across the membranes. MIP channels play significant role in plant-fungi symbiotic relationship and are believed to be important in host-pathogen interactions in human fungal diseases. In plants, at least five major MIP subfamilies have been identified. Fungal MIP subfamilies include orthodox aquaporins and five subgroups within aquaglyceroporins. XIP subfamily is common to both plants and fungi. In this study, we have investigated the extent of diversity in fungal MIPs and explored further evolutionary relationships with the plant MIP counterparts.ResultsWe have extensively analyzed the available fungal genomes and examined nearly 400 fungal MIPs. Phylogenetic analysis and homology modeling exhibit the existence of a new MIP cluster distinct from any of the known fungal MIP subfamilies. All members of this cluster are found in microsporidia which are unicellular fungal parasites. Members of this family are small in size, charged and have hydrophobic residues in the aromatic/arginine selectivity filter and these features are shared by small and basic intrinsic proteins (SIPs), one of the plant MIP subfamilies. We have also found two new subfamilies (δ and γ2) within the AQGP group. Fungal AQGPs are the most diverse and possess the largest number of subgroups. We have also identified distinguishing features in loops E and D in the newly identified subfamilies indicating their possible role in channel transport and gating.ConclusionsFungal SIP-like MIP family is distinct from any of the known fungal MIP families including orthodox aquaporins and aquaglyceroporins. After XIPs, this is the second MIP subfamily from fungi that may have possible evolutionary link with a plant MIP subfamily. AQGPs in fungi are more diverse and possess the largest number of subgroups. The aromatic/arginine selectivity filter of SIP-like fungal MIPs and the δ AQGPs are unique, hydrophobic in nature and are likely to transport novel hydrophobic solutes. They can be attractive targets for developing anti-fungal drugs. The evolutionary pattern shared with their plant counterparts indicates possible involvement of new fungal MIPs in plant-fungi symbiosis and host-pathogen interactions.

High accumulation of arsenic in the esophagus of mice after exposure to arsenite.

Arsenic-induced toxicity appears to be dependent on the tissue- or cell-specific accumulation of this metalloid. An early study showed that arsenic was retained in the esophagus as well as the liver, kidney cortex and skin of marmosets after intraperitoneal administration of (74)As-arsenite. However, there is little available information regarding the distribution of arsenic in the esophagus. Here, we compared the retention of arsenic in the esophagus, liver, lung, kidney and heart in mice intraperitoneally administered 1 or 5mg/kg sodium arsenite (As(III)) daily for 3 or 7days. The results showed that the arsenic concentration was highest in the esophagus. We compared the mRNA levels of aquaglyceroporin (AQP) 3, AQP7 and AQP9, which are responsible for arsenic influx, and those of multidrug-resistance protein (MRP) 1 and MRP2, which are responsible for arsenic efflux. The levels of AQP3 mRNA in the esophagus were much higher than those in liver, lung and heart, while the mRNA levels of MRP2 were very low in the esophagus. In addition, we found extremely low expression of Nrf2 in the esophagus at the basal and under the activated conditions, which might have resulted in low levels of glutamyl-cysteine ligase catalytic and modulatory subunits, and subsequently in the low levels of glutathione. Thus, the highest retention of arsenic was detected in the esophagus after intraperitoneal administration of As(III) to mice, and this appeared to result from multiple factors, including high expression of AQP3, low expression of MRP2, low capacity of glutathione synthesis and low activation of Nrf2.

Trypanosomatid Aquaporins: Roles in Physiology and Drug Response

In the class Kinetoplastida, we find an order of parasitic protozoans classified as Trypanosomatids. Three major pathogens form part of this order, Trypanosoma cruzi, Trypanosoma brucei, and Leishmania, which are responsible for disease and fatalities in millions of humans worldwide, especially in non-industrialized countries in tropical and sub-tropical regions. In order to develop new drugs and treatments, the physiology of these pathogenic protozoans has been studied in detail, specifically the significance of membrane transporters in host parasites interactions. Aquaporins and Aquaglyceroporins (AQPs) are a part of the major intrinsic proteins (MIPs) super-family. AQPs are characterized for their ability to facilitate the diffusion of water (aquaporin), glycerol (aquaglyceroporin), and other small-uncharged solutes. Furthermore, AQPs have been shown to allow the ubiquitous passage of some metalloids, such as trivalent arsenic and antimony. These trivalent metalloids are the active ingredient of a number of chemotherapeutic agents used against certain cancers and protozoan parasitic infections. Recently, the importance of the AQPs not only in osmotic adaptations but also as a factor in drug resistance of the trypanosomatid parasites has been reported. In this review, we will describe the physiological functions of aquaporins and their effect in drug response across the different trypanosomatids.

Flow cytometry-assisted rapid isolation of recombinant Plasmodium berghei parasites exemplified by functional analysis of aquaglyceroporin

The most critical bottleneck in the generation of recombinant Plasmodium berghei parasites is the mandatory in vivo cloning step following successful genetic manipulation. This study describes a new technique for rapid selection of recombinant P. berghei parasites. The method is based on flow cytometry to isolate isogenic parasite lines and represents a major advance for the field, in that it will speed the generation of recombinant parasites as well as cut down on animal use significantly. High expression of GFP during blood infection, a prerequisite for robust separation of transgenic lines by flow cytometry, was achieved. Isogenic recombinant parasite populations were isolated even in the presence of a 100-fold excess of wild-type (WT) parasites. Aquaglyceroporin (AQP) loss-of-function mutants and parasites expressing a tagged AQP were generated to validate this approach. aqp− parasites grow normally within the WT phenotypic range during blood infection of NMRI mice. Similarly, colonization of the insect vector and establishment of an infection after mosquito transmission were unaffected, indicating that AQP is dispensable for life cycle progression in vivo under physiological conditions, refuting its use as a suitable drug target. Tagged AQP localized to perinuclear structures and not the parasite plasma membrane. We suggest that flow-cytometric isolation of isogenic parasites overcomes the major roadblock towards a genome-scale repository of mutant and transgenic malaria parasite lines.

A Novel Aquaporin 3 in Killifish (Fundulus heteroclitus) Is Not An Arsenic Channel

The Atlantic killifish (Fundulus heteroclitus) is a model environmental organism that has an extremely low assimilation rate of environmental arsenic. As a first step in elucidating the mechanism behind this phenomenon, we used quantitative real-time PCR to identify aquaglyceroporins (AQPs), which are arsenite transporters, in the killifish gill. A novel homolog killifish AQP3 (kfAQP3a) was cloned from the killifish gill, and a second homolog was identified as the consensus from a transcriptome database (kfAQP3b). The two were 99% homologous to each other, 98% homologous to a previously identified killifish AQP3 from embryos (kfAQP3ts), and 78% homologous to hAQP3. Expression of kfAQP3a in Xenopus oocytes significantly enhanced water, glycerol, and urea transport. However, kfAQP3a expressed in HEK293T cells did not transport significant amounts of arsenic. All sequence motifs thought to confer the ability of AQP3 to transport solutes were conserved in kfAQP3a, kfAQP3b, and kfAQP3ts; however, the C-terminal amino acids were different in kfAQP3a versus the other two homologs. Replacement of the three C-terminal amino acids of kfAQP3 (GKS) with the three C-terminal amino acids of kfAQP3b and kfAQP3ts (ANC) was sufficient to enable kfAQP3a to robustly transport arsenic. Thus, the C-terminus of kfAQP3b and kfAQP3ts confers arsenic selectivity in kfAQP3. Moreover, kfAQP3a, the only AQP expressed in killifish gill, is the first aquaglyceroporin identified that does not transport arsenic, which may explain, in part, why killifish poorly assimilate arsenic and are highly tolerant to environmental arsenic.

Insulin- and Leptin-Mediated Control of Aquaglyceroporins in Human Adipocytes and Hepatocytes Is Mediated via the PI3K/Akt/mTOR Signaling Cascade

Glycerol constitutes an important metabolite for the control of lipid accumulation and glucose homeostasis. The impact of obesity and obesity-associated type 2 diabetes as well as the potential regulatory role of insulin and leptin on aquaglyceroporins (AQP) 3, 7, and 9 were analyzed. The tissue distribution and expression of AQP in biopsies of omental and sc adipose tissue as well as liver were analyzed in lean and obese Caucasian volunteers (n = 63). The effect of insulin (1, 10, and 100 nmol/liter) and leptin (0.1, 1, and 10 nmol/liter) on the expression of the glycerol channels was determined in vitro in human omental adipocytes and HepG2 hepatocytes. The translocation of AQP in response to insulin and isoproterenol was analyzed by immunocytochemistry. In addition to the well-known expression of AQP7 in adipose tissue, AQP3 and AQP9 were also expressed in both omental and sc adipose tissue. Obese type 2 diabetes patients showed higher expression of AQP in visceral adipose tissue and lower expression of AQP7 in sc adipose tissue and hepatic AQP9. The staining of AQP9 in the plasma membrane of adipocytes was reinforced by insulin, whereas isoproterenol induced the translocation of AQP3 and AQP7 from the lipid droplets to the plasma membrane. Insulin up-regulated all AQP, whereas leptin up-regulated AQP3 and down-regulated AQP7 and AQP9 in adipocytes and hepatocytes. These effects were abrogated by both the phosphatidylinositol 3-kinase inhibitor wortmannin and the mammalian target of rapamycin inhibitor rapamycin. Our findings show, for the first time, that insulin and leptin regulate the AQP through the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin pathway in human visceral adipocytes and hepatocytes. AQP3 and AQP7 may 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.

Arsenate transport by sodium/phosphate cotransporter type IIb

Arsenic is a metalloid that causes the dysfunction of critical enzymes, oxidative stress, and malignancies. In recent years several transporters of As III have been identified, including aquaglyceroporins (AQP) and multidrug resistance proteins (MRP). As V transport, however, has not been sufficiently studied because it has been assumed that arsenate is taken up by mammalian cells through inorganic phosphate (Pi) transporters. In this paper we have analyzed the role of Pi transporters in the uptake of arsenate by directly using 73As V as a radiotracer in phosphate transporter-expressing Xenopus laevis oocytes. The affinities of Pi transporters for H 3AsO 4 were lower than the affinities for Pi. NaPiIIa, NaPiIIc, Pit1, and Pit2 showed a K m for arsenate that was > 1 mM (i.e., at least ten times lower than the affinities for Pi). The NaPiIIb isoform showed the highest affinity for As V in mouse (57 μM), rat (51 μM), and human (9.7 μM), which are very similar to the affinities for Pi. Therefore, NaPiIIb can have a prominent role in the toxicokinetics of arsenic following oral exposure to freshwater or food contaminated with As V.

Assessing aquaglyceroporin gene status and expression profile in antimony-susceptible and -resistant clinical isolates of Leishmania donovani from India

Clinical resistance to pentavalent antimonials results from an interplay between uptake, efflux and sequestration in Leishmania. Aquaglyceroporins (AQPs) have been shown to facilitate uptake of trivalent metalloids. Down-regulation of AQP1 in Leishmania results in resistance to trivalent antimony, whereas overexpression of AQP1 in drug-resistant parasites can reverse the resistance. The present work investigates the role of AQP1 in monitoring antimonial resistance in Indian leishmaniasis. Susceptibility to trivalent antimony as determined in vitro with intracellular amastigotes from both visceral leishmaniasis (VL) and post-kala-azar dermal leishmaniasis (PKDL) patients correlated well with the clinical response. Higher accumulation of trivalent antimony (SbIII) was observed in all susceptible isolates compared with resistant isolates. Reduced accumulation of SbIII correlated, with a few exceptions, with down-regulation of AQP1 RNA as determined by real-time PCR. Cloning and sequencing of the AQP1 gene from both VL and PKDL isolates showed sequence variation in four of the clinical isolates. None of the isolates had an alteration of Glu152 and Arg230, which have been previously shown to affect metalloid transport. Transfection of the AQP1 gene in a sodium antimony gluconate-resistant field isolate conferred susceptibility to the resistant isolate. Our studies indicate genetic variation in VL and PKDL isolates. Down-regulation of AQP1 correlates well with clinical drug resistance in a majority of Indian VL and PKDL isolates. AQP1 gene expression at both the genetic and transcriptional level showed positive correlation with SbIII accumulation, with some exceptions.

Aquaglyceroporins are involved in uptake of arsenite into murine gastrointestinal tissues

Aquaglyceroporins (AQGPs) are members of aquaporin (AQP) family and belong to a subgroup of this water channel family; they are transmembrane proteins that transport water as well as glycerol and other solutes of small molecules. Recent studies have also identified that AQGPs are important transporters of trivalent metalloid in some mammalian cells. However, the uptake routes of arsenite in mammals are still less defined. In this study, to understand the routes of arsenite intake in mammals, mice were treated with Hg(II), glycerol, and As(III) and uptake of As(III) into the gastrointestinal tissues was measured. The level of inorganic arsenic (iAs) in gastrointestinal tissues after As(III) stimulation was much higher than Hg(II) +As(III) or glycerol+As(III) group. RT-PCR results showed that AQGPs were extensively expressed in gastrointestinal tissues of mice. We also treated Caco-2 cells with Hg(II) and As(III); the level of iAs in a group treated with Hg(II)+As(III) decreased compared with As(III)-treated group. Our results suggested that AQGPs could be important transporters in arsenite uptake into gastrointestinal tissues of mice, but more data are need to prove if AQGPs is the only pathway involved in As transport in mammals or just one of them.

Two distinct arsenite-resistant variants of Leishmania amazonensis take different routes to achieve resistance as revealed by comparative transcriptomics

Genome-wide search for the genes involved in arsenite resistance in two distinct variants A and A′ of Leishmania amazonensis revealed that the two variants used two different mechanisms to achieve resistance, eventhough these two variants were derived from the same clone and selected against arsenite under the same conditions. In variant A, the variant with DNA amplification, the biochemical pathways for detoxification of oxidative stress, the energy generation system to support the biochemical and physiological needs of the variant for DNA and protein synthesis and the arsenite translocating system to dispose arsenite are among the primary biochemical events that are upregulated under the arsenite stress to gain resistance. In variant A′, the variant without DNA amplification, the upregulation of aquaglyceroporin (AQP) gene and the high level of resistance to arsenate point to the direction that the resistance gained by the variant is due to arsenate which is probably oxidized from arsenite in the arsenite solution used for selection and the maintenance of the cell culture. As a result of the AQP upregulation for arsenite disposal, a different set of biochemical pathways for detoxification of oxidative stress, energy generation and cellular signaling are upregulated to sustain the growth of the variant to gain resistance to arsenate. From current evidences, reactive oxygen species (ROS) overproduced by the parasite soon after exposure to arsenite appear to play an instrumental role in both variants to initiate the subsequent biochemical events that allow the same clone of L. amazonensis to take two totally different routes to diverge into two different variants.

Enhanced Expression of Multidrug resistance-associated Protein 2 and Reduced Expression of Aquaglyceroporin 3 in an Arsenic-resistant Human Cell Line

Arsenic-resistant cells (R15), derived from a human lung adenocarcinoma cell line (CL3), were 10-fold more resistant to sodium arsenite (As(III)). Because R15 cells accumulated less arsenic than parental CL3 cells, this arsenic resistance may be due to higher efflux and/or lower uptake of As(III). We therefore compared expression of the multidrug resistance-associated proteins MRP1, MRP2, and MRP3 in these two cell lines. MRP2 expression was 5-fold higher in R15 cells than in CL3 cells, whereas MRP1 and MRP3 expression levels were similar. Furthermore, verapamil and cyclosporin A, inhibitors of multidrug resistance transporters, significantly reduced the efflux of arsenic from R15. Thus, increased arsenic extrusion by MRP2 may contribute to arsenic resistance in R15 cells. We also examined the expression of several aquaglyceroporins (AQPs), which mediate As(III) uptake by cells. Little AQP7 or AQP9 mRNA was detected by reverse transcription-PCR in either cell line, whereas AQP3 mRNA expression was 2-fold lower in R15 cells than in CL3 cells. When AQP3 expression in CL3 cells was knocked down by RNA interference, CL3 cells accumulated less arsenic and became more resistant to As(III). Conversely, overexpression of AQP3 in human embryonic kidney 293T cells increased arsenic accumulation, and the cells were more susceptible to As(III) than 293T cells transfected with vector alone. These results suggest that AQP3 is involved in As(III) accumulation. Taken together, our results suggest that enhanced expression of MRP2 and lower expression of AQP3 are responsible for lower arsenic accumulation in arsenic-resistant R15 cells.

Structure, Transport and Regulation of the NIP subfamily of plant multifunctional aquaglyceroporins

Structure, Transport and Regulation of the NIP subfamily of plant multifunctional aquaglyceroporins