Unraveling the Aquaporin-3 Inhibitory Effect of Rottlerin by Experimental and Computational Approaches.

The natural polyphenolic compound Rottlerin (RoT) showed anticancer properties in a variety of human cancers through the inhibition of several target molecules implicated in tumorigenesis, revealing its potential as an anticancer agent. Aquaporins (AQPs) are found overexpressed in different types of cancers and have recently emerged as promising pharmacological targets. Increasing evidence suggests that the water/glycerol channel aquaporin-3 (AQP3) plays a key role in cancer and metastasis. Here, we report the ability of RoT to inhibit human AQP3 activity with an IC50 in the micromolar range (22.8 ± 5.82 µM for water and 6.7 ± 2.97 µM for glycerol permeability inhibition). Moreover, we have used molecular docking and molecular dynamics simulations to understand the structural determinants of RoT that explain its ability to inhibit AQP3. Our results show that RoT blocks AQP3-glycerol permeation by establishing strong and stable interactions at the extracellular region of AQP3 pores interacting with residues essential for glycerol permeation. Altogether, our multidisciplinary approach unveiled RoT as an anticancer drug against tumors where AQP3 is highly expressed providing new information to aquaporin research that may boost future drug design.

Regulation of aquaporin-3 water permeability by hyaluronan.

Hyaluronan (HA) is a major component in the extracellular matrix and is responsible for maintaining the water content of the skin. However, the function and moisturizing mechanism at the atomic level of HA remain only partially understood. Investigating the interactions of HA and other skin components can help us understand how the former moisturizes the skin. Considering that aquaporin-3 (AQP3) is a protein responsible for transmembrane water transport in the human skin, we have, therefore, investigated the interactions of AQP3 and HA with different molecular weights using molecular dynamics simulations in the present work. Our results indicate that HA can adsorb onto AQP3 and decrease water mobility around the latter. In addition, the permeation rate of water through AQP3 can also be decreased by HA, and this phenomenon is particularly obvious for small molecular HA. Moreover, we found that large molecular HA can link two adjacent membranes in the extracellular matrix, increasing the adhesion between the membranes in the periplasm. The results of the present study indicate that HA is a natural regulator of AQP3, revealing the synergetic function of HA and AQP3 in the extracellular matrix of the skin.

The Aquaporin-3-Inhibiting Potential of Polyoxotungstates.

Polyoxometalates (POMs) are of increasing interest due to their proven anticancer activities. Aquaporins (AQPs) were found to be overexpressed in tumors bringing particular attention to their inhibitors as anticancer drugs. Herein, we report for the first time the ability of polyoxotungstates (POTs), such as of Wells-Dawson P2W18, P2W12, and P2W15, and Preyssler P5W30 structures, to affect aquaporin-3 (AQP3) activity and impair melanoma cell migration. The tested POTs were revealed to inhibit AQP3 function with different effects, with P2W18, P2W12, and P5W30 being the most potent (50% inhibitory concentration (IC50) = 0.8, 2.8, and 3.2 µM), and P2W15 being the weakest (IC50 > 100 µM). The selectivity of P2W18 toward AQP3 was confirmed in yeast cells transformed with human aquaglyceroporins. The effect of P2W12 and P2W18 on melanoma cells that highly express AQP3 revealed an impairment of cell migration between 55% and 65% after 24 h, indicating that the anticancer properties of these compounds may in part be due to the blockage of AQP3-mediated permeability. Altogether, our data revealed that P2W18 strongly affects AQP3 activity and cancer cell growth, unveiling its potential as an anticancer drug against tumors where AQP3 is highly expressed.

Constructing Supported Cell Membranes with Controllable Orientation.

Membrane proteins play important roles in various cellular processes. Methods that can retain their structure and membrane topology information during their characterization are desirable for understanding their structure-function behavior. Here, we use giant plasma membrane vesicles (GPMVs) to form the supported cell membrane and develop a blotting method to control the orientation of the deposited cell membrane in order to study membrane proteins from either the extracellular or the cytoplasmic sides. We show that the membrane orientation can be retained in the directly-deposited membrane and the deposited membrane on mica can be blotted onto glass to reverse the membrane orientation. We used Aquaporin 3 (AQP3), an abundant native transmembrane protein in Hela cells, as a target to examine the cell membrane orientation in the directly-deposited and reversed membrane platforms. The immunostaining of antibodies targeting either the cyto-domain or ecto-domain of AQP3 shows that the intracellular side of the cell membrane faced the bulk aqueous environment when the GPMVs spontaneously ruptured on the support and that the membrane orientation was reversed after blotting. With this blotting method, we can thus control the orientation of the supported cell membrane to study membrane protein functions and structures from either side of the cell plasma membrane.

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.

Gain-of-function mutations in Aqp3a influence zebrafish pigment pattern formation through the tissue environment.

The development of the pigmentation pattern in zebrafish is a tightly regulated process that depends on both the self-organizing properties of pigment cells and extrinsic cues from other tissues. Many of the known mutations that alter the pattern act cell-autonomously in pigment cells, and our knowledge about external regulators is limited. Here, we describe novel zebrafish mau mutants, which encompass several dominant missense mutations in Aquaporin 3a (Aqp3a) that lead to broken stripes and short fins. A loss-of-function aqp3a allele, generated by CRISPR-Cas9, has no phenotypic consequences, demonstrating that Aqp3a is dispensable for normal development. Strikingly, the pigment cells from dominant mau mutants are capable of forming a wild-type pattern when developing in a wild-type environment, but the surrounding tissues in the mutants influence pigment cell behaviour and interfere with the patterning process. The mutated amino acid residues in the dominant alleles line the pore surface of Aqp3a and influence pore permeability. These results demonstrate an important effect of the tissue environment on pigment cell behaviour and, thereby, on pattern formation.

Aquaglyceroporins 3 and 7 in bull spermatozoa: identification, localisation and their relationship with sperm cryotolerance.

The present study aimed to determine the localisation of aquaglyceroporins 3 (AQP3) and 7 (AQP7) in bull spermatozoa and their relationship with the sperm cell's resilience to withstand cryopreservation (i.e. cryotolerance). A total of 18 bull ejaculates were cryopreserved and their sperm quality analysed before and after freeze-thawing. The presence and localisation of AQP3 and AQP7 was determined through immunoblotting and immunocytochemistry. AQP3 was found in the mid-piece and AQP7 in the mid-piece and post-acrosomal region of bull spermatozoa. Immunoblotting showed specific signal bands at 30 and 60kDa for AQP3 and at 25kDa for AQP7. Neither the relative abundance of AQP3 and AQP7 nor their localisation patterns was altered by cryopreservation but individual differences between bull ejaculates were found in immunoblots. In order to determine whether these individual differences were related to sperm cryotolerance, bull ejaculates were classified as having good (GFE) or poor freezability (PFE) on the basis of their sperm quality after thawing. While the relative abundance of AQP3 before cryopreservation did not differ between ejaculates with GFE and PFE, the abundance of AQP7 was higher in GFE than in PFE ejaculates. This finding was further confirmed through principal component and linear regression analyses. In conclusion, the relative abundance of AQP7 in fresh semen may be used as a marker to predict bull sperm cryotolerance.

Expression, localization and possible functions of aquaporins 3 and 8 in rat digestive system.

Although aquaporins (AQPs) play important roles in transcellular water movement, their precise quantification and localization remains controversial. We investigated expression levels and localizations of AQP3 and AQP8 and their possible functions in the rat digestive system using real-time polymerase chain reactions, western blot analysis and immunohistochemistry. We investigated the expression levels and localizations of AQP3 and AQP8 in esophagus, forestomach, glandular stomach, duodenum, jejunum, ileum, proximal and distal colon, and liver. AQP3 was expressed in the basolateral membranes of stratified epithelia (esophagus and forestomach) and simple columnar epithelia (glandular stomach, ileum, and proximal and distal colon). Expression was particularly abundant in the esophagus, and proximal and distal colon. AQP8 was found in the subapical compartment of columnar epithelial cells of the jejunum, ileum, proximal colon and liver; the most intense staining occurred in the jejunum. Our results suggest that AQP3 and AQP8 play significant roles in intestinal function and/or fluid homeostasis and may be an important subject for future investigation of disorders that involve disruption of intestinal fluid homeostasis, such as inflammatory bowel disease and irritable bowel syndrome.

Exploring the gating mechanisms of aquaporin-3: new clues for the design of inhibitors?

The pH gating of human AQP3 and its effects on both water and glycerol permeabilities have been fully characterized for the first time using a human red blood cell model (hRBC). For comparison, the effects of pH on the gating of rat AQP3 have also been characterized in yeast. The obtained results highlight similarities as well as differences between the two isoforms. In addition, we investigated the molecular mechanism of hAQP3 pH gating in silico, which may disclose new pathways to AQP regulation by small molecule inhibitors, and therefore may be important for drug development.

The AQP-3 water channel is a pivotal modulator of glycerol-induced chloride channel activation in nasopharyngeal carcinoma cells.

Aquaporin (AQP) and chloride channels are ubiquitous in virtually all living cells, playing pivotal roles in cell proliferation, migration and apoptosis. We previously reported that AQP-3 aquaglyceroporin and ClC-3 chloride channels could form complexes to regulate cell volume in nasopharyngeal carcinoma cells. In this study, the roles of AQP-3 in their hetero-complexes were further investigated. Glycerol entered the cells via AQP-3 and induced two different Cl(-) currents through cell swelling-dependent or -independent pathways. The swelling-dependent Cl(-) current was significantly inhibited by pretreatment with CuCl2 and AQP-3-siRNA. After siRNA-induced AQP-3 knock-down, the 140 mM glycerol isoosmotic solution swelled cells by 22% (45% in AQP-3-intact cells) and induced a smaller Cl(-) current; this current was smaller than that activated by 8% cell volume swelling, which induced by the 140 mM glycerol hyperosmotic solution in AQP-3-intact cells. This suggests that the interaction between AQP-3 and ClC-3 plays an important role in cell volume regulation and that AQP-3 may be a modulator that opens volume-regulated chloride channels. The swelling-independent Cl(-) current, which was activated by extracellular glycerol, was reduced by CuCl2 and AQP-3-siRNA pretreatment. Dialyzing glycerol into cells via the pipette directly induced the swelling-independent Cl(-) current; however this current was blocked by AQP-3 down-regulation, suggesting AQP-3 is essential for the opening of chloride channels. In conclusion, AQP-3 is the pathway for water, glycerol and other small solutes to enter cells, and it may be an essential modulator for the gating of chloride channels.

Structural Determinants of Oligomerization of the Aquaporin-4 Channel.

The aquaporin (AQP) family of integral membrane protein channels mediate cellular water and solute flow. Although qualitative and quantitative differences in channel permeability, selectivity, subcellular localization, and trafficking responses have been observed for different members of the AQP family, the signature homotetrameric quaternary structure is conserved. Using a variety of biophysical techniques, we show that mutations to an intracellular loop (loop D) of human AQP4 reduce oligomerization. Non-tetrameric AQP4 mutants are unable to relocalize to the plasma membrane in response to changes in extracellular tonicity, despite equivalent constitutive surface expression levels and water permeability to wild-type AQP4. A network of AQP4 loop D hydrogen bonding interactions, identified using molecular dynamics simulations and based on a comparative mutagenic analysis of AQPs 1, 3, and 4, suggest that loop D interactions may provide a general structural framework for tetrameric assembly within the AQP family.

The inhibition of glycerol permeation through aquaglyceroporin-3 induced by mercury(II): A molecular dynamics study.

Mercurial compounds are known to inhibit water permeation through aquaporins (AQPs). Although in the last years some hypotheses were proposed, the exact mechanism of inhibition is still an open question and even less is known about the inhibition of the glycerol permeation through aquaglyceroporins. Molecular dynamics (MD) simulations of human aquaporin-3 (AQP3) have been performed up to 200ns in the presence of Hg(2+) ions. For the first time, we have observed the unbiased passage of a glycerol molecule from the extracellular to cytosolic side. Moreover, the presence of Hg(2+) ions covalently bound to Cys40 leads to a collapse of the aromatic/arginine selectivity filter (ar/R SF), blocking the passage of both glycerol and water. Interestingly, the local conformational changes of the protein follow mercury coordination by water and by aminoacidic donor atoms. Overall, the obtained results are important to improve the design of selective AQP inhibitors for future therapeutic and imaging applications.

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.

Easy measurement of diffusion coefficients of EGFP-tagged plasma membrane proteins using k-Space Image Correlation Spectroscopy.

Lateral diffusion and compartmentalization of plasma membrane proteins are tightly regulated in cells and thus, studying these processes will reveal new insights to plasma membrane protein function and regulation. Recently, k-Space Image Correlation Spectroscopy (kICS)(1) was developed to enable routine measurements of diffusion coefficients directly from images of fluorescently tagged plasma membrane proteins, that avoided systematic biases introduced by probe photophysics. Although the theoretical basis for the analysis is complex, the method can be implemented by nonexperts using a freely available code to measure diffusion coefficients of proteins. kICS calculates a time correlation function from a fluorescence microscopy image stack after Fourier transformation of each image to reciprocal (k-) space. Subsequently, circular averaging, natural logarithm transform and linear fits to the correlation function yields the diffusion coefficient. This paper provides a step-by-step guide to the image analysis and measurement of diffusion coefficients via kICS. First, a high frame rate image sequence of a fluorescently labeled plasma membrane protein is acquired using a fluorescence microscope. Then, a region of interest (ROI) avoiding intracellular organelles, moving vesicles or protruding membrane regions is selected. The ROI stack is imported into a freely available code and several defined parameters (see Method section) are set for kICS analysis. The program then generates a "slope of slopes" plot from the k-space time correlation functions, and the diffusion coefficient is calculated from the slope of the plot. Below is a step-by-step kICS procedure to measure the diffusion coefficient of a membrane protein using the renal water channel aquaporin-3 tagged with EGFP as a canonical example.

Measuring localization and diffusion coefficients of basolateral proteins in lateral versus basal membranes using functionalized substrates and kICS analysis.

Micropatterning enabled semiquantitation of basolateral proteins in lateral and basal membranes of the same cell. Lateral diffusion coefficients of basolateral aquaporin-3 (AQP3-EGFP) and EGFP-AQP4 were extracted from "lateral" and "basal" membranes using identical live-cell imaging and k-space Image Correlation Spectroscopy (kICS). To simultaneously image proteins in "lateral" and "basal" membranes, micropatterning with the extracellular domain of E-cadherin and collagen, to mimic cell-cell and cell-extracellular matrix (ECM) adhesion, respectively, was used. In kidney collecting duct principal cells AQP3 localizes lateral and basal whereas AQP4 localizes mainly basal. On alternating stripes of E-cadherin and collagen, AQP3-EGFP was predominantly localized to "lateral" compared to "basal" membranes, whereas Orange-AQP4 was evenly distributed. Average diffusion coefficients were extracted via kICS analysis of rapid time-lapse sequences of AQP3-EGFP and EGFP-AQP4 on uniform substrates of either E-cadherin or collagen. AQP3-EGFP was measured to 0.022±0.010µm(2)/s on E-cadherin and 0.019±0.004µm(2)/s on collagen, whereas EGFP-AQP4 was measured to 0.044±0.009µm(2)/s on E-cadherin and 0.037±0.009µm(2)/s on collagen, thus, diffusion did not differ between substrates. Cholesterol depletion by methyl-beta-cyclodextrin (MBCD) reduced the AQP3-EGFP diffusion coefficient by 43% from 0.024±0.007µm(2)/s (water) to 0.014±0.003µm(2)/s (MBCD) (p<0.05) on collagen surfaces, and by 41% from 0.023±0.011µm(2)/s (water) to 0.014±0.005µm(2)/s (MBCD) (p<0.05) on E-cadherin surfaces. Thus, protein patterning enables the semiquantitation of protein distribution between the "lateral" and "basal" membranes as well as measurements of lateral diffusion coefficients.

Preparative scale production and functional reconstitution of a human aquaglyceroporin (AQP3) using a cell free expression system.

Understanding the selectivity of aquaporin (AQP) membrane channels and exploiting their biotechnological potential will require structural and functional studies of wild type and modified proteins; however, expression systems have not previously yielded AQPs in the necessary milligrams quantities. Cell free (CF) systems have emerged in recent years as fast, efficient and versatile technologies for the production of high quality membrane proteins. Here, we establish a convenient method to synthesize large amounts of functional human aquaglyceroporin 3 protein (AQP3), an AQP of physiological relevance conducting glycerol and some small neutral solutes besides water. Milligram amounts of AQP3 were produced as a histidine-tagged protein (hAQP3-6His) in an Escherichia coli extract-based CF system in the presence of the non-ionic detergent Brij-98. The recombinant AQP3 was purified by affinity chromatography, incorporated into liposomes and evaluated functionally by stopped-flow light scattering. Correct protein folding was indicated by the high glycerol and water permeability exhibited by the hAQP3-6His proteoliposomes as compared to empty control liposomes. Functionality of hAQP3-6His was further confirmed by the strong inhibition of the glycerol and water permeability by phloretin and HgCl2, respectively, two blockers of AQP3. Fast and convenient CF production of functional AQP3 may serve as basis for further structural/functional assessment of aquaglyceroporins and help boosting the AQP-based biomimetic technologies.

Immunolocalization of water channel aquaporins in human knee articular cartilage with intact and early degenerative regions.

Aquaporins (AQPs), a family of water channel proteins expressed in various cells and tissues, serve as physiological pathways of water and small solute transport. Articular cartilage is avascular tissue with unique biomechanical structure, a major component of which is "water". Our objective is to investigate the immunolocalization and expression pattern changes of AQPs in articular cartilage with normal and early degenerative regions in the human knee joint, which is the joint most commonly involved in osteoarthritis (OA). Two isoforms (AQPs 1 and 3) of AQPs were examined by immunohistochemical analyses using isoform-specific antibodies with cartilage samples from OA patients undergoing total knee arthroplasty. AQP 1 and AQP 3 were expressed in human knee articular cartilage and were localized in chondrocytes, both in the intact and early degenerative cartilage regions. Compared to the intact cartilage, both AQP 1 and AQP 3 immunopositive cells were observed at the damaged surface area in the degenerative region. These findings suggest that these AQPs play roles in metabolic water regulation in articular cartilage of load bearing joints and that they are responsible for OA onset.

Targeting aquaporin function: potent inhibition of aquaglyceroporin-3 by a gold-based compound.

Aquaporins (AQPs) are membrane channels that conduct water and small solutes such as glycerol and are involved in many physiological functions. Aquaporin-based modulator drugs are predicted to be of broad potential utility in the treatment of several diseases. Until today few AQP inhibitors have been described as suitable candidates for clinical development. Here we report on the potent inhibition of AQP3 channels by gold(III) complexes screened on human red blood cells (hRBC) and AQP3-transfected PC12 cells by a stopped-flow method. Among the various metal compounds tested, Auphen is the most active on AQP3 (IC(50) = 0.8±0.08 µM in hRBC). Interestingly, the compound poorly affects the water permeability of AQP1. The mechanism of gold inhibition is related to the ability of Au(III) to interact with sulphydryls groups of proteins such as the thiolates of cysteine residues. Additional DFT and modeling studies on possible gold compound/AQP adducts provide a tentative description of the system at a molecular level. The mapping of the periplasmic surface of an homology model of human AQP3 evidenced the thiol group of Cys40 as a likely candidate for binding to gold(III) complexes. Moreover, the investigation of non-covalent binding of Au complexes by docking approaches revealed their preferential binding to AQP3 with respect to AQP1. The high selectivity and low concentration dependent inhibitory effect of Auphen (in the nanomolar range) together with its high water solubility makes the compound a suitable drug lead for future in vivo studies. These results may present novel metal-based scaffolds for AQP drug development.

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.

Compartmentalization of aquaporins in the human intestine.

Improper localization of water channel proteins called aquaporins (AQP) induce mucosal injury which is implicated in Crohn's disease and ulcerative colitis. The amino acid sequences of AQP3 and AQP10 are 79% similar and belong to the mammalian aquaglyceroporin subfamily. AQP10 is localized on the apical compartment of the intestinal epithelium called the glycocalyx while AQP3 is selectively targeted to the basolateral membrane. Despite the high sequence similarity and evolutionary relatedness, the molecular mechanism involved in the polarity, selective targeting and function of AQP3 and AQP10 in the intestine is largely unknown. Our hypothesis is that the differential polarity and selective targeting of AQP3 and AQP10 in the intestinal epithelial cells is influenced by amino acid signal motifs. We performed sequence and structural alignments to determine differences in signals for localization and posttranslational glycosylation. The basolateral sorting motif "YRLL" is present in AQP3 but absent in AQP10; while Nglycosylation signals are present in AQP10 but absent in AQP3. Furthermore, the C-terminal region of AQP3 is longer compared to AQP10. The sequence and structural differences between AQP3 and AQP10 provide insights into the differential compartmentalization and function of these two aquaporins commonly expressed in human intestines.