Molecular basis of pH and Ca2+ regulation of aquaporin water permeability

Aquaporins facilitate the diffusion of water across cell membranes. We previously showed that acid pH or low Ca(2+) increase the water permeability of bovine AQP0 expressed in Xenopus oocytes. We now show that external histidines in loops A and C mediate the pH dependence. Furthermore, the position of histidines in different members of the aquaporin family can "tune" the pH sensitivity toward alkaline or acid pH ranges. In bovine AQP0, replacement of His40 in loop A by Cys, while keeping His122 in loop C, shifted the pH sensitivity from acid to alkaline. In the killifish AQP0 homologue, MIPfun, with His at position 39 in loop A, alkaline rather than acid pH increased water permeability. Moving His39 to His40 in MIPfun, to mimic bovine AQP0 loop A, shifted the pH sensitivity back to the acid range. pH regulation was also found in two other members of the aquaporin family. Alkaline pH increased the water permeability of AQP4 that contains His at position 129 in loop C. Acid and alkaline pH sensitivity was induced in AQP1 by adding histidines 48 (in loop A) and 130 (in loop C). We conclude that external histidines in loops A and C that span the outer vestibule contribute to pH sensitivity. In addition, we show that when AQP0 (bovine or killifish) and a crippled calmodulin mutant were coexpressed, Ca(2+) sensitivity was lost but pH sensitivity was maintained. These results demonstrate that Ca(2+) and pH modulation are separable and arise from processes on opposite sides of the membrane.     

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.     

Light inactivation of water transport and protein-protein interactions of aquaporin-Killer Red chimeras

Aquaporins (AQPs) have a broad range of cellular and organ functions; however, nontoxic inhibitors of AQP water transport are not available. Here, we applied chromophore-assisted light inactivation (CALI) to inhibit the water permeability of AQP1, and of two AQP4 isoforms (M1 and M23), one of which (M23) forms aggregates at the cell plasma membrane. Chimeras containing Killer Red (KR) and AQPs were generated with linkers of different lengths. Osmotic water permeability of cells expressing KR/AQP chimeras was measured from osmotic swelling-induced dilution of cytoplasmic chloride, which was detected using a genetically encoded chloride-sensing fluorescent protein. KR-AQP1 red fluorescence was bleached rapidly (~10% per second) by wide-field epifluorescence microscopy. After KR bleaching, KR-AQP1 water permeability was reduced by up to 80% for the chimera with the shortest linker. Remarkably, CALI-induced reduction in AQP4-KR water permeability was approximately twice as efficient for the aggregate-forming M23 isoform; this suggests intermolecular CALI, which was confirmed by native gel electrophoresis on cells coexpressing M23-AQP4-KR and myc-tagged M23-AQP4. CALI also disrupted the interaction of AQP4 with a neuromyelitis optica autoantibody directed against an extracellular epitope on AQP4. CALI thus permits rapid, spatially targeted and irreversible reduction in AQP water permeability and interactions in live cells. Our data also support the utility of CALI to study protein-protein interactions as well as other membrane transporters and receptors.     

Water permeability and characterization of aquaporin-11

The water permeability of aquaporin-11 (AQP11), which has a cysteine substituted for an alanine at a highly conserved asparagine-proline-alanine (NPA) motif in the water channel family, is controversial. Our previous study, however, showed that AQP11 is water permeable in proteoliposomes in which AQP11 molecules were reconstituted after purification with Fos-choline 10, which is the most suitable detergent available for stable solubilization of AQP11. In our previous study, we were unable to exclude the effect of the detergent on the water conductance. Therefore, in the present study, we measured the water permeability of AQP11 without detergent using vesicles that directly formed from Sf9 cell membranes expressing AQP11 molecules. The water permeability of AQP11 was 8-fold lower than that of AQP1 and 3-fold higher than that of mock-infected cell membrane, and was reversibly inhibited by mercury ions. Considering the slow but constant water permeable functions of AQP11, we performed homology modeling to search for a common structural feature. When comparing our model with those of other AQP structures, we found that Tyr83 facing the channel pore might be a key amino acid residue that decreases the water permeation of AQP11. Our findings indicate that AQP11 could be involved in slow but constant water movement across the membrane.     

Comparative simulations of aquaporin family: AQP1, AQPZ, AQP0 and GlpF

Molecular dynamics simulations were performed for four members of the aquaporin family (AQP1, AQPZ, AQP0, and GlpF) in the explicit membrane environment. The single-channel water permeability, pf, was evaluated to be GlpF approximately AQPZ > AQP1 > AQP0, while their relative pore sizes were GlpF > AQP1 > AQPZ > AQP0. This relation between pf and pore size indicates that water permeability was determined not only by the channel radius, but also another competing factor. Analysis of water dynamics revealed that this factor was the single-file nature of water transport.     

Annexin A4 reduces water and proton permeability of model membranes but does not alter aquaporin 2-mediated water transport in isolated endosomes

Annexin A4 (Anx4) belongs to a ubiquitous family of Ca2+-dependent membrane-binding proteins thought to be involved in membrane trafficking and membrane organization within cells. Anx4 localizes to the apical region in epithelia; however, its physiological role is unclear. We show that Anx4 exhibited binding to liposomes (phosphatidylcholine:phosphatidylserine, 1:1) in the presence of Ca2+ and binding was reversible with EDTA. Anx4 binding resulted in liposome aggregation and a reduction in membrane water permeability of 29% (P < 0.001) at 25 degrees C. These effects were not seen in the presence of Ca2+ or Anx4 alone and were reversible with EDTA. Measurements of membrane fluidity made by monitoring fluorescence anisotropy of 2-(12-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dodecanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine (NBD-HPC) demonstrated that Anx4 binding rigidified the outer leaflet of the bilayer (P < 0.001), thus providing a molecular explanation for the inhibition of water flux. To determine whether Anx4 would produce similar effects on physiological membranes we constructed liposomes which recapitulated the lipid composition of the inner leaflet of the MDCK apical membrane. These membranes exhibited reductions to water permeability upon Anx4 binding (19.5% at 25 degrees C, 31% at 37 degrees C; P < 0.01 and P < 0.001, respectively) and to proton permeability (15% at 25 degrees C, 19.5% at 37 degrees C; P < 0.05). Since our in vitro experiments indicated an effect on membrane permeability, we examined localization of Anx4 in the kidney collecting duct, a region of the nephron responsible for concentrating urine through water reabsorbtion. Anx4 was shown to colocalize apically with aquaporin 2 (AQP2) in collecting duct epithelia. To test for the existence of a functional interaction between Anx4 and AQP2 we isolated AQP2-containing endosomes and exposed them to Anx4/Ca2+. Water flux rates were unchanged, indicating Anx4 does not directly regulate AQP2. We conclude that Anx4 can alter the physical properties of membranes by associating with them and regulate passive membrane permeability to water and protons. These properties represent important new functions for Anx4.     

Influence of the Cytoplasmic Domains of Aquaporin-4 on Water Conduction and Array Formation

Phosphorylation of Ser180 in cytoplasmic loop D has been shown to reduce the water permeability of aquaporin (AQP) 4, the predominant water channel in the brain. However, when the structure of the S180D mutant (AQP4M23S180D), which was generated to mimic phosphorylated Ser180, was determined to 2.8 Å resolution using electron diffraction patterns, it showed no significant differences from the structure of the wild-type channel. High-resolution density maps usually do not resolve protein regions that are only partially ordered, but these can sometimes be seen in lower-resolution density maps calculated from electron micrographs. We therefore used images of two-dimensional crystals and determined the structure of AQP4M23S180D at 10 Å resolution. The features of the 10-Å density map are consistent with those of the previously determined atomic model; in particular, there were no indications of any obstruction near the cytoplasmic pore entrance. In addition, water conductance measurements, both in vitro and in vivo, show the same water permeability for wild-type and mutant AQP4M23, suggesting that the S180D mutation neither reduces water conduction through a conformational change nor reduces water conduction by interacting with a protein that would obstruct the cytoplasmic channel entrance. Finally, the 10-Å map shows a cytoplasmic density in between four adjacent tetramers that most likely represents the association of four N termini. This finding supports the critical role of the N terminus of AQP4 in the stabilization of orthogonal arrays, as well as their interference through lipid modification of cysteine residues in the longer N-terminal isoform.     

Quaternary ammonium compounds as water channel blockers. Specificity, potency, and site of action

Excessive water uptake through Aquaporins (AQP) can be life-threatening and reversible AQP inhibitors are needed. Here, we determined the specificity, potency, and binding site of tetraethylammonium (TEA) to block Aquaporin water permeability. Using oocytes, externally applied TEA blocked AQP1/AQP2/AQP4 with IC50 values of 1.4, 6.2, and 9.8 microM, respectively. Related tetraammonium compounds yielded some (propyl) or no (methyl, butyl, or pentyl) inhibition. TEA inhibition was lost upon a Tyr to Phe amino acid switch in the external water pore of AQP1/AQP2/AQP4, whereas the water permeability of AQP3 and AQP5, which lack a corresponding Tyr, was not blocked by TEA. Consistent with experimental data, multi-nanosecond molecular dynamics simulations showed one stable binding site for TEA, but not tetramethyl (TMA), in AQP1, resulting in a nearly 50% water permeability inhibition, which was reduced in AQP1-Y186F due to effects on the TEA inhibitory binding region. Moreover, in the simulation TEA interacted with charged residues in the C (Asp128) and E (Asp185) loop, and the A(Tyr37-Asn42-Thr44) loop of the neighboring monomer, but not directly with Tyr186. The loss of TEA inhibition in oocytes expressing properly folded AQP1-N42A or -T44A is in line with the computationally predicted binding mode. Our data reveal that the molecular interaction of TEA with AQP1 differs and is about 1000-fold more effective on AQPs than on potassium channels. Moreover, the observed experimental and simulated similarities open the way for rational design and virtual screening for AQP-specific inhibitors, with quaternary ammonium compounds in general, and TEA in particular as a lead compound.     

The structure of aquaporins

The ubiquitous members of the aquaporin (AQP) family form transmembrane pores that are either exclusive for water (aquaporins) or are also permeable for other small neutral solutes such as glycerol (aquaglyceroporins). The purpose of this review is to provide an overview of our current knowledge of AQP structures and to describe the structural features that define the function of these membrane pores. The review will discuss the mechanisms governing water conduction, proton exclusion and substrate specificity, and how the pore permeability is regulated in different members of the AQP family.     

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

Background information. Mercurials inhibit AQPs (aquaporins), and site‐directed mutagenesis has identified Cys¹⁸⁹ 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 Cys¹⁸⁹ 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 rAPR4 (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 μM 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 Cys¹⁷⁸, 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.     

Aquaporin-4 (AQP4) Associations and Array Dynamics Probed by Photobleaching and Single-molecule Analysis of Green Fluorescent Protein-AQP4 Chimeras

The plasma membrane assembly of aquaporin-4 (AQP4) water channels into orthogonal arrays of particles (OAPs) involves interactions of AQP4 N-terminal domains. To study in live cells the site of OAP assembly, the size and dynamics of plasma membrane OAPs, and the heterotetrameric associations of AQP4, we constructed green fluorescent protein (GFP)-labeled AQP4 “long” (M1) and “short” (M23) isoforms in which GFP was inserted at the cytoplasm-facing N or C terminus or between Val-141 and Val-142 in the second extracellular loop of AQP4. The C-terminal and extracellular loop GFP insertions did not interfere with the rapid unrestricted membrane diffusion of GFP-labeled M1 or the restricted diffusion and OAP assembly of GFP-labeled M23. Photobleaching of brefeldin A-treated cells showed comparable and minimally restricted diffusion of M1 and M23, indicating that OAP assembly occurs post-endoplasmic reticulum. Single-molecule step photobleaching and intensity analysis of GFP-labeled M1 in the absence versus presence of excess unlabeled M1 or M23 with an OAP-disrupting mutation indicated heterotetrameric AQP4 association. Time-lapse total internal reflection fluorescence imaging of M23 in live cells at 37 °C indicated that OAPs diffuse slowly (D ∼ 10⁻¹² cm²/s) and rearrange over tens of minutes. Our biophysical measurements in live cells thus reveal extensive AQP4 monomer-monomer and tetramer-tetramer interactions.     

Implications of the aquaporin-4 structure on array formation and cell adhesion

Aquaporin-4 (AQP4) is the predominant water channel in the mammalian brain and an important drug target for treatment of cerebral edema, bipolar disorder and mesial temporal lobe epilepsy. We determined the AQP4 structure by electron crystallography of double-layered, two-dimensional (2D) crystals. The structure allows us to discuss how the expression ratio between the long and short AQP4 splicing variant can determine the size of in vivo orthogonal arrays. Furthermore, AQP4 contains a short 3(10) helix in an extracellular loop, which mediates weak but specific interactions between AQP4 molecules in adjoining membranes. This finding suggests a previously unexpected role for AQP4 in cell adhesion. This notion was corroborated by expression of AQP4 in L-cells, which resulted in clustering of the cells. Our AQP4 structure thus enables us to propose models for the size regulation of orthogonal arrays and channel-mediated cell adhesion.     

Noncanonical binding of calmodulin to aquaporin-0: implications for channel regulation

Aquaporins (AQPs) are a family of ubiquitous membrane channels that conduct water across cell membranes. AQPs form homotetramers containing four functional and independent water pores. Aquaporin-0 (AQP0) is expressed in the eye lens, where its water permeability is regulated by calmodulin (CaM). Here we use a combination of biochemical methods and NMR spectroscopy to probe the interaction between AQP0 and CaM. We show that CaM binds the AQP0 C-terminal domain in a calcium-dependent manner. We demonstrate that only two CaM molecules bind a single AQP0 tetramer in a noncanonical fashion, suggesting a form of cooperativity between AQP0 monomers. Based on these results, we derive a structural model of the AQP0/CaM complex, which suggests CaM may be inhibitory to channel permeability by capping the vestibules of two monomers within the AQP0 tetramer. Finally, phosphorylation within AQP0's CaM binding domain inhibits the AQP0/CaM interaction, suggesting a temporal regulatory mechanism for complex formation.     

Vasopressin-dependent water permeability of the basolateral membrane of the kidney outer medullary collecting duct in postnatal ontogenesis in rats

Kidneys of new-born animals are resistant to arginine vasopressin (AVP). The ability of the hormone to regulate water permeability of the collecting duct can be seen from weaning period, probably due to the maturation of the intracellular signaling pathway. The purpose of the present work was to investigate the effect of V2 receptor agonist dDAVP on the water permeability of OMCD basolateral membrane in 10-, 22- and 60-day old Wistar rats. We also estimated ontogenetic gene expression of AQP2, AQP3, AQP4 and V2 receptor. Osmotic water permeability (Pf) of the basolateral membrane of microdissected OMCD was measured under control conditions and after incubation with the agonist V2 receptor desmopressin (dDAVP; 10(-7) M). Water permeability in 10- and 22-day old rats under control conditions were significantly higher than in adults. Desmopressin stimulated significant increase of this parameter in 22-day old pups (Pf = = 125 +/- 4.85; Pf = 174 +/- 8.2 microns/s, p < 0.001) and adult rats (Pf = 100.5 +/- 7.38; Pf = 178.8 +/- 9.54 microns/s, p < 0.001). Osmotic water permeability of the OMCD basolateral membrane in 10-day old rats does not depend on dDAVP (Pf = 172.5 +/- 23.8; Pf = 164.8 +/- 34 microns/s). With the RT-PCR, we observed a gradual increase of AQP2 and V2 receptor genes expression during postnatal ontogenesis. The gene expression of AQP3 and AQP4 remained unchanged during postnatal ontogenesis. In general, the water permeability of the OMCD basolateral membrane of rats can be stimulated by AVP since the 22nd day of postnatal life. The water permeability of the OMCD basolateral membrane under control conditions gradually decreased during postnatal development, while gene expression of AQP3 and AQP4 was unchanged. The mechanism of this decrease remains to be established.     

Detection of aquaporin-2 in the plasma membranes of oocytes: a novel isolation method with improved yield and purity

Aquaporin-2 (AQP2) water channel mutations cause autosomal recessive and dominant nephrogenic diabetes insipidus (NDI). Expressed in oocytes, a mutant in dominant (AQP2-E258K), but not in recessive (AQP2-R187C), NDI conferred a specific dominant-negative effect on wild-type (wt) AQP2 water permeability (Pf) only at low expression levels. Since at these levels, the yield of conventional-isolated plasma membranes was too low, an improved technique to semiquantify AQP2 in the plasma membrane was needed. Antibodies against the C-loop of AQP2 were not applicable since they were unspecific and introduction of a tag into this loop caused misfolding and ER retardation. Membrane-impermeable biotin analogues turned out to label intracellular AQP2 proteins. Therefore, a method has been developed which generates a high yield of nearly pure plasma membranes, which enables semiquantification of plasma membrane proteins expressed at low levels in oocytes. Our new method allows for phenotype-genotype correlation studies in a wide range of channelopathies.     

Expression and immunolocalization of aquaporin water channels in rat exocrine pancreas

Both the acinar and ductal cells of the pancreas secrete a near-isotonic fluid and may thus be sites of aquaporin (AQP) water channel expression. Northern blot analysis of mRNA from whole rat pancreas revealed high levels of AQP1 and AQP8 expression, whereas lower levels of AQP4 and AQP5 expression were just detectable by RT-PCR Southern blot analysis. Immunohistochemistry showed that AQP1 is localized in the microvasculature, whereas AQP8 is confined to the apical pole of the acinar cells. No labeling of acinar, ductal, or vascular tissue was detected with antibodies to AQP2-7. With immunoelectron microscopy, AQP8 labeling was observed not only at the apical membrane of the acinar cells but also among small intracellular vesicles in the subapical cytoplasm, suggesting that there may be regulated trafficking of AQP8 to the apical plasma membrane. To evaluate the contribution of AQPs to the membrane water permeability, video microscopy was used to measure the swelling of acinar cells in response to hypotonic stress. Osmotic water permeability was reduced by 90% following exposure to Hg(2+). Since AQP8 is confined to the apical membrane, the marked effect of Hg(2+) suggests that other water channels may be expressed in the basolateral membrane.     

Identification of a novel aquaporin, AQP12, expressed in pancreatic acinar cells

Members of the aquaporin (AQP) water channel family are widely distributed in various tissues and contribute to the water permeability of epithelial and endothelial cells. Currently 11 members of the AQP family (AQP0-10) have been reported in mammals. Here we report the identification of AQP12, which we found by performing a BLAST program search. Northern blot analysis revealed that AQP12 was specifically expressed in the pancreas. Further analysis by in situ hybridization and RT-PCR studies showed that AQP12 was selectively localized in the acinar cells of the pancreas. To analyze the cellular localization and function of AQP12, we expressed AQP12 in Xenopus oocytes and cultured mammalian cells. Immunocytochemistry revealed that AQP12 was not targeted to the plasma membrane. The selective localization of AQP12 in pancreatic acinar cells and possibly in the intracellular organelles suggests a role of AQP12 in digestive enzyme secretion such as maturation and exocytosis of secretory granules.     

Transmembrane helix 5 is critical for the high water permeability of aquaporin

Aquaporin-2 (AQP2), a vasopressin-regulated water channel, plays a major role in urinary concentration. AQP2 and the major intrinsic protein (MIP) of lens fiber are highly homologous (58% amino acid identity) and share a topology of six transmembrane helices connected by five loops (loops A-E). Despite the similarities of these proteins, however, the water channel activity of AQP2 is much higher than that of MIP. To determine the site responsible for this gain of activity in AQP2, several parts of MIP were replaced with the corresponding parts of AQP2. When expressed in Xenopus oocytes, the osmotic water permeability (P(f)) of MIP and AQP2 was 48 and 245 x 10(-)(4) cm/s, respectively. Substitutions in loops B-D failed to increase P(f), whereas substitution of loop E significantly increased P(f) 1.5-fold. A similar increase in P(f) was observed with the substitution of the front half of loop E. P(f) measurements taken in a yeast vesicle expression system also confirmed that loop E had a complementary effect, whereas loops B-D did not. However, P(f) values of the loop E chimeras were only approximately 30% of that of AQP2. Simultaneous exchanges of loop E and a distal half of transmembrane helix 5 just proximal to loop E increased P(f) to the level of that of AQP2. Replacement of helix 5 alone stimulated P(f) 2.7-fold. Conversely, P(f) was decreased by 73% when helix 5 of AQP2 was replaced with that of MIP. Moreover, P(f) was stimulated 2.6- and 3.3-fold after helix 5 of AQP1 and AQP4 was spliced into MIP, respectively. Our findings suggested that the distal half of helix 5 is necessary for maximum water channel activity in AQP. We speculate that this portion contributes to the formation of the aqueous pore and the determination of the flux rate.     

Functional characterization of a novel aquaporin from Dictyostelium discoideum amoebae implies a unique gating mechanism

The social amoeba Dictyostelium discoideum is a widely used model organism for studying basic functions of protozoan and metazoan cells, such as osmoregulation and cell motility. There is evidence from other species that cellular water channels, aquaporins (AQP), are central to both processes. Yet, data on D. discoideum AQPs is almost absent. Despite cloning of two putative D. discoideum AQPs, WacA, and AqpA, water permeability has not been shown. Further, WacA and AqpA are expressed at the late multicellular stage and in spores but not in amoebae. We cloned a novel AQP, AqpB, from amoeboidal D. discoideum cells. Wild-type AqpB was impermeable to water, glycerol, and urea when expressed in Xenopus laevis oocytes. Neither stepwise truncation of the N terminus nor selected point mutations activated the water channel. However, mutational truncation by 12 amino acids of an extraordinary long intracellular loop induced water permeability of AqpB, hinting at a novel gating mechanism. This AqpB mutant was inhibited by mercuric chloride, confirming the presence of a cysteine residue in the selectivity filter as predicted by our structure model. We detected AqpB by Western blot analysis in a glycosylated and a non-glycosylated form throughout all developmental stages. When expressed in D. discoideum amoebae, AqpB-GFP fusion constructs localized to vacuolar structures, to the plasma membrane, and to lamellipodia-like membrane protrusions. We conclude that the localization pattern in conjunction with channel gating may be indicative of AqpB functions in osmoregulation as well as cell motility of D. discoideum.