Mutational widening of constrictions in a formate-nitrite/H+ transporter enables aquaporin-like water permeability and proton conductance.

The unrelated protein families of the microbial formate-nitrite transporters (FNTs) and aquaporins (AQP) likely adapted the same protein fold through convergent evolution. FNTs facilitate weak acid anion/H+ cotransport, whereas AQP water channels strictly exclude charged substrates including protons. The FNT channel-like transduction pathway bears two lipophilic constriction sites that sandwich a highly conserved histidine residue. Because of lacking experiments, the function of these constrictions is unclear, and the protonation status of the central histidine during substrate transport remains a matter of debate. Here, we introduced constriction-widening mutations into the prototypical FNT from Escherichia coli, FocA, and assayed formate/H+ transport properties, water/solute permeability, and proton conductance. We found that enlargement of these constrictions concomitantly decreased formate/formic acid transport. In contrast to wildtype FocA, the mutants were unable to make use of a transmembrane proton gradient as a driving force. A construct in which both constrictions were eliminated exhibited water permeability, similar to AQPs, although accompanied by a proton conductance. Our data indicate that the lipophilic constrictions mainly act as barriers to isolate the central histidine from the aqueous bulk preventing protonation via proton wires. These results are supportive of an FNT transport model in which the central histidine is uncharged, and weak acid substrate anion protonation occurs in the vestibule regions of the transporter before passing the constrictions.

The Plasmodium Lactate/H+ Transporter PfFNT Is Essential and Druggable In Vivo.

Malaria parasites in the blood stage express a single transmembrane transport protein for the release of the glycolytic end product l-lactate/H+ from the cell. This transporter is a member of the strictly microbial formate-nitrite transporter (FNT) family and a novel putative drug target. Small, drug-like FNT inhibitors potently block lactate transport and kill Plasmodium falciparum parasites in culture. The protein structure of Plasmodium falciparum FNT (PfFNT) in complex with the inhibitor has been resolved and confirms its previously predicted binding site and its mode of action as a substrate analog. Here, we investigated the mutational plasticity and essentiality of the PfFNT target on a genetic level, and established its in vivo druggability using mouse malaria models. We found that, besides a previously identified PfFNT G107S resistance mutation, selection of parasites at 3 × IC50 (50% inhibitory concentration) gave rise to two new point mutations affecting inhibitor binding: G21E and V196L. Conditional knockout and mutation of the PfFNT gene showed essentiality in the blood stage, whereas no phenotypic defects in sexual development were observed. PfFNT inhibitors mainly targeted the trophozoite stage and exhibited high potency in P. berghei- and P. falciparum-infected mice. Their in vivo activity profiles were comparable to that of artesunate, demonstrating strong potential for the further development of PfFNT inhibitors as novel antimalarials.

Structure memes: Intuitive visualization of sequence logo and subfamily logo information in a 3D protein-structural context.

The number of available protein sequences covering virtually all known species is tremendous and ever growing due to the feasibility of the underlying nucleotide sequencing. The speed at which protein structures are being determined is increasing, and as a result of refined cryo-electron microscopy the proportion of solved membrane protein folds is expanding. Sequence data are used to illustrate evolution and to group proteins into families with various levels of subfamilies. Structure data of prototypical proteins provide insight into function brought about by an interplay of specific amino acid residues that are dispersed throughout the sequence. Visually combining rich sequence information with structure data in an intuitively comprehensible way would enhance the process of elucidating key protein aspects regarding evolution, sequence relations, and function. Here, a method is described that projects the information contained in sequence logos and subfamily logos onto protein structures. The amino acid composition at a site is encoded by a mix color in the red-yellow-blue space and the information content is presented by the radius of a sphere at the α-carbon position. The resulting display is termed "structure meme." The underlying sequence and atom coordinate data are retained in the file for simple retrieval on demand using a molecular structure visualization program. Structure memes are recognizable and convey extensive information in a human-discernable way that requires little training.

Mechanism of formate-nitrite transporters by dielectric shift of substrate acidity.

Bacterial formate-nitrite transporters (FNTs) regulate the metabolic flow of small, weak mono-acids. Recently, the eukaryotic PfFNT was identified as the malaria parasite's lactate transporter and novel drug target. Despite crystal data, central mechanisms of FNT gating and transport remained unclear. Here, we show elucidation of the FNT transport mechanism by single-step substrate protonation involving an invariant lysine in the periplasmic vestibule. Opposing earlier gating hypotheses and electrophysiology reports, quantification of total uptake by radiolabeled substrate indicates a permanently open conformation of the bacterial formate transporter, FocA, irrespective of the pH Site-directed mutagenesis, heavy water effects, mathematical modeling, and simulations of solvation imply a general, proton motive force-driven FNT transport mechanism: Electrostatic attraction of the acid anion into a hydrophobic vestibule decreases substrate acidity and facilitates protonation by the bulk solvent. We define substrate neutralization by proton transfer for transport via a hydrophobic transport path as a general theme of the Amt/Mep/Rh ammonium and formate-nitrite transporters.

Degraded Arabinogalactans and Their Binding Properties to Cancer-Associated Human Galectins.

Galectins represent ß-galactoside-binding proteins with numerous functions. Due to their role in tumor progression, human galectins-1, -3 and -7 (Gal-1, -3 and -7) are potential targets for cancer therapy. As plant derived glycans might act as galectin inhibitors, we prepared galactans by partial degradation of plant arabinogalactan-proteins. Besides commercially purchased galectins, we produced Gal-1 and -7 in a cell free system and tested binding capacities of the galectins to the galactans by biolayer-interferometry. Results for commercial and cell-free expressed galectins were comparable confirming functionality of the cell-free produced galectins. Our results revealed that galactans from Echinacea purpurea bind to Gal-1 and -7 with KD values of 1-2 µM and to Gal-3 slightly stronger with KD values between 0.36 and 0.70 µM depending on the sensor type. Galactans from the seagrass Zostera marina with higher branching of the galactan and higher content of uronic acids showed stronger binding to Gal-3 (0.08-0.28 µM) compared to galactan from Echinacea. The results contribute to knowledge on interactions between plant polysaccharides and galectins. Arabinogalactan-proteins have been identified as a new source for production of galactans with possible capability to act as galectin inhibitors.

Formate-nitrite transporters carrying nonprotonatable amide amino acids instead of a central histidine maintain pH-dependent transport.

Microbial formate-nitrite transporter-type proteins (FNT) exhibit dual transport functionality. At neutral pH, electrogenic anion currents are detectable, whereas upon acidification transport of the neutral, protonated monoacid predominates. Physiologically, FNT-mediated proton co-transport is vital when monocarboxylic acid products of the energy metabolism, such as l-lactate, are released from the cell. Accordingly, Plasmodium falciparum malaria parasites can be killed by small-molecule inhibitors of PfFNT. Two opposing hypotheses on the site of substrate protonation are plausible. The proton relay mechanism postulates proton transfer from a highly conserved histidine centrally positioned in the transport path. The dielectric slide mechanism assumes decreasing acidity of substrates entering the lipophilic vestibules and protonation via the bulk water. Here, we defined the transport mechanism of the FNT from the amoebiasis parasite Entamoeba histolytica, EhFNT, and also show that BtFdhC from Bacillus thuringiensis is a functional formate transporter. Both FNTs carry a nonprotonatable amide amino acid, asparagine or glutamine, respectively, at the central histidine position. Despite having a nonprotonatable residue, EhFNT displayed the same substrate selectivity for larger monocarboxylates including l-lactate, a low substrate affinity as is typical for FNTs, and, strikingly, proton motive force-dependent transport as observed for PfFNT harboring a central histidine. These results argue against a proton relay mechanism, indicating that substrate protonation must occur outside of the central histidine region, most likely in the vestibules. Furthermore, EhFNT is the sole annotated FNT in the Entamoeba genome suggesting that it could be a putative new drug target with similar utility as that of the malarial PfFNT.

The intracellular parasite Toxoplasma gondii harbors three druggable FNT-type formate and l-lactate transporters in the plasma membrane.

Toxoplasma gondii is a globally prevalent parasitic protist. It is well-known for its ability to infect almost all nucleated vertebrate cells, which is reflected by its unique metabolic architecture. Its fast-growing tachyzoite stage catabolizes glucose via glycolysis to yield l-lactate as a major by-product that must be exported from the cell to prevent toxicity; the underlying mechanism remains to be elucidated, however. Herein, we report three formate-nitrite transporter (FNT)-type monocarboxylate/proton symporters located in the plasma membrane of the T. gondii tachyzoite stage. We observed that all three proteins transport both l-lactate and formate in a pH-dependent manner and are inhibited by 2-hydroxy-chromanones (a class of small synthetic molecules). We also show that these compounds pharmacologically inhibit T. gondii growth. Using a chemical biology approach, we identified the critical residues in the substrate-selectivity region of the parasite transporters that determine differential specificity and sensitivity toward both substrates and inhibitors. Our findings further indicate that substrate specificity in FNT family proteins from T. gondii has evolved such that a functional repurposing of prokaryotic-type transporters helps fulfill a critical metabolic role in a clinically important parasitic protist. In summary, we have identified and characterized the lactate transporters of T. gondii and have shown that compounds blocking the FNTs in this parasite can inhibit its growth, suggesting that these transporters could have utility as potential drug targets.

Substrate-analogous inhibitors exert antimalarial action by targeting the Plasmodium lactate transporter PfFNT at nanomolar scale.

Resistance against all available antimalarial drugs calls for novel compounds that hit unexploited targets in the parasite. Here, we show that the recently discovered Plasmodium falciparum lactate/proton symporter, PfFNT, is a valid druggable target, and describe a new class of fluoroalkyl vinylogous acids that potently block PfFNT and kill cultured parasites. The original compound, MMV007839, is derived from the malaria box collection of potent antimalarials with unknown targets and contains a unique internal prodrug principle that reversibly switches between a lipophilic transport form and a polar, substrate-analogous active form. Resistance selection of cultured P. falciparum parasites with sub-lethal concentrations of MMV007839 produced a single nucleotide exchange in the PfFNT gene; this, and functional characterization of the resulting PfFNT G107S validated PfFNT as a novel antimalarial target. From quantitative structure function relations we established the compound binding mode and the pharmacophore. The pharmacophore largely circumvents the resistance mutation and provides the basis for a medicinal chemistry program that targets lactate and proton transport as a new mode of antimalarial action.

Electrostatic attraction of weak monoacid anions increases probability for protonation and passage through aquaporins.

A positive electrostatic field emanating from the center of the aquaporin (AQP) water and solute channel is responsible for the repulsion of cations. At the same time, however, a positive field will attract anions. In this regard, l-lactate/lactic acid permeability has been shown for various isoforms of the otherwise highly water and neutral substrate selective AQP family. The structural requirements rendering certain AQPs permeable for weak monoacids and the mechanism of conduction have remained unclear. Here, we show by profiling pH-dependent substrate permeability, measurements of media alkalization, and proton decoupling that AQP9 acts as a channel for the protonated, neutral monocarboxylic acid species. Intriguingly, the obtained permeability rates indicate an up to 10 times higher probability of passage via AQP9 than given by the fraction of the protonated acid substrate at a certain pH. We generated AQP9 point mutants showing that this effect is independent from properties of the channel interior but caused by the protein surface electrostatics. Monocarboxylic acid-conducting AQPs thus employ a mechanism similar to the family of formate-nitrite transporters for weak monoacids. On a more general basis, our data illustrate semiquantitatively the contribution of surface electrostatics to the interaction of charged molecule substrates or ligands with target proteins, such as channels, transporters, enzymes, or receptors.

Pentamidine Is Not a Permeant but a Nanomolar Inhibitor of the Trypanosoma brucei Aquaglyceroporin-2.

The chemotherapeutic arsenal against human African trypanosomiasis, sleeping sickness, is limited and can cause severe, often fatal, side effects. One of the classic and most widely used drugs is pentamidine, an aromatic diamidine compound introduced in the 1940s. Recently, a genome-wide loss-of-function screen and a subsequently generated trypanosome knockout strain revealed a specific aquaglyceroporin, TbAQP2, to be required for high-affinity uptake of pentamidine. Yet, the underlying mechanism remained unclear. Here, we show that TbAQP2 is not a direct transporter for the di-basic, positively charged pentamidine. Even though one of the two common cation filters of aquaglyceroporins, i.e. the aromatic/arginine selectivity filter, is unconventional in TbAQP2, positively charged compounds are still excluded from passing the channel. We found, instead, that the unique selectivity filter layout renders pentamidine a nanomolar inhibitor of TbAQP2 glycerol permeability. Full, non-covalent inhibition of an aqua(glycero)porin in the nanomolar range has not been achieved before. The remarkable affinity derives from an electrostatic interaction with Asp265 and shielding from water as shown by structure-function evaluation and point mutation of Asp265. Exchange of the preceding Leu264 to arginine abolished pentamidine-binding and parasites expressing this mutant were pentamidine-resistant. Our results indicate that TbAQP2 is a high-affinity receptor for pentamidine. Taken together with localization of TbAQP2 in the flagellar pocket of bloodstream trypanosomes, we propose that pentamidine uptake is by endocytosis.

Reducing isoform complexity of human tetraspanins by optimized expression in Dictyostelium discoideum enables high-throughput functional read-out.

The human tetraspanin family of scaffold proteins comprises 33 isoforms. Being integral membrane proteins, they organize a so-called tetraspanin web via homomeric and heteromeric protein-protein interactions with integrins, immunoglobulins, growth factors, receptor tyrosine kinases, proteases, signaling proteins, and viral capsid proteins. Tetraspanins promote cellular effects, such as adhesion, migration, invasion, signaling, membrane fusion, protein trafficking, cancer progression, and infections. The ubiquitous expression of multiple tetraspanin isoforms and partner proteins hampers specific interaction studies. Here, we evaluated Dictyostelium discoideum as a non-mammalian expression system for human tetraspanins. Using high-content imaging we quantified tetraspanins in D. discoideum via fusion with green fluorescent protein. Three human tetraspanins, CD9, CD81, and CD151, served as test cases for which optimizations were carried out. We swapped the GFP domain between the N- and C-termini, added a Kozak sequence, and partially or fully adapted of the codon usage. This way, CD81 and CD151 were successfully produced. A conformation specific antibody further confirmed correct folding of CD81 and flow cytometry indicated an intracellular localization. Based on these data, we envision a D. discoideum-based co-expression platform with human partner proteins for studying tetraspanin interactions and their selective druggability on a large scale without the interference of endogenous human proteins.

High-level cell-free production of the malarial lactate transporter PfFNT as a basis for crystallization trials and directional transport studies.

The malaria parasite Plasmodium falciparum relies on the function of channel and transport proteins for the uptake of nutrients and the release of metabolic waste products. Inhibition of vital transport processes is an unexploited means for developing novel antimalarial drugs. The recently discovered plasmodial lactate transporter, PfFNT, represents a promising new drug target since the parasite's energy generation by anaerobic glycolysis depends on the rapid secretion of lactate. Yet, membrane proteins, in particular those of malaria parasites, are notoriously difficult to produce and purify in the native, functional form hampering crystallization and biophysical studies. Here, we show synthesis of milligram quantities of correctly folded PfFNT in a cell-free system. Solubilized PfFNT maintained its oligomeric, largely SDS-resistant quaternary structure and appears suitable for setting up crystallization trials. After reconstitution into proteoliposomes, PfFNT was functional as a transporter for formate, acetate, and lactate as determined by a light-scattering assay. Analysis of the accessibility of a protease cleavage site at the N-terminus revealed an even outside-in orientation of the total proteoliposomal PfFNT population that may be due to membrane curvature restrictions. Contrary to previous studies using heterologous expression in cell systems with oppositely oriented PfFNT, the proteoliposomes eventually allow for biophysical transport studies in the native, physiological direction.

The amoeboidal Dictyostelium aquaporin AqpB is gated via Tyr216 and aqpB gene deletion affects random cell motility.

BACKGROUND INFORMATION: Regulation of aquaporin (AQPs) water channels mostly occurs on the transcriptional level or by intracellular trafficking. Direct AQP gating is comparatively rare and is described mainly for fungi and plants. Earlier, we identified a gated water-specific AQP in Dictyostelium discoideum, AqpB, which is opened by truncation of an extended intracellular loop D (AqpB Δ208-219). RESULTS: We show that Tyr216 of loop D is a key residue in the gating mechanism possibly involving phosphorylation. Mutation of Tyr216 to aspartate or glutamate initiated water permeability (Pf  = 69 µm/s) to the same extent as AqpB Δ208-219, whereas neither replacement of Tyr216 by a positive arginine (Y216R) nor introduction of a negative charge in a neighbouring position (T217D) opened the channel. We overexpressed AqpB wildtype and AqpB Δ208-219 as GFP fusion constructs in Dictyostelium amoebae and found that the truncated, permanently open AqpB yielded cells with reduced capability to cope with hypotonic stress. Genetic deletion of aqpB yielded a strain with significantly reduced speed of random motility (4.5 µm/min vs. 6.3 µm/min of wildtype cells). Yet, chemotaxis towards folate and cAMP was unaffected. In this context, we identified a second, so-far uncharacterised amoeboidal AQP, AqpD, whose role in directed cell locomotion needs to be established. CONCLUSION: Our data add to the completion of the Dictyostelium model for cell motility and show that knowledge on the expression, permeability properties and localisation of amoeboidal AQPs are crucial for further development of the system.

Parasite aquaporins: current developments in drug facilitation and resistance.

BACKGROUND: Although being situated in a niche, research on parasite aquaporins is a lively field that has provided new insight into basic aquaporin structure-function relationships and physiological roles of water and solute transport. Moreover, it bears the potential to find novel approaches to antiparasitic chemotherapy. SCOPE OF REVIEW: Here, we summarize the current knowledge about the structure and substrate selectivity of aquaporins from protozoan and helminth parasites, review the current views on their physiological roles, and discuss their potency for chemotherapy. MAJOR CONCLUSIONS: Parasite aquaporins fulfill highly diverse tasks in the physiology of the various organisms, yet their general protein structure is well conserved. Aquaporins are directly (antimonials) and indirectly (melarsoprol, pentamidine) linked to the uptake of antiparasitic drugs. Unfortunately, drug-like aquaporin inhibitors are still missing. GENERAL SIGNIFICANCE: Aquaporins expression levels determine the degree of parasite resistance against certain drugs. Further studies on parasite aquaporins may provide data about overcoming drug resistance mechanisms or even spark novel treatments. This article is part of a Special Issue entitled Aquaporins.

The arginine-facing amino acid residue of the rat aquaporin 1 constriction determines solute selectivity according to its size and lipophilicity.

Aquaporins (AQP) are transmembrane channels for small, predominantly uncharged solutes. Their selectivity is partly determined by the aromatic/arginine constriction. Ammonia is similar in size and polarity to water, yet a subset of aquaporins distinguishes between the two. We mutated the constriction of water-selective rat AQP1 to mimic that of the ammonia-permeable human AQP8 by replacing Phenylalanine 56 with histidine, Histidine 180 with isoleucine, and Cysteine 189 with glycine, alone and in combination. Only AQP1 mutants including the H180I exchange increased the ammonia and methylamine tolerance of yeast. In a second set of mutations, we replaced Histidine 180 with alanine, leucine, methionine, phenylalanine, asparagine or glutamine. AQP1 H180A was equivalent to AQP1 H180I. AQP1 H180L increased ammonia but not methylamine tolerance of yeast. AQP1 mutants with methionine, phenylalanine, asparagine or glutamine in place of Histidine 180, increased neither ammonia nor methylamine tolerance of yeast. All mutants conducted water, as judged by osmotic assays with yeast sphaeroplasts. We propose that the arginine-facing amino acid residue is the most versatile selector of aquaporin constrictions, excluding Escherichia coli glycerol facilitator-type aquaporins.

Specific aquaporins increase the ammonia tolerance of a Saccharomyces cerevisiae mep1-3 fps1 deletion strain.

Abstract Aquaporins (AQPs) are channel proteins which facilitate the bidirectional membrane permeation of small neutral molecules such as water and glycerol. A convenient way to characterize their permeability is by growth of transformed Saccharomyces cerevisiae deletion strains on nutrient-limited substrates. We selected a yeast strain deficient in its endogenous ammonium transporters Mep1-3 and aquaglyceroporin Fps1 in order to study the ammonia permeability of heterologously expressed AQPs. Surprisingly, AQP-expression improved yeast growth at high, not low, concentrations of unprotonated ammonia. At neutral or mildly alkaline pH, ammonia concentrations above 10 µM decreased the growth rate and especially the number of yeast cell duplications, but did not affect the lag phase. AQP-expression raised the threshold to about 100 µM. The exchange of ammonium ions for amino acids or urea did not completely abolish this effect. AQPs capable of rescuing growth had a selectivity filter wide enough to permit passage of molecules larger than water but smaller than glycerol. It appears that the endogenous aquaglyceroporin Fps1 may, under alkaline conditions, be beneficial to yeast by facilitating the membrane permeation of an as yet unidentified molecule other than glycerol.

Osmotic water transport in aquaporins: evidence for a stochastic mechanism.

Abstract We test a novel, stochastic model of osmotic water transport in aquaporins. A solute molecule present at the pore mouth can either be reflected or permeate the pore. We assume that only reflected solute molecules induce osmotic transport of water through the pore, while permeating solute molecules give rise to no water transport. Accordingly, the rate of water transport is proportional to the reflection coefficient σ, while the solute permeability, P(S), is proportional to 1 - σ. The model was tested in aquaporins heterologously expressed in Xenopus oocytes. A variety of aquaporin channel sizes and geometries were obtained with the two aquaporins AQP1 and AQP9 and mutant versions of these. Osmotic water transport was generated by adding 20 mM of a range of different-sized osmolytes to the outer solution. The osmotic water permeability and the reflection coefficient were measured optically at high resolution and compared to the solute permeability obtained from short-term uptake of radio-labelled solute under isotonic conditions. For each type of aquaporin there was a linear relationship between solute permeability and reflection coefficient, in accordance with the model. We found no evidence for coupling between water and solute fluxes in the pore. In confirmation of molecular dynamic simulations, we conclude that the magnitude of the osmotic water permeability and the reflection coefficient are determined by processes at the arginine selectivity filter located at the outward-facing end of the pore.

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.

Molar concentrations of sorbitol and polyethylene glycol inhibit the Plasmodium aquaglyceroporin but not that of E. coli: involvement of the channel vestibules.

The aquaglyceroporins of Escherichia coli, EcGlpF, and of Plasmodium falciparum, PfAQP, are probably the best characterized members of the solute-conducting aquaporin (AQP) subfamily. Their crystal structures have been elucidated and numerous experimental and theoretical analyses have been conducted. However, opposing reports on their rates of water permeability require clarification. Hence, we expressed EcGlpF and PfAQP in yeast, prepared protoplasts, and compared water and glycerol permeability of both aquaglyceroporins in the presence of different osmolytes, i.e. sucrose, sorbitol, PEG300, and glycerol. We found that water permeability of PfAQP strongly depends on the external osmolyte, with full inhibition by sorbitol, and increasing water permeability when glycerol, PEG300, and sucrose were used. EcGlpF expression did not enhance water permeability over that of non-expressing control protoplasts regardless of the osmolyte. Glycerol permeability of PfAQP was also inhibited by sorbitol, but to a smaller extent, whereas EcGlpF conducted glycerol independently of the osmolyte. Mixtures of glycerol and urea passed PfAQP equally well under isosmotic conditions, whereas under hypertonic conditions in a countercurrent with water, glycerol was clearly preferred over urea. We conclude that PfAQP has high and EcGlpF low water permeability, and explain the inhibiting effect of sorbitol on PfAQP by its binding to the extracellular vestibule. The preference for glycerol under hypertonic conditions implies that in a physiological setting, PfAQP mainly acts as a water/glycerol channel rather than a urea facilitator.

Concerted action of two cation filters in the aquaporin water channel.

Aquaporin (AQP) facilitated water transport is common to virtually all cell membranes and is marked by almost perfect specificity and high flux rates. Simultaneously, protons and cations are strictly excluded to maintain ionic transmembrane gradients. Yet, the AQP cation filters have not been identified experimentally. We report that three point mutations turned the water-specific AQP1 into a proton/alkali cation channel with reduced water permeability and the permeability sequence: H(+) >>K(+) >Rb(+) >Na(+) >Cs(+) >Li(+). Contrary to theoretical models, we found that electrostatic repulsion at the central asn-pro-ala (NPA) region does not suffice to exclude protons. Full proton exclusion is reached only in conjunction with the aromatic/arginine (ar/R) constriction at the pore mouth. In contrast, alkali cations are blocked by the NPA region but leak through the ar/R constriction. Expression of alkali-leaking AQPs depolarized membrane potentials and compromised cell survival. Our results hint at the alkali-tight but solute-unselective NPA region as a feature of primordial channels and the proton-tight and solute-selective ar/R constriction variants as later adaptations within the AQP superfamily.