Aquaporins (AQP) conduct small, uncharged molecules, such as water (orthodox AQPs), ammonia (aquaammoniaporins) or glycerol (aquaglyceroporins). The physiological functions of AQPs are involved in osmotic volume regulation or the transport of biochemical precursors and metabolic waste products. The recent identification of hydrogen peroxide (H 2 O 2 ) as a permeant of certain AQPs suggests additional roles in mitigating oxidative stress or enabling paracrine H 2 O 2 signalling. Yet, an analysis of the structural requirements of the H 2 O 2 permeability of AQPs is missing. We subjected a representative set of wild-type and mutant AQPs to a newly established quantitative phenotypic assay. We confirmed high H 2 O 2 permeability of the human aquaammoniaporin AQP8 and found intermediate H 2 O 2 permeability of the prototypical orthodox water channel AQP1 from the rat. Differences from an earlier report showing an absence of H 2 O 2 permeability of human AQP1 can be explained by expression levels. By generating point mutations in the selectivity filter of rat orthodox aquaporin AQP1, we established a correlation of H 2 O 2 permeability primarily with water permeability and secondarily with the pore diameter. Even the narrowest pore of the test set (i.e. rat orthodox aquaporin AQP1 H180F with a pore diameter smaller than that of natural orthodox AQPs) conducted water and H 2 O 2 . We further found that H 2 O 2 permeability of the aquaglyceroporin from the malaria parasite Plasmodium falciparum was lower despite its wider pore diameter. The data suggest that all water-permeable AQPs are H 2 O 2 channels, yet H 2 O 2 permeability varies with the isoform. Thus, generally, AQPs must be considered as putative players in situations of oxidative stress (e.g. in Plasmodium-infected red blood cells, immune cells, the cardiovascular system or cells expressing AQP8 in their mitochondria).

Human aquaporin-1 (hAQP1) is a water channel found in many tissues and potentially involved in several human pathologies. Selective inhibitors of hAQP1 are discussed as novel treatment opportunities for glaucoma, brain edema, inflammatory pain, and certain types of cancer. However, only very few potent and chemically attractive blockers have been reported to date. In this study we present three novel hAQP1 blockers that have been identified by virtual screening and inhibit water flux through hAQP1 in Xenopus laevis oocyte swelling assays at low micromolar concentrations. The newly discovered compounds display no chemical similarity to hitherto known hAQP1 blockers and bind at the extracellular entrance of the channel, close to the ar/R selectivity filter. Futhermore, mutagenesis studies showed that Lys36, which is not conserved among the hAQP family, is crucially involved in binding and renders the discovered compounds suitable as leads for the development of selective hAQP1 inhibitors.A quaporins (AQPs) are passive membrane channels that, in many species, facilitate highly efficient yet strictly selective permeation of water and small solutes across lipid bilayers. AQPs can be divided into two major subclasses, the aquaporin subfamiliy that selectively transports water and the aquaglyceroporin family that in addition transports small uncharged molecules such as glycerol.

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.

Aquaporins enable the passage of a diverse set of solutes besides water. Many novel aquaporin permeants, such as antimonite and arsenite, silicon, ammonia, and hydrogen peroxide, have been described very recently. By the same token, the number of available aquaporin sequences has rapidly increased. Yet, sequence analyses and structure models cannot reliably predict permeability properties. Even the contribution to pore selectivity of individual residues in the channel layout is not fully understood. Here, we describe and discuss established in vitro assays for water and solute permeability. Measurements of volume change due to flux along osmotic or chemical gradients yield quantitative biophysical data, whereas phenotypic growth assays can hint at the relevance of aquaporins in the physiological setting of a certain cell. We also summarize data on the modification of pore selectivity of the prototypical water-specific mammalian aquaporin-1. We show that replacing residues in the pore constriction region allows ammonia, urea, glycerol, and even protons to pass the aquaporin pore.

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.