Stopped-flow Light Scattering Analysis of Red Blood Cell Glycerol Permeability

Stopped-Flow Light Scattering (SFLS) is a method devised to analyze the kinetics of fast chemical reactions that result in a significant change of the average molecular weight and/or in the shape of the reaction substrates. Several modifications of the original stopped-flow system have been made leading to a significant extension of its technical applications. One of these modifications allows the biophysical characterization of the water and solute permeability of biological and artificial membranes.Here, we describe a protocol of SFLS to measure the glycerol permeability of isolated human red blood cells (RBCs) and evaluate the pharmacokinetics properties (selectivity and potency) of isoform-specific inhibitors of AQP3, AQP7 and AQP9, three mammalian aquaglyceroporins allowing transport of glycerol across membranes. Suspensions of RBCs (1% hematocrit) are exposed to an inwardly directed gradient of 100 mM glycerol in a SFLS apparatus at 20 degrees C and the resulting changes in scattered light intensity are recorded at a monochromatic wavelength of 530 nm for 120 s. The SFLS apparatus is set up to have a dead time of 1.6-ms and 99% mixing efficiency in less than 1 ms. Data are fitted to a single exponential function and the related time constant (tau, seconds) of the cell-swelling phase of light scattering corresponding to the osmotic movement of water that accompanies the entry of glycerol into erythrocytes is measured. The coefficient of glycerol permeability (P-gly, cm/s) of RBCs is calculated with the following equation:P-gly = 1/[(S/V)tau]where tau (s) is the fitted exponential time constant and S/V is the surface-to-volume ratio (cm(-1)) of the analyzed RBC specimen. Pharmacokinetics of the isoform-specific inhibitors of AQP3, AQP7 and AQP9 are assessed by evaluating the extent of RBC P-gly values resulting after the exposure to serial concentrations of the blockers

Putative ligand binding sites of two functionally characterized bark beetle odorant receptors

Background: Bark beetles are major pests of conifer forests, and their behavior is primarily mediated via olfaction. Targeting the odorant receptors (ORs) may thus provide avenues towards improved pest control. Such an approach requires information on the function of ORs and their interactions with ligands, which is also essential for understanding the functional evolution of these receptors. Hence, we aimed to identify a high-quality complement of ORs from the destructive spruce bark beetle Ips typographus (Coleoptera, Curculionidae, Scolytinae) and analyze their antennal expression and phylogenetic relationships with ORs from other beetles. Using 68 biologically relevant test compounds, we next aimed to functionally characterize ecologically important ORs, using two systems for heterologous expression. Our final aim was to gain insight into the ligand-OR interaction of the functionally characterized ORs, using a combination of computational and experimental methods. Results: We annotated 73 ORs from an antennal transcriptome of I. typographus and report the functional characterization of two ORs (ItypOR46 and ItypOR49), which are responsive to single enantiomers of the common bark beetle pheromone compounds ipsenol and ipsdienol, respectively. Their responses and antennal expression correlate with the specificities, localizations, and/or abundances of olfactory sensory neurons detecting these enantiomers. We use homology modeling and molecular docking to predict their binding sites. Our models reveal a likely binding cleft lined with residues that previously have been shown to affect the responses of insect ORs. Within this cleft, the active ligands are predicted to specifically interact with residues Tyr84 and Thr205 in ItypOR46. The suggested importance of these residues in the activation by ipsenol is experimentally supported through site-directed mutagenesis and functional testing, and hydrogen bonding appears key in pheromone binding. Conclusions: The emerging insight into ligand binding in the two characterized ItypORs has a general importance for our understanding of the molecular and functional evolution of the insect OR gene family. Due to the ecological importance of the characterized receptors and widespread use of ipsenol and ipsdienol in bark beetle chemical communication, these ORs should be evaluated for their potential use in pest control and biosensors to detect bark beetle infestations

A structural preview of aquaporin 8 via homology modeling of seven vertebrate isoforms

Abstract Background Aquaporins (AQPs) facilitate the passage of small neutral polar molecules across membranes of the cell. In animals there are four distinct AQP subfamilies, whereof AQP8 homologues constitute one of the smallest subfamilies with just one member in man. AQP8 conducts water, ammonia, urea, glycerol and H2O2 through various membranes of animal cells. This passive channel has been connected to a number of phenomena, such as volume change of mitochondria, ammonia neurotoxicity, and mitochondrial dysfunction related to oxidative stress. Currently, there is no experimentally determined structure of an AQP8, hence the structural understanding of this subfamily is limited. The recently solved structure of the plant AQP, AtTIP2;1, which has structural and functional features in common with AQP8s, has opened up for construction of homology models that are likely to be more accurate than previous models. Results Here we present homology models of seven vertebrate AQP8s. Modeling based on the AtTIP2;1 structure alone resulted in reasonable models except for the pore being blocked by a phenylalanine that is not present in AtTIP2;1. To achieve an open pore, these models were supplemented with models based on the bacterial water specific AQP, EcAqpZ, creating a chimeric monomeric model for each AQP8 isoform. The selectivity filter (also named the aromatic/arginine region), which defines the permeant substrate profile, comprises five amino acid residues in AtTIP2;1, including a histidine coming from loop C. Compared to AtTIP2;1, the selectivity filters of modelled AQP8s only deviates in that they are slightly more narrow and more hydrophobic due to a phenylalanine replacing the histidine from loop C. Interestingly, the models do not exclude the existence of a side pore beneath loop C similar to that described in the structure of AtTIP2;1. Conclusions Our models concur that AQP8s are likely to have an AtTIP2;1-like selectivity filter. The detailed description of the expected configuration of residues in the selectivity filters of AQP8s provides an excellent starting point for planning of as well as rationalizing the outcome of mutational studies. Our strategy to compile hybrid models based on several templates may prove useful also for other AQPs for which structural information is limited

Endogenous insensitivity to the Orco agonist VUAA1 reveals novel olfactory receptor complex properties in the specialist fly Mayetiola destructor

Insect olfactory receptors are routinely expressed in heterologous systems for functionalcharacterisation. It was recently discovered that the essential olfactory receptor co-receptor (Orco)of the Hessian fly, Mayetiola destructor (Mdes), does not respond to the agonist VUAA1, whichactivates Orco in all other insects analysed to date. Here, using a mutagenesis-based approach weidentified three residues in MdesOrco, located in different transmembrane helices as supported by 3Dmodelling, that confer sensitivity to VUAA1. Reciprocal mutations in Drosophila melanogaster (Dmel)and the noctuid moth Agrotis segetum (Aseg) Orcos diminish sensitivity of these proteins to VUAA1.Additionally, mutating these residues in DmelOrco and AsegOrco compromised odourant receptor (OR)dependent ligand-induced Orco activation. In contrast, both wild-type and VUAA1-sensitive MdesOrcowere capable of forming functional receptor complexes when coupled to ORs from all three species,suggesting unique complex properties in M. destructor, and that not all olfactory receptor complexesare “created” equal

Additional file 3: of A structural preview of aquaporin 8 via homology modeling of seven vertebrate isoforms

Figure S3. Structural alignment of AQP8 models. The seven AQP8 models and the main template AtTIP2;1 are shown in ribbon representation, and the five residues of the selectivity filters are depicted as sticks. The highest variability is found in loop regions, especially in loop C. Still the part of the loop contributing to the selectivity filter is consistently modelled in to a AtTIP2;1-like structure. a Side view of models and the template. b View of opposite side relative (a). c Top view of the selectivity filter. AtTIP2;1 – green, HsAQP8 – slate blue, BtAQP8 – cyan, RnAQP8 – orange, FpAQP8 – magenta, CpAQP8 – greenblue, XtAQP8 – yellow, SsAQP8b – salmon. (TIFF 883 kb

Additional file 2: of A structural preview of aquaporin 8 via homology modeling of seven vertebrate isoforms

Figure S2. Cartoon representation highlighting parts of hybrid models that are based on the structure of EcAqpZ. All seven final monomeric composite models are aligned, and regions that are initially modelled on EcAqpZ are marked in magenta. Residues at the five positions of the selectivity filter of HsAQP8, as well as the phenylalanine (F85 in HsAQP8) that occludes the pore in models solely relying on AtTIP2;1, are depicted as sticks. The extent of the EcAqpZ based structure in the chimeric models varies but as a minimum consist of the complete helix 2 (H2) and the consecutive residues up to and including the phenylalanine corresponding to F85 in HsAQP8. (TIFF 703 kb

Single amino acid substitutions in the selectivity filter render NbXIP1;1α aquaporin water permeable

BACKGROUND: Aquaporins (AQPs) are integral membrane proteins that facilitate transport of water and/or other small neutral solutes across membranes in all forms of life. The X Intrinsic Proteins (XIPs) are the most recently recognized and the least characterized aquaporin subfamily in higher plants. XIP1s have been shown to be impermeable to water but permeable to boric acid, glycerol, hydrogen peroxide and urea. However, uncertainty regarding the determinants for selectivity and lack of an activity that is easy to quantify have hindered functional investigations. In an effort to resolve these issues, we set out to introduce water permeability in Nicotiana benthamiana XIP1;1α (NbXIP1;1α), by exchanging amino acid residues of predicted alternative aromatic/arginine (ar/R) selectivity filters of NbXIP1;1α for residues constituting the water permeable ar/R selectivity filter of AtTIP2;1.RESULTS: Here, we present functional results regarding the amino acid substitutions in the putative filters as well as deletions in loops C and D of NbXIP1;1α. In addition, homology models were created based on the high resolution X-ray structure of AtTIP2;1 to rationalize the functional properties of wild-type and mutant NbXIP1;1α. Our results favour Thr 246 rather than Val 242 as the residue at the helix 5 position in the ar/R filter of NbXIP1;1α and indicate that the pore is not occluded by the loops when heterologously expressed in Pichia pastoris. Moreover, our results show that a single amino acid substitution in helix 1 (L79G) or in helix 2 (I102H) is sufficient to render NbXIP1;1α water permeable. Most of the functional results can be rationalized from the models based on a combination of aperture and hydrophobicity of the ar/R filter.CONCLUSION: The water permeable NbXIP1;1α mutants imply that the heterologously expressed proteins are correctly folded and offer means to explore the structural and functional properties of NbXIP1;1α. Our results support that Thr 246 is part of the ar/R filter. Furthermore, we suggest that a salt bridge to an acidic residue in helix 1, conserved among the XIPs in clade B, directs the orientation of the arginine in the ar/R selectivity filter and provides a novel approach to tune the selectivity of AQPs

Additional file 6: of A structural preview of aquaporin 8 via homology modeling of seven vertebrate isoforms

Text. The PDB IDs of the structures used for the alignment. 1FQY, 1FX8, 1H6I, 1IH5, 1J4N, 1LDA, 1LDF, 1LDI, 1RC2, 1SOR, 1YMG, 1Z98, 2ABM, 2B5F, 2B6O, 2B6P, 2C32, 2D57, 2EVU, 2F2B, 2O9D, 2O9E, 2O9F, 2O9G, 2W1P, 2W2E, 2ZZ9, 3C02, 3CLL, 3CN5, 3CN6, 3D9S, 3GD8, 3LLQ, 3M9I, 3NE2, 3NK5, 3NKA, 3NKC, 3ZOJ, 4CSK, 4IA4, 4JC6, 4NEF, 4OJ2, 5BN2, 5C5X, 5DYE, 5I32. (DOCX 12 kb