Two distinct aquaporin 0s required for development and transparency of the zebrafish lens.

PURPOSE: AQP0, formerly known as MIP26, likely has multiple separate functions in the mammalian lens, including water transport, formation of thin junctions, and interactions with other lens components. Although mammalian genomes contain only one Aqp0 gene, the zebrafish genome contains two, Aqp0a and Aqp0b, and the putative multiple functions of the single mammalian protein may be divided between these two genes. The purpose of this study was to exploit this gene duplication and divergence to illuminate the multiple functions of AQP0 in the lens. METHODS: Wholemount in situ hybridization and Western blot analyses were used to determine the expression pattern of Aqp0a and Aqp0b. The role of both proteins was studied in vivo by microinjection of antisense morpholino oligonucleotides in zebrafish. The water permeability of both proteins was tested using the Xenopus oocyte swelling assay and a yeast shrinkage assay. RESULTS: Both genes, like their mammalian counterpart, are expressed in the lens. Morpholino knock-down of either gene alone led to cataract formation, indicating that both genes are necessary for normal lens development and transparency. Full-length Aqp0a is a functional water channel when expressed in Xenopus oocytes and in yeast, whereas Aqp0b was not. However, the addition of an HA-tag at its N terminus converted Aqp0b to a water channel in Xenopus oocytes. CONCLUSIONS: These results suggest that Aqp0a is the primary water channel of the lens and that Aqp0b, though possibly a secondary water channel, has an unidentified function in the lens.

Phosphorylation determines the calmodulin-mediated Ca2+ response and water permeability of AQP0.

In Xenopus oocytes, the water permeability of AQP0 (P(f)) increases with removal of external calcium, an effect that is mediated by cytoplasmic calmodulin (CaM) bound to the C terminus of AQP0. To investigate the effects of serine phosphorylation on CaM-mediated Ca(2+) regulation of P(f), we tested the effects of kinase activation, CaM inhibition, and a series of mutations in the C terminus CaM binding site. Calcium regulation of AQP0 P(f) manifests four distinct phenotypes: Group 1, with high P(f) upon removal of external Ca(2+) (wild-type, S229N, R233A, S235A, S235K, K238A, and R241E); Group 2, with high P(f) in elevated (5 mm) external Ca(2+) (S235D and R241A); Group 3, with high P(f) and no Ca(2+) regulation (S229D, S231N, S231D, S235N, and S235N/I236S); and Group 4, with low P(f) and no Ca(2+) regulation (protein kinase A and protein kinase C activators, S229D/S235D and S235N/I236S). Within each group, we tested whether CaM binding mediates the phenotype, as shown previously for wild-type AQP0. In the presence of calmidazolium, a CaM inhibitor, S235D showed high P(f) and no Ca(2+) regulation, suggesting that S235D still binds CaM. Contrarily, S229D showed a decrease in recruitment of CaM, suggesting that S229D is unable to bind CaM. Taken together, our results suggest a model in which CaM acts as an inhibitor of AQP0 P(f). CaM binding is associated with a low P(f) state, and a lack of CaM binding is associated with a high P(f) state. Pathological conditions of inappropriate phosphorylation or calcium/CaM regulation could induce P(f) changes contributing to the development of a cataract.

Zinc modulation of water permeability reveals that aquaporin 0 functions as a cooperative tetramer.

We previously showed that the water permeability of AQP0, the water channel of the lens, increases with acid pH and that His40 is required (Németh-Cahalan, K.L., and J.E. Hall. 2000. J. Biol. Chem. 275:6777-6782; Németh-Cahalan, K.L., K. Kalman, and J.E. Hall. 2004. J. Gen. Physiol. 123:573-580). We have now investigated the effect of zinc (and other transition metals) on the water permeability of AQP0 expressed in Xenopus oocytes and determined the amino acid residues that facilitate zinc modulation. Zinc (1 mM) increased AQP0 water permeability by a factor of two and prevented any additional increase induced by acid pH. Zinc had no effect on water permeability of AQP1, AQP4 or MIPfun (AQP0 from killifish), or on mutants of AQP1 and MIPfun with added external histidines. Nickel, but not copper, had the same effect on AQP0 water permeability as zinc. A fit of the concentration dependence of the zinc effect to the Hill equation gives a coefficient greater than three, suggesting that binding of more than one zinc ion is necessary to enhance water permeability. His40 and His122 are necessary for zinc modulation of AQP0 water permeability, implying structural constraints for zinc binding and functional modulation. The change in water permeability was highly sensitive to a coinjected zinc-insensitive mutant and a single insensitive monomer completely abolished zinc modulation. Our results suggest a model in which positive cooperativity among subunits of the AQP0 tetramer is required for zinc modulation, implying that the tetramer is the functional unit. The results also offer the possibility of a pharmacological approach to manipulate the water permeability and transparency of the lens.

Transformation of plasmid DNA into E. coli using the heat shock method.

Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. It consists of inserting a foreign plasmid or ligation product into bacteria. This video protocol describes the traditional method of transformation using commercially available chemically competent bacteria from Genlantis. After a short incubation in ice, a mixture of chemically competent bacteria and DNA is placed at 42 degrees C for 45 seconds (heat shock) and then placed back in ice. SOC media is added and the transformed cells are incubated at 37 degrees C for 30 min with agitation. To be assured of isolating colonies irrespective of transformation efficiency, two quantities of transformed bacteria are plated. This traditional protocol can be used successfully to transform most commercially available competent bacteria. The turbocells from Genlantis can also be used in a novel 3-minute transformation protocol, described in the instruction manual.

AQP0-LTR of the Cat Fr mouse alters water permeability and calcium regulation of wild type AQP0.

Aquaporin 0 (AQP0) is the major intrinsic protein of the lens and its water permeability can be modulated by changes in pH and Ca2+. The Cataract Fraser (Cat Fr) mouse accumulates an aberrant AQP0 (AQP0-LTR) in sub-cellular compartments resulting in a congenital cataract. We investigated the interference of AQP0-LTR with normal function of AQP0 in three systems. First, we created a transgenic mouse expressing AQP0 and AQP0-LTR in the lens. Expression of AQP0 did not prevent the congenital cataract but improved the size and transparency of the lens. Second, we measured water permeability of AQP0 co-expressed with AQP0-LTR in Xenopus oocytes. A low expression level of AQP0-LTR decreased the water permeability of AQP0, and a high expression level eliminated its calcium regulation. Third, we studied trafficking of AQP0 and AQP0-LTR in transfected lens epithelial cells. At low expression level, AQP0-LTR migrated with AQP0 toward the cell membrane, but at high expression level, it accumulated in sub-cellular compartments. The deleterious effect of AQP0-LTR on lens development may be explained by lowering water permeability and abolishing calcium regulation of AQP0. This study provides the first evidence that calcium regulation of AQP0 water permeability may be crucial for maintaining normal lens homeostasis and development.

The architecture of a water-selective pore in the lipid bilayer visualized by electron crystallography in vitreous ice.

The water-selective pathway through aquaporin 1 (AQP1) membrane channel has been visualized by fitting an atomic model to a 3.7 A resolution three-dimensional density map. This map was determined by analysing images and electron diffraction patterns of lipid-reconstituted two-dimensional crystals of AQP1 preserved in vitrified buffer in the absence of any additive. The aqueous pathway in a monomer is characterized by a size-selective, to approximately 4.0 +/- 0.5 A wide pore that spans a length of to approximately 18 and bends by to approximately 25 degrees as it traverses the bilayer. This narrow pore is connected by wide, funnel-shaped openings at the extracellular and cytoplasmic faces, and is outlined mostly by hydrophobic residues interspersed with short stretches of polar amino acids, which results in relatively inert pathway conducive to diffusion-limited water flow. Although not visible at the current resolution, the 3D structure suggests putative binding sites for water molecules in the size-selective pore.

Role of C-terminal domain and transmembrane helices 5 and 6 in function and quaternary structure of major intrinsic proteins: analysis of aquaporin/glycerol facilitator chimeric proteins.

We previously observed that aquaporins and glycerol facilitators exhibit different oligomeric states when studied by sedimentation on density gradients following nondenaturing detergent solubilization. To determine the domains of major intrinsic protein (MIP) family proteins involved in oligomerization, we constructed protein chimeras corresponding to the aquaporin AQPcic substituted in the loop E (including the proximal part of transmembrane domain (TM) 5) and/or the C-terminal part (including the distal part of TM 6) by the equivalent domain of the glycerol channel aquaglyceroporin (GlpF) (chimeras called AGA, AAG, and AGG). The analogous chimeras of GlpF were also constructed (chimeras GAG, GGA, and GAA). cRNA corresponding to all constructs were injected into Xenopus oocytes. AQPcic, GlpF, AAG, AGG, and GAG were targeted to plasma membranes. Water or glycerol membrane permeability measurements demonstrated that only the AAG chimera exhibited a channel function corresponding to water transport. Analysis of all proteins expressed either in oocytes or in yeast by velocity sedimentation on sucrose gradients following solubilization by 2% n-octyl glucoside indicated that only AQPcic and AAG exist in tetrameric forms. GlpF, GAG, and GAA sediment in a monomeric form, whereas GGA and AGG were found mono/dimeric. These data bring new evidence that, within the MIP family, aquaporins and GlpFs behave differently toward nondenaturing detergents. We demonstrate that the C-terminal part of AQPcic, including the distal half of TM 6, can be substituted by the equivalent domain of GlpF (AAG chimera) without modifying the transport specificity. Our results also suggest that interactions of TM 5 of one monomer with TM 1 of the adjacent monomer are crucial for aquaporin tetramer stability.

Functional characterization of a microbial aquaglyceroporin.

The major intrinsic proteins (MIPs) constitute a widespread membrane channel family essential for osmotic cell equilibrium. The MIPs can be classified into three functional subgroups: aquaporins, glycerol facilitators and aquaglyceroporins. Bacterial MIP genes have been identified in archaea as well as in Gram-positive and Gram-negative eubacteria. However, with the exception of Escherichia coli, most bacterial MIPs have been analysed by sequence homology. Since no MIP has yet been functionally characterized in Gram-positive bacteria, we have studied one of these members from Lactococcus lactis. This MIP is shown to be permeable to glycerol, like E. coli GlpF, and to water, like E. coli AqpZ. This is the first characterization of a microbial MIP that has a mixed function. This result provides important insights to reconstruct the evolutionary history of the MIP family and to elucidate the molecular pathway of water and other solutes in these channels.

A functional water channel protein in the pathogenic bacterium Brucella abortus.

The gene for a new bacterial aquaporin, AqpX, was cloned from the pathogenic Gram-negative bacterium Brucella abortus. The gene was mapped on the large chromosome of B. abortus. It is flanked by one upstream and two downstream copies of the Brucella repeated sequence Bru-RS. Prediction from the nucleotide sequence indicated that the protein is a member of the MIP family, which comprises channels for water and/or solute transport. Expression in Xenopus oocytes and cryoelectron microscopy of Escherichia coli cells transformed with the aqpX gene confirmed that the protein is an efficient water channel. Glycerol uptake experiments in E. coli also showed that the protein is not able to transport glycerol.