Valérie Lagrée

Switch from an Aquaporin to a Glycerol Channel by Two Amino Acids Substitution

The MIP (major intrinsic protein) proteins constitute a channel family of currently 150 members that have been identified in cell membranes of organisms ranging from bacteria to man. Among these proteins, two functionally distinct subgroups are characterized: aquaporins that allow specific water transfer and glycerol channels that are involved in glycerol and small neutral solutes transport. Since the flow of small molecules across cell membranes is vital for every living organism, the study of such proteins is of particular interest. For instance, aquaporins located in kidney cell membranes are responsible for reabsorption of 150 liters of water/day in adult human. To understand the molecular mechanisms of solute transport specificity, we analyzed mutant aquaporins in which highly conserved residues have been substituted by amino acids located at the same positions in glycerol channels. Here, we show that substitution of a tyrosine and a tryptophan by a proline and a leucine, respectively, in the sixth transmembrane helix of an aquaporin leads to a switch in the selectivity of the channel, from water to glycerol.

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 Yeast Recombinant Aquaporin Mutant That Is Not Expressed or Mistargeted in Xenopus Oocyte Can Be Functionally Analyzed in Reconstituted Proteoliposomes

We have recently identified AQPcic (foraquaporin cicadella), an insect aquaporin found in the digestive tract of homopteran insects and involved in the elimination of water ingested in excess with the dietary sap (Le Cahérec, F., Deschamps, S., Delamarche, C., Pellerin, I., Bonnec, G., Guillam, M. T., Gouranton, J., Thomas, D., and Hubert, J. F. (1996) Eur. J. Biochem.241, 707–715). Like many other aquaporins, AQPcic is inhibited by mercury reagents. In this study, we have demonstrated that residue Cys82 is essential for mercury inhibition. Another mutant version of AQPcic (AQP-C134S), expression of which in Xenopus laevis failed to produce an active molecule, was successfully expressed in Saccharomyces cerevisiae. Using stopped-flow analysis of reconstituted proteoliposomes, we demonstrated that the biological activity and Hg sensitivity of yeast-expressed wild type and mutant type AQPcic was readily assessed. Therefore, we propose that the yeast system is a valid alternative to Xenopus oocytes for studying particular mutants of aquaporin.

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.

Different Behaviours of MIP Proteins in N-Lauroylsarcosine

Purification of MIP proteins is of fundamental interest for further reconstitution in artificial lipid bilayers and determination of functional properties or structural analysis by cryoelectron microscopy of 2D-crystals. It has been shown that native or recombinant AQP1 can be purified by a few steps procedure using N-lauroylsarcosine (NLS) as detergent (Smith & Agre, 1991). A first extraction with NLS solubilizes almost all the membranous proteins, except AQP1 which remains in 100,000g pellet. The aquaporin can then be obtained at a good degree of purity by resuspending the NLS insoluble extract in n-octyl glucoside (OG). We successfully used this procedure to purify the insect aquaporin expressed in yeast. This protocol allows the protein to exhibit water channel properties in proteoliposomes and to retain its homotetrameric native state (Lagree et al., 1998, 1999). In the MIP family, we observed that the E. coli glycerol facilitator, GlpF, is monomeric in the non-denaturing detergents OG (2%, w/v) and Triton X-100 (1%, v/v), while aquaporins are tetrameric. We thus hypothesised the existence of a relationship between the functional specificity and the quaternary structural organisation of MIPs. As mentioned earlier, NLS resistance is a well established physicochemical characteristic of the aquaporins AQP1 and AQPcic. In the present study, we analysed the behaviour in 2% NLS (w/v) of the solute facilitator GlpF and several mutants of AQPcic.

A Water Channel Network in Cell Membranes of the Filter Chamber of Homopteran Insects

Water is the most ubiquitous molecule in the living cell and movement of water across the cell membrane accompanies fundamental cell functions. All biological membranes exhibit some water permeability as a result of diffusion through the lipid bilayer and osmotic gradients constitute the driving force for water flow. Osmotic water permeability is therefore of the highest relevance. However, some cells have the ability to transport water across their cell membrane at greatly accelerated rates, for example mammalian red blood cells, epithelial cells of the renal proximal tubules. Water permeability in such cells is simply too high to be accounted for by lipid-mediated diffusion, thus leading biophysicists to predict that water-selective channels must exist. The search for water channel began not surprisingly in tissues that had been already identified from physiological studies as having high water permeabilities. But the molecular basis of water channels remained elusive for a long time, since many attempts to determine its structure by biochemical approaches and expression cloning were unsuccessful. The reasons of this failure were linked to the inability of the water channel to be labeled by its substrate, the lack of highly specific inhibitors and the basal diffusional permeability of cell membranes.

Study of fast water movements in bacteria by cryoelectron microscopy

Movement of water into or out of cells is a fundamental process of life found throughout nature. However the molecular pathway of this transport remained elusive until discovery of the aquaporins, a large family of water channel proteins. In bacteria, the osmotic movement of water across the cytoplasmic membrane is one of the mechanisms triggered to maintain the cell turgor, a function essential for growth and survival. The first known prokaryotic aquaporin water channel gene, aqpZ, has been reported in Escherichia coli (Calamita et al., 1995) and functionally characterized (Borgnia et al., 1999; Delamarche et al., 1999). This indicates that, in spite of the high surface-to-volume ratio characterizing bacteria, the simple diffusion of water through the membrane lipids could not be always sufficient to preserve turgor. Presently, several microbial members of the MIP family have been identified by sequence homology, but only few microbial MIPs have been functionally studied (Maurel et al., 1994).