BACKGROUND INFORMATION ABOUT THE AQUAPORIN TMR PROJECT
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BACKGROUND INFORMATION ABOUT THE AQUAPORIN TMR PROJECT |
3. SCIENTIFIC ORIGINALITY OF THE PROJECT
4. COLLECTIVE EXPERIENCE OF THE RESEARCH TEAMS
Water is the major constituent of all living cells and of the environment which surrounds them. Water transport across cell membranes is therefore of crucial significance for the whole of biology, but especially for those physiological processes which involve fluid transport including: osmoregulation and water conservation by the kidney, fluid secretion and absorption by epithelia of the gastrointestinal, respiratory and reproductive tracts and their associated exocrine glands, and the formation of cerebrospinal fluid, aqueous humour, amniotic fluid and sweat. In many cells, water movement across the cell membrane is achieved by simple diffusion through the lipid bilayer. However, in tissues involved in fluid transport, specific membrane proteins function as molecular water channels, the so-called aquaporins.
Aquaporins belong to the ancient MIP family of channel-like proteins, named after the first member of the family to be cloned, the Major Intrinsic Protein of the eye lens. More than 50 members of this family have now been identified in bacteria, yeast, plants, insects and vertebrates. The discovery of aquaporins caused a break-through in understanding the physiology of water transport in the kidney and the pathophysiological basis of certain inherited and acquired disorders of water homeostasis. For example, the Nijmegen-group first reported that mutations in the gene coding for aquaporin-2 cause nephrogenic diabetes insipidus, NDI, a hereditary disease in which the kidney fails to respond to the antidiuretic hormone, vasopressin. Furthermore, the Aarhus-group has shown that changes in aquaporin-2 expression underly acquired disorders of water balance. The significance of aquaporins is likely to be more widespread, as more members of the family are discovered and as more information on hormonal regulation of these proteins becomes available.
Of critical importance for water balance and disorders of water balance are the effects of agents (e.g. antidiuretics, corticosteroids, and vasopressin) which modulate aquaporin expression and function. For example, expression of certain aquaporins, that have recently been shown to be essential during development of kidney and lung, is induced by corticosteroid treatment. This may represent a future therapy for treatment of premature infants having immature lung and kidney function.
Gaining a better insight into the molecular details of the water pore structure will enable us, in collaboration with the pharmaceutical industry, to develop highly selective inhibitors which could be valuable as powerful diuretics. One company, with whom we are collaborating, sees aquaporins in human sweat glands as potential targets for antiperspirants. Induction of expression of a plant root aquaporin by root-knot nematodes causes untold damage to crops, which might be prevented by specific aquaporin blockers. In bacteria, aquaporins may provide a target for novel antimicrobial agents.
Water transport at the molecular level represents an exciting new field of fundamental research. Studies are now required to reveal the physiological and medical significance of aquaporins. There are strong groups in the USA and Japan working in this field. For the EU to remain competitive and explore the potential exploitation of aquaporins by pharmaceutical and cosmetic industries (where the EU is especially strong), European expertise must be coordinated. Within Europe, groups in the Netherlands, England, Denmark, France, Italy and Sweden participate extensively in pioneering studies on water transport and the characterization of aquaporins. These groups possess distinct and complementary expertise in molecular and cell biology, structural biology and cell physiology. The purpose of this proposal is to unite these groups into an effective and efficient working network. By focusing on water channel biology, this network will remain at the forefront of this rapidly-expanding field of bioscience and medicine and thereby discover new knowledge, provide a resource for EU industries, and establish an excellent training ground for young scientists, not only in the precise topic of this programme but in the general field of the Molecular Physiology of membrane transport proteins.
The scientific aims of the project are:
to understand the fundamental molecular mechanisms responsible for water transport through cellular membranes and epithelia
to determine the molecular bases of the pathogenesis of diseases related to disturbed water homeostasis
to devise strategies for therapeutic intervention
In the course of the project we will attract further interest and support from the pharmaceutical and cosmetic industries. Furthermore, by capitalizing on the complementary training opportunities available in the six laboratories, we will produce young scientists who will be motivated, trained and equipped to tackle fundamental and applied research on the molecular physiology of membrane transport processes.
To achieve these general aims, our specific objectives are as follows.
A. Identification and characterization of novel aquaporins
In addition to the six mammalian aquaporins (AQPs) that have already been described, it is predicted that many more exist, particularly in non-renal epithelia. For example, although one aquaporin (AQP5) that is specific to secretory epithelia has been identified in salivary and lacrimal glands, this isoform is not present in other exocrine glands. Since the known mammalian aquaporins show a high level of structural homology in those parts of the protein that are thought to form the aqueous pore, it is possible to search for other, novel members of the family using a homology cloning strategy. Therefore we will search for novel aquaporins in exocrine glands, as well as in regions of the brain, gastrointestinal tract and nephron where water fluxes are large but where channels have not yet been identified. Additionally, an AQP4-related protein detected in skeletal muscle by the Bari-group will be characterized. Attention will focus mainly on tissues where channels may be involved in disease processes or where they might be targets for novel pharmaceuticals or cosmetics.
B. Investigation of the relationships between aquaporin channel structure and function
Aquaporins vary in their substrate specificity. Some are highly selective to water, while others are also able to transport small neutral molecules such as glycerol and urea. To identify the molecular features of the channels that determine this selectivity, we will test current models for the structure of aquaporins and other MIP family members, especially glycerol facilitators and channels of broader specificity, by in vitro mutagenesis, and by analysis of naturally occurring mutations of the human aquaporins. This information will allow the functional classification of new members of the family on the basis of their amino acid sequence. We will also investigate the three-dimensional structure of aquaporin water channels and their subunit composition. This will be of vital importance for the design of specific aquaporin blockers.
C. Analysis of the mechanisms by which aquaporins reach their target membranes
In different tissues, aquaporins are preferentially located within different cellular domains. For example, large amounts of AQP1 are present in both apical and basolateral membranes of kidney proximal tubules. By contrast, in the principal cells of the collecting duct, AQP2 is confined to the apical membrane while AQP3 and AQP4 are located exclusively in the basolateral membrane. The targetting signals thus appear to be contained in the aquaporin protein itself. We will, therefore, characterize the molecular determinants of aquaporin sorting and targetting to specific plasma membrane domains. Identifying these signals is of primary importance for human health, since all mis-sense mutants of AQP2 that have been identified in NDI are impaired in their routing to the plasma membrane. In order to develop therapeutic strategies to relieve these impairments in intracellular transport, it is essential to identify the sorting signals in the aquaporin protein, the cell organelle in which the mutant aquaporin accumulates, and the proteins with which the mutant interacts in that organelle.
D. Elucidation of the mechanisms through which hormones regulate aquaporins
AQP2 is the predominant vasopressin-regulated water channel. The regulation of this channel is essential for the maintenance of body water balance. We will characterize the signaling pathways involved in the regulation of (1) aquaporin gene expression and (2) aquaporin function, including membrane targetting and in situ activation. Success in this objective will represent a break-through in understanding body water balance and the pathogenesis of kidney diseases and other water balance disorders associable with common disease, like congestive heart failure and liver cirrhosis. An important specific objective is to obtain a detailed characterization of the targetting receptors in the vesicles that carry aquaporins to the plasma membrane and those in the apical plasma membrane with which the vesicles fuse. While most of these questions relate mainly to AQP2, we will also examine the possibility that other known aquaporins, and novel members of the family, are hormonally regulated.
E. Determination of the physiological roles of aquaporins in vivo
While the role of AQP2 in vasopressin-regulated water absorption in the renal collecting duct is undisputed, mutations in the AQP1 gene, which result in the complete absence of AQP1 from the body, surprisingly cause no apparent clinical symptoms. To evaluate the physiological role of aquaporins therefore involves not only determining the localization of newly identified channels at a cellular and subcellular level, but also physiological studies to determine their contribution to fluid transport at both the cellular and organ level. This will require the development of appropriate physiological models as well as strategies for perturbing channel expression and function.
F. Development of strategies for screening drugs or cosmetics which modify aquaporins
Aquaporins are known to be of particular importance in the function of the kidney and various fluid-secreting tissues. This makes them promising targets for the development of novel pharmaceuticals and cosmetics. In order to screen and test such products, we will develop model systems such as transfected mammalian cell lines, yeast strains and bacterial strains. We will use these models to identify potential targets in the molecular structure of the aquaporins and to devise a rationale for designing drugs and cosmetics that will modify water transport in specific tissues and organs.
3. SCIENTIFIC ORIGINALITY OF THE PROJECT
A. Identification and characterization of novel aquaporins in water-transporting tissues
The first water channel was discovered by chance when Peter Agre and associates cloned CHIP28, which proved to be a member of the ancient MIP-family. By homology cloning, 50 full sequences of MIP-like proteins from bacteria to mammals have now been isolated and characterized. Phylogenetic analysis suggests that all current MIP-family members are derived from two divergent bacterial ancestors, one a water channel, the other a glycerol facilitator.
In mammals, six MIP family members have so far been identified as functional aquaporins. AQP0 (MIP26) is expressed exclusively in the eye lens and mutations in its gene have been shown to cause cataract. AQP1 (CHIP28) is expressed in a wide range of tissues, especially kidney, lung, eye and blood vessels. AQP2 is localized exclusively in the principal cells of the renal collecting duct which are the target cells for vasopressin-dependent water conservation. AQP3 is also expressed in the kidney and in other tissues, and appears to function as both a water channel and a glycerol facilitator. AQP4 is most highly expressed in the brain but it is also present in the kidney, lung and airways. AQP5 was cloned from rat salivary glands and is also found in the lacrimal glands and lung. In this proposal, the cloning of new MIP-family proteins will be pursued in tissues where physiological and pathophysiological evidence predicts the presence of aquaporins. The identification of new aquaporins will enable us to delineate their physiological role and should allow us to elucidate the pathogenesis of water balance disorders such as brain edema, anhydrosis, xerostomia, lung edema etc.
B. Investigation of the relationships between aquaporin channel structure and function
AQPs occur as homotetramers in the membrane, but a monomer is the functional unit. As far as their monomeric structure is concerned, aquaporins and other MIP-like proteins have six bilayer-spanning hydrophobic domains which are connected by three extracellular loops (A, C and E) and two intracellular loops (B and D). The N- and C- terminal regions are intracellular. On the basis of site-directed mutagenesis studies, Jung et al. have proposed an "hour-glass" model for the structure of AQP1 in which loops B and E fold back into the membrane and together form the water pore. Analysis of 8 mutant AQP2 proteins in NDI patients by the Nijmegen-group supports this "hour-glass" model, since mutations in the B and E loops result in non-functional water channels whereas mutations in loop C and D result in water channels which are functionally normal. The impaired trafficking of these recessive AQP2 mutants is the cause of NDI. This poses serious problems for the functional analysis of both naturally-occurring mutants and laboratory-generated mutants and chimeras. Development of novel blockers of aquaporins for scientific, pharmaceutical and cosmetic applications will require detailed information about the molecular structure of the water pore within the aquaporin protein. Only AQP1 has been amenable to a high resolution electron microscopy and electron diffraction, because only AQP1 could be purified in sufficiently-large quantities. To overcome problems in trafficking and availability of low amounts of protein, the Saclay-group has developed a new protein expression system based on a yeast secretory mutant which will allow both structural and functional studies of a wide range of wild-type and mutant MIP family proteins.
The molecular basis of the differing selectivities of the various MIP family members to water and other small nonelectrolytes remains a mystery. For example, the bacterial AqpZ, which is studied by the Bari-group, is water selective, while the well-studied E. coli glycerol facilitator (GlipF) does not transport water et all. One of the three MIP-family members in yeast, Fps1p, has been characterised by the Göteborg-group and shown to be a glycerol channel, which opens and closes rapidly with changes in environmental osmolarity. Fps1p has a long N-terminal extension which is essential for closing the channel. By performing domain swapping experiments between aquaporins and other members of the MIP family, we expect to identify the regions of the proteins that confer channel selectivity and, in the case of Fps1p, channel gating activity. Success in these experiments would then lead to a strategy for the design of specific aquaporin inhibitors based on the N-terminal extension of Fps1p.
C. Analysis of the mechanisms by which aquaporins reach their target membranes
Structurally similar aquaporins are sorted between apical and basolateral membrane domains of collecting duct principal cells, but the mechanisms are unknown. Upon expression in Xenopus oocytes and MDCK cells, AQP2 mutants, encoded by patients that recessively inherit NDI, accumulate in the endoplasmic reticulum. From studies of mutant proteins in other diseases, like the cystic fibrosis transmembrane conductance regulator (CFTR) mutant F508, it is known that this accumulation is often a consequence of disturbed binding to chaperones that guide the folding process. The folding efficiency can sometimes be increased by growing cells expressing the mutant at low temperatures, in the presence of osmolytes or by overexpression of chaperones with which the mutant interacts. The identification of ways to correct the misrouting of otherwise functional proteins might result in treatments to relieve patients with this kind of NDI or disorders with similar causes.
D. Elucidation of the mechanisms through which hormones regulate aquaporins
We and others have shown that AQP2 is the predominant vasopressin-regulated water channel of the kidney collecting duct and is essential for regulation of body water balance. AQP2 activity is determined both by transcriptional regulation of AQP2 expression and by vasopressin-regulated trafficking of vesicles containing AQP2 to the plasma membrane. The first issue will be studied by the Bari-group, using a cell line with endogenous AQP2 expression, and by the Aarhus-group. The Aarhus-group has already shown that, in collecting duct cells, the regulation of trafficking may involve the targetting receptors VAMP2 in the vesicles and syntaxin-4 in the apical plasma membrane. Our objective is to determine the role of SNARE proteins and tagmins, the molecular switches for vesicle docking and fusion, in vasopressin regulation of AQP2. These studies will be done in vivo and with AQP2-transfected MDCK and CD8 cell lines, which both exert proper vasopressin-regulated routing and water permeability of AQP2. The expected results will have important implications for elucidating the pathogenesis of several common urinary concentrating defects seen in primary kidney diseases and also in common diseases of the heart and liver which have associated secondary renal concentrating defects.
E. Determination of the physiological roles of aquaporins in vivo
The role of AQP1 as the primary route for water reabsorption in the renal proximal tubule has been cast into doubt by the identification of people who lack the protein and yet show no obvious clinical consequences. Other important specific functions attributed to aquaporins - such as the proposed roles of AQP4 in osmoreception and AQP5 in exocrine secretion - have not yet been directly assessed, either because naturally-occurring mutants have not been found or because of the lack of specific aquaporin blockers for physiological studies. Development of mouse "knockout" models will enable us to delineate the physiological relevance of aquaporins and may lead to the attribution of other human diseases to aquaporin mutations.
F. Development of strategies for screening drugs or cosmetics which modify aquaporins
Water or glycerol transport measurements in the available AQP1 or AQP2-transfected cell lines (see 3D), in AqpZ-expressing bacteria and in yeast cells that express MIP proteins have already been set up and will be extended to model systems in which putative drugs or cosmetics can be tested. High-resolution structure of MIP proteins (see 3B) and the availability of model systems to test the effects of drugs or cosmetics on the function of these proteins will provide the tools for the development of channel blockers that might be exploited by industry.
4. COLLECTIVE EXPERIENCE OF THE RESEARCH TEAMS
Summary
Expertise: molecular biology and genetics, functional expression in oocytes and cell lines, western blotting, immunocytochemistry
Specific roles: isolation/characterization of novel aquaporins, analysis of AQP2 mutants, generation of knock-out mice, identification of routing signals
Collaboration: Aarhus (immunocytochemistry of AQP1,2 in cell lines; Saclay (water transport through AQP2 cell line)
The Manchester-group
Expertise: patch-clamp electrophysiology, microfluorometry, fluorescence/video-imaging.
Specific roles: isolation/characterization of aquaporins in exocrine glands, pancreas; generation/analysis of knock-out mice
Collaboration: Nijmegen (provided protocols and aquaporin cDNA probes), Aarhus (immunocytochemistry)
The Aarhus-group
Expertise: immunocytochemistry at LM and EM level, freeze-fracture electron microscopy, subcellular fractionation, animal water metabolisms
specific roles: (sub)cellular localization of aquaporins, hormonal regulation/sorting of AQPs in vivo, generation/analysis of knock-out mice
Collaboration: Nijmegen (immunocytochemistry of AQP1,2 in cell lines)
Expertise: Molecular biology, yeast expression, water/solute transport in oocytes/cells/tissues, immunocytochemistry, freeze-fracture microscopy
specific roles: isolation/characterization of aquaporins, set up of yeast expression/test system,
Collaboration: Nijmegen (water transport AQP2 cell line); Bari (freeze-fracture microscopy training); Göteborg (students training in functional characterization in yeast)
The Bari-group
Expertise: Molecular/Cell biology, immunocytochemistry, biophysical measurements
Specific roles: isolation/characterizationof aquaporins, identification of routing signals, hormo-nal regulation/sorting/modulation, generation/analysis of knock-out bacteria
Collaboration: Saclay (training in freeze-fracture immunoelectron microscopy)
Expertise: yeast genetics and molecular biology, molecular genetics, mutagenesis, biochemical analysis, expression and structural analysis of membrane proteins.
Specific roles: set up of yeast expression/tests systems, studies on the model yeast, membrane protein biochemistry and structural analysis.
Collaboration: Aarhus; Saclay (Ph.D. student worked in Göteborg)