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Oligomerization of water and solute channels of the major intrinsic protein (MIP) family

2001; Elsevier BV; Volume: 60; Issue: 2 Linguagem: Inglês

10.1046/j.1523-1755.2001.060002422.x

1523-1755

Laurence Duchesne, Stéphane Deschamps, Isabelle Pellerin, Valérie Lagrée, Alexandrine Froger, Daniel Thomas, Patrick Bron, Christian Delamarche, Jean‐François Hubert,

Erythrocyte Function and Pathophysiology

Oligomerization of water and solute channels of the major intrinsic protein (MIP) family. Water and small solute fluxes through cell membranes are ensured in many tissues by selective pores that belong to the major intrinsic protein family (MIP). This family includes the water channels or aquaporins (AQP) and the neutral solute facilitators such as the glycerol facilitator (GlpF). We have compared the characteristics of representatives of each subfamily. Following solubilization in the nondenaturing detergents n-octyl-glucoside (OG) and Triton X-100 (T-X100), AQPs remain in their native homotetrameric state, while GlpF always behaves as a monomer. Solute facilitators are fully solubilized by the detergent N-lauroyl sarcosine (NLS), while AQPs are not. Analyses of mutants and chimeras demonstrate a close correlation between the water transport function and the resistance to NLS solubilization. Thus, AQPs and solute facilitators exhibit different behaviors in mild detergents; this could reflect differences in quaternary organization within the membranes. We propose that the oligomerization state or the strength of self-association is part of the mechanisms used by MIP proteins to ensure solute selectivity. Oligomerization of water and solute channels of the major intrinsic protein (MIP) family. Water and small solute fluxes through cell membranes are ensured in many tissues by selective pores that belong to the major intrinsic protein family (MIP). This family includes the water channels or aquaporins (AQP) and the neutral solute facilitators such as the glycerol facilitator (GlpF). We have compared the characteristics of representatives of each subfamily. Following solubilization in the nondenaturing detergents n-octyl-glucoside (OG) and Triton X-100 (T-X100), AQPs remain in their native homotetrameric state, while GlpF always behaves as a monomer. Solute facilitators are fully solubilized by the detergent N-lauroyl sarcosine (NLS), while AQPs are not. Analyses of mutants and chimeras demonstrate a close correlation between the water transport function and the resistance to NLS solubilization. Thus, AQPs and solute facilitators exhibit different behaviors in mild detergents; this could reflect differences in quaternary organization within the membranes. We propose that the oligomerization state or the strength of self-association is part of the mechanisms used by MIP proteins to ensure solute selectivity. Water and small solute movements across the cell membranes are necessary for fundamental cell function such as reabsorption, secretion, or homeostasis behavior. The flow of small molecules into and out of cells is mediated by various classes of membrane proteins: pumps, transporters, and channels. Among such proteins, the major intrinsic protein (MIP) family represents a specific membranous channels group. To date, more than 350 MIP family members have been identified essentially from amino acid sequences homologies1.Heymann J.B. Engel A. Structural clues in the sequences of the aquaporins.J Mol Biol. 2000; 295: 1039-1053https://doi.org/10.1006/jmbi.1999.3413Crossref PubMed Scopus (133) Google Scholar. However, functional data subdivided the family into three major groups. Aquaporins (AQPs) are water-selective channels, glycerol facilitators (GlpF) allow glycerol and small solute transport, and aquaglyceroporins transport both molecules. The primary structures of these proteins are similar (250 to 290 amino acids), and the high conservation throughout the MIP family may indicate a common fold: a NH2 cytosolic portion followed by a hydrophobic stretch of six transmembrane helices Figure 1a. Among highly conserved amino acids in the family, two repetitions of Asp, Pro, Ala residues (NPA box) localized in the B and E loops draw the sequence signature of the family Figure 1b. The folding of these two loops in the membrane bilayer is proposed to be responsible of the pore formation and solute movement across the membrane2.Murata K. Mitsuoka K. Hiral T. et al.Structural determinants of water permeation through aquaporin-1.Nature. 2000; 407: 599-605https://doi.org/10.1038/35036519Crossref PubMed Scopus (1435) Google Scholar,3.Fu D. Libson A. Miercke L. et al.Structure of a glycerol-conducting channel and the basis for its selectivity.Science. 2000; 290: 481-486https://doi.org/10.1126/science.290.5491.481Crossref PubMed Scopus (882) Google Scholar. Interestingly, AQPs are widely distributed in bacteria, plants, and animals, while GlpFs have been characterized only within microorganisms such as bacteria or yeast. In mammals, ten MIPs have been cloned and functionally characterized. Some of them, such as AQP1, AQP3, and AQP4, are widely distributed in the body4.King L.S. Agre P. Pathophysiology of the aquaporin water channels.Annu Rev Physiol. 1996; 58: 619-648Crossref PubMed Scopus (458) Google Scholar. In contrast, AQP0, AQP2, and AQP6 are tissue specific5.Deen P.M. Verdijk M.A. Knoers N.V. et al.Requirement of human renal water channel aquaporin-2 for vasopressin-dependent concentration of urine.Science. 1994; 264: 92-95Crossref PubMed Scopus (763) Google Scholar, 6.Ma T. Yang B. Verkman A.S. Gene structure, cDNA cloning, and expression of a mouse mercurial-insensitive water channel.Genomics. 1996; 33: 288-382https://doi.org/10.1006/geno.1996.0214Crossref Scopus (37) Google Scholar, 7.Mulders S.M. Preston G.M. Deen P.M. et al.Water channel properties of major intrinsic protein of lens.J Biol Chem. 1995; 270: 9010-9016https://doi.org/10.1074/jbc.270.15.9010Crossref PubMed Scopus (213) Google Scholar. To date, the regulation of MIP expression is poorly documented except for AQP2, the vasopressin-regulated water channel expressed selectively in the principal cells of the renal collecting duct. Activation of both expression and apical membrane integration of AQP2 induced by vasopressin is responsible for water reabsorption and urine concentration in the kidney5.Deen P.M. Verdijk M.A. Knoers N.V. et al.Requirement of human renal water channel aquaporin-2 for vasopressin-dependent concentration of urine.Science. 1994; 264: 92-95Crossref PubMed Scopus (763) Google Scholar. An epithelial complex highly specialized in water transfer is present in the digestive tract of homopteran sap sucking insects such as Cicadella: the “filter chamber,” which allows rapid sap concentration and water movement8.Gouranton J. Ultrastructures en rapport avec un transit d'eau: Etude de la “chambre filtrante” de Cicadella viridis L. (Homoptera, Jassidae).J Microsc. 1969; 7: 574-599Google Scholar. The large excess of water ingested in the sap is rapidly transferred from the initial midgut to the terminal midgut or Malpighian tubules, down a transepithelial osmotic gradient Figure 2. Analysis of membranes in this water-shunting complex revealed the presence of a major 25 kD protein that was associated in homotetramers and organized in regular arrays of particles in the cell membranes Figure 29.Hubert J.F. Thomas D. Cavalier A. Gouranton J. Structural and biochemical observations on specialized membranes of the “filter chamber,” a water-shunting complex in sap-sucking homopteran insects.Biol Cell. 1989; 66: 155-163Crossref PubMed Scopus (20) Google Scholar,10.Beuron F. Le Cahérec F. Guillam M.T. et al.Structural analysis of a MIP family protein from the digestive tract of Cicadella viridis.J Biol Chem. 1995; 270: 17414-17422https://doi.org/10.1074/jbc.270.29.17414Crossref PubMed Scopus (67) Google Scholar. We have cloned the corresponding cDNA and showed that it encodes a 255 amino acid polypeptide that we named AQPcic (for AQuaPorin of Cicadella). This protein, which is specifically expressed in filter chamber cells, exhibits a high sequence homology with the MIP family members11.Le Cahérec F. Deschamps S. Delamarche C. et al.Molecular cloning and characterization of an insect aquaporin: Functional comparison with aquaporin-1.Eur J Biochem. 1996; 241: 707-715Crossref PubMed Scopus (73) Google Scholar,12.Le Cahérec F. Bron P. Verbavatz J.M. et al.Incorporation of proteins into Xenopus oocytes by proteoliposome microinjection: Functional characterization of a novel aquaporin.J Cell Sci. 1996; 109: 1285-1295Crossref PubMed Google Scholar. Overexpression and functional studies of AQPcic in Xenopus oocytes or in Saccharomyces cerevisiae have clearly demonstrated the selective water channel function of the protein11.Le Cahérec F. Deschamps S. Delamarche C. et al.Molecular cloning and characterization of an insect aquaporin: Functional comparison with aquaporin-1.Eur J Biochem. 1996; 241: 707-715Crossref PubMed Scopus (73) Google Scholar,13.Lagrée V. Pellerin I. Hubert J.F. et al.A yeast recombinant aquaporin mutant that is not expressed or mistargeted in Xenopus oocyte can be functionally analysed in reconstituted proteoliposomes.J Biol Chem. 1998; 273: 12422-12426https://doi.org/10.1074/jbc.273.20.12422Crossref PubMed Scopus (23) Google Scholar. Figure 1 A) Topological model of the major intrinsic protein (MIP) family with six transmembrane domains and NPA boxes localized in loops B and E. (B) The two NPA-containing loops folding in the lipid bilayer are structural components for the pore formation. To investigate the molecular basis of MIP protein solute selectivity, we analyzed the structural characteristics of different functional members of the family. Human AQP1 and AQPcic, as representative members of the AQP family, were compared with the GlpF of Escherichia coli. This article reports the results of our work on AQP1, AQPcic, and GlpF proteins overexpressed in yeast, bacteria, or Xenopus oocytes. Several observations have clearly demonstrated the homotetrameric organization of AQPs in either native membranes10.Beuron F. Le Cahérec F. Guillam M.T. et al.Structural analysis of a MIP family protein from the digestive tract of Cicadella viridis.J Biol Chem. 1995; 270: 17414-17422https://doi.org/10.1074/jbc.270.29.17414Crossref PubMed Scopus (67) Google Scholar, 14.Engel A. Fujiyoshi Y. Agre P. The importance of aquaporin water channel protein structures.EMBO J. 2000; 19 (review): 800-806https://doi.org/10.1093/emboj/19.5.800Crossref PubMed Scopus (113) Google Scholar, 15.Verbavatz J.M. Brown D. Sabolic I. et al.Tetrameric assembly of CHIP28 water channels in liposomes and cell membranes: A freeze-fracture study.J Cell Biol. 1993; 123: 605-618Crossref PubMed Scopus (218) Google Scholar, 16.Lagrée V. Froger A. Deschamps S. et al.Oligomerization state of water channels and glycerol facilitators: Involvement of loop E.J Biol Chem. 1998; 273: 33949-33953https://doi.org/10.1074/jbc.273.51.33949Crossref PubMed Scopus (54) Google Scholar or proteoliposomes after reconstitution with purified proteins12.Le Cahérec F. Bron P. Verbavatz J.M. et al.Incorporation of proteins into Xenopus oocytes by proteoliposome microinjection: Functional characterization of a novel aquaporin.J Cell Sci. 1996; 109: 1285-1295Crossref PubMed Google Scholar,15.Verbavatz J.M. Brown D. Sabolic I. et al.Tetrameric assembly of CHIP28 water channels in liposomes and cell membranes: A freeze-fracture study.J Cell Biol. 1993; 123: 605-618Crossref PubMed Scopus (218) Google Scholar. Hydrodynamic studies performed in our laboratory have shown that the AQPs AQP1 and AQPcic always remain in their native homotetrameric state when solubilized in 1% Triton X-100 or 2% n-octyl-glucoside (OG). The same quaternary structure was determined for recombinant AQPs10.Beuron F. Le Cahérec F. Guillam M.T. et al.Structural analysis of a MIP family protein from the digestive tract of Cicadella viridis.J Biol Chem. 1995; 270: 17414-17422https://doi.org/10.1074/jbc.270.29.17414Crossref PubMed Scopus (67) Google Scholar,16.Lagrée V. Froger A. Deschamps S. et al.Oligomerization state of water channels and glycerol facilitators: Involvement of loop E.J Biol Chem. 1998; 273: 33949-33953https://doi.org/10.1074/jbc.273.51.33949Crossref PubMed Scopus (54) Google Scholar. In contrast, the E. coli GlpF always behaves as a monomer in these nondenaturing detergents, suggesting that AQPs and GlpF have a different oligomerization state in cellular membranes16.Lagrée V. Froger A. Deschamps S. et al.Oligomerization state of water channels and glycerol facilitators: Involvement of loop E.J Biol Chem. 1998; 273: 33949-33953https://doi.org/10.1074/jbc.273.51.33949Crossref PubMed Scopus (54) Google Scholar. These results were corroborated by an in vivo analysis of the particle size distribution in membranes of Xenopus oocytes overexpressing AQPcic or GlpF. The size of the intramembraneous particles was investigated by freeze fracture of oocyte plasma membranes. When AQPcic was overexpressed in these cells, orthogonal particles arrays identical to those observed in native membranes were specifically detected. The main diameter of these particles was 8.2 nm and corresponded to the tetrameric quaternary structure of the AQP, as previously measured in native insect membranes. Similar results have been observed previously for AQP117.Eskandari S. Wright E.M. Kreman M. et al.Structural analysis of cloned plasma membrane proteins by freeze-fracture electron microscopy.Proc Natl Acad Sci USA. 1998; 95: 11235-11240https://doi.org/10.1073/pnas.95.19.11235Crossref PubMed Scopus (161) Google Scholar. Conversely, the functional expression of GlpF correlated to the presence of a specifically induced subpopulation of membranous particles in which the diameter size of 5.8 nm best fits with a monomeric form of GlpF protein18.Bron P. Lagrée V. Froger A. et al.Oligomerization state of MIP proteins expressed in Xenopus oocytes as revealed by freeze-fracture electron-microscopy analysis.J Struct Biol. 1999; 128: 287-296https://doi.org/10.1006/jsbi.1999.4196Crossref PubMed Scopus (27) Google Scholar. Moreover, such differences in organization between AQPs and GlpF in lipid bilayers were suggested by analyzing the behavior of the proteins toward 2% of the nondenaturing detergent, N-lauroyl sarcosine (NLS). Native AQPs, like AQP1 from red blood cells or AQPcic from insect membranes, are particularly resistant to solubilization by NLS Figure 3. This property allows an efficient first step of AQP purification12.Le Cahérec F. Bron P. Verbavatz J.M. et al.Incorporation of proteins into Xenopus oocytes by proteoliposome microinjection: Functional characterization of a novel aquaporin.J Cell Sci. 1996; 109: 1285-1295Crossref PubMed Google Scholar,19.Zeidel M.L. Ambudkar S.V. Smith B.L. Agre P. Reconstitution of functional water channels in liposomes containing purified red cell CHIP28 protein.Biochemistry. 1992; 31: 7436-7440Crossref PubMed Scopus (518) Google Scholar. This notable resistance to NLS solubilization is also observed when AQPs are overexpressed in yeast or oocyte cells13.Lagrée V. Pellerin I. Hubert J.F. et al.A yeast recombinant aquaporin mutant that is not expressed or mistargeted in Xenopus oocyte can be functionally analysed in reconstituted proteoliposomes.J Biol Chem. 1998; 273: 12422-12426https://doi.org/10.1074/jbc.273.20.12422Crossref PubMed Scopus (23) Google Scholar,20.Duchesne L. Hubert J.F. Lagrée V. et al.Differential behaviour of aquaporins and GlpF in N-lauroyl sarcosine detergent.in: Hohmann S. Nielson S. Molecular Biology and Physiology of Water and Solute Transport. Kluwer Academic/Plenium, New York2000: 23-28Crossref Google Scholar. In contrast, the GlpF, either native or overexpressed in yeast and oocyte, is always fully solubilized by NLS. Our data demonstrate that, despite a high degree of homology, AQPs and GlpFs have a differential comportment in nondenaturing detergents and probably have a different organization within cell membranes. From these observations, we propose that oligomeric state or strength of self-association of MIP proteins could be a key element in transport selectivity and/or in regulation of MIP properties. However, a recent three-dimensional crystallographic analysis of the E. coli GlpF indicates that histidine-tagged GlpF crystallizes as a symmetric arrangement of four channels3.Fu D. Libson A. Miercke L. et al.Structure of a glycerol-conducting channel and the basis for its selectivity.Science. 2000; 290: 481-486https://doi.org/10.1126/science.290.5491.481Crossref PubMed Scopus (882) Google Scholar. The authors nicely demonstrate that the nature of the pore, within the monomer, is closely responsible for the selective transport of glycerol molecules and water exclusion. Indeed, in the three-dimensional structure, a clear difference of nature and configuration of amino acids surrounding the solute pore is observed when compared with amino acids surrounding the AQP1 water pore2.Murata K. Mitsuoka K. Hiral T. et al.Structural determinants of water permeation through aquaporin-1.Nature. 2000; 407: 599-605https://doi.org/10.1038/35036519Crossref PubMed Scopus (1435) Google Scholar. Nevertheless, the oligomeric organization of GlpF in native bacterial membranes remains unsolved. Some data indicate that the oligomeric state of membrane proteins observed following crystallization does not necessarily correlate with their native organization in the lipid bilayer21.Shi D. Lewis M.R. Young H.S. Stokes D.L. Three-dimensional crystals of Ca2+-ATPase from sarcoplasmic reticulum: Merging electron diffraction tilt series and imaging the (h, k, 0) projection.J Mol Biol. 1998; 284: 1547-1564https://doi.org/10.1006/jmbi.1998.2283Crossref PubMed Scopus (16) Google Scholar, 22.Auer M. Scarborough G.A. Kuhlbrandt W. Three-dimensional map of the plasma membrane H+-ATPase in the open conformation.Nature. 1998; 392: 840-843https://doi.org/10.1038/33967Crossref PubMed Scopus (185) Google Scholar, 23.Mascher E. Lundahl P. The human red cell glucose transporter in octyl glucoside: High specific activity of monomers in the presence of membrane lipids.Biochim Biophys Acta. 1988; 945: 350-359Crossref PubMed Scopus (37) Google Scholar. Moreover, as deduced from structural analysis, the interfaces between GlpF subunits are almost as hydrophobic as the exterior, suggesting that a monomer could be stable in the membrane3.Fu D. Libson A. Miercke L. et al.Structure of a glycerol-conducting channel and the basis for its selectivity.Science. 2000; 290: 481-486https://doi.org/10.1126/science.290.5491.481Crossref PubMed Scopus (882) Google Scholar. In addition, a recent analysis using chemical cross-linking and mass spectrometry on purified GlpF has confirmed our main observations with a major fraction of GlpF being organized in monomers24.Manley D.M. McComb M.E. Perreault H. et al.Secondary structure and oligomerization of the E. coli glycerol facilitator.Biochemistry. 2000; 39: 12303-12311https://doi.org/10.1021/bi000703tCrossref PubMed Scopus (26) Google Scholar. The presence of a small fraction of GlpF oligomers observed by those authors after the chemical cross-link on native membranes suggests that folded GlpF could have a propensity for self-association. These data may reflect a significant physiological mechanism24.Manley D.M. McComb M.E. Perreault H. et al.Secondary structure and oligomerization of the E. coli glycerol facilitator.Biochemistry. 2000; 39: 12303-12311https://doi.org/10.1021/bi000703tCrossref PubMed Scopus (26) Google Scholar. Multiple sequence alignments of MIP proteins allowed us to identify five major positions corresponding to amino acid residues that are highly conserved in AQPs or in GlpFs, but with very different physicochemical properties between the two subgroups25.Froger A. Tallur B. Thomas D. Delamarche C. Prediction of functional residues in water channels and related proteins.Protein Sci. 1998; 7: 1458-1468Crossref PubMed Scopus (189) Google Scholar. Four or five of these amino acids are located either in the loop E or at the upper part of the sixth transmembrane domain. We constructed mutants of AQPcic in which four of these discriminating amino acids were substituted by the amino acids for GlpF Figure 3. Mutations in these characteristic positions abolished or modified solute transport, indicating a direct or indirect involvement of these amino acids in the functional properties of protein. In particular, substitution of two of these amino acids (AQP-Y222P/W223L) induced the transformation of AQP into a glycerol channel26.Lagrée V. Froger A. Deschamps S. et al.Switch from an aquaporin to a glycerol channel by two amino-acids substitution.J Biol Chem. 1999; 274: 6817-6819https://doi.org/10.1074/jbc.274.11.6817Crossref PubMed Scopus (90) Google Scholar. Analysis of both oligomeric states in OG and NLS solubilizations of AQPcic or GlpF mutants strengthens our hypothesis on the relationship between oligomerization and function of MIP proteins. Indeed, the functional water channels, AQP1, AQPcic, and AQP-C134S, exhibit a tetrameric oligomerization state in OG and are NLS-resistant. AQP-C134S is a mistargeted mutant in oocytes but is expressed as a functional water channel in yeast cells13.Lagrée V. Pellerin I. Hubert J.F. et al.A yeast recombinant aquaporin mutant that is not expressed or mistargeted in Xenopus oocyte can be functionally analysed in reconstituted proteoliposomes.J Biol Chem. 1998; 273: 12422-12426https://doi.org/10.1074/jbc.273.20.12422Crossref PubMed Scopus (23) Google Scholar. Conversely, GlpF, AQP-S205D, AQP-Y222P, and AQP-Y222P/W223L, which are unable to transport water, are in a monomeric form in OG and are soluble in NLS detergent. In contrast, the nonfunctional mutants AQP-A209K and -W223L remain tetrameric, like the wild-type AQP, but have lost the particular comportment toward NLS, suggesting a modification in the protein organization into the membrane. These data indicate that the loss of water channel function of AQPcic mutants is closely correlated to the modification of oligomeric organization in OG and/or resistance to NLS solubilization. Interestingly, AQP-Y222P/W223L mutants are permeable to glycerol, monomeric in OG, and soluble in 2% NLS as the wild-type GlpF. However, according to the three-dimensional structures of AQP1 and GlpF, the Y222P-W223L positions do not correspond to areas directly involved in the structure of the water or glycerol pores2.Murata K. Mitsuoka K. Hiral T. et al.Structural determinants of water permeation through aquaporin-1.Nature. 2000; 407: 599-605https://doi.org/10.1038/35036519Crossref PubMed Scopus (1435) Google Scholar,3.Fu D. Libson A. Miercke L. et al.Structure of a glycerol-conducting channel and the basis for its selectivity.Science. 2000; 290: 481-486https://doi.org/10.1126/science.290.5491.481Crossref PubMed Scopus (882) Google Scholar. These two positions are located at the junction between loop E and the sixth transmembrane domain on the extracellular side of the protein. Substitution of two amino acids in this critical zone, especially by introducing a proline residue in the primary structure of AQPcic, likely induces distal structural rearrangements that lead to the switch from a water to a glycerol channel. The importance of the sixth transmembrane domain in function and selectivity of MIP family proteins was investigated by analyzing the truncated forms of AQPcic or AQPcic/GlpF chimeras Figure 3. Deletion of the C-terminal cytoplasmic domain of AQPcic (AQP-245Z) had no effect on the AQP function, but deletion of the entire sixth transmembrane domain (AQP-216Z) led to a monomeric protein that was soluble in NLS like the nonfunctional mutants of AQPcic. To investigate the involvement of this domain in MIP properties, substitutions of half of the sixth transmembrane domains of AQPcic (AAG chimera) and GlpF (GGA chimera) were performed. In AQPcic, substitution was performed in position 228 in the primary structure, after the amino acids Y222 and W223, which already have been identified to be involved in AQPcic function. In GlpF, the substitution was performed after the residue 242 Figure 3. Interestingly, we noted that if the AAG chimera exhibited an oligomerization state and functional characteristics identical to the wild-type AQPcic, the GGA chimera remained monomeric but unable to transport glycerol. Our data show that the presence of half of the sixth transmembrane domain of GlpF in the AQPcic protein is compatible with tetrameric association and water channel function. This observation is in agreement with structural data showing that the lower half portion of the sixth transmembrane domain of AQP1 does not contain amino acids facing the inside of the aqueous pore in the membrane2.Murata K. Mitsuoka K. Hiral T. et al.Structural determinants of water permeation through aquaporin-1.Nature. 2000; 407: 599-605https://doi.org/10.1038/35036519Crossref PubMed Scopus (1435) Google Scholar. However, the amino acids present in the sixth transmembrane domain of GlpF retain the ability to fold AQPcic in an active structural conformation. In contrast, the presence of half of the sixth transmembrane domain of AQPcic in GlpF abolishes the glycerol transport function of GlpF, suggesting an important but different involvement of this domain in the MIP function. Crystallographic analysis of AQPcic and AAG, now in progress in our laboratory, may explain the importance of this domain in the protein organization and/or function. Together these data show that, despite their high homology in primary and tertiary structures, AQPs and solute facilitators exhibit different behaviors in vitro in mild detergents like OG or NLS, or in vivo when expressed in Xenopus oocytes. The close correlation observed between the water transport function of some MIP proteins and their differential comportment in detergent certainly reflects differences in their quaternary structures in cell membranes. We propose that the oligomerization state or strength of self-association is part of the mechanisms used by MIP proteins to ensure selectivity. Determination of molecular motifs involved in detergent binding and in the interactions between monomers is currently under investigation.