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Molecular physiology of water transport: Molecular structure and function of MIP family members
253
Biology of the Cell (1997) 89, 253-254 o Elsevier, Paris
Proceedings
Molecular physiology of water transport: Molecular structure and function of MIP family members Mark L Zeidel Rena/-ElectrolyteDivision and Laboratory of Cell Biology, University of Pittsburgh Medical Center, A91 9 Sciafe Hall, 3550 Terrace Street, Pittsburgh, PA 15261, USA
The participants in the conference gathered to try to integrate the available functional and structural data into a model of the water pore running through the aquaporin molecule. Because the vast majority of the functional and all of the structural data currently available were obtained with AQPl, this protein served as the focal point of discussion. However, given the high degree of homology among AQP’s, especially in critical membranespanning domains, it appears likely that the major features of AQPl pore function will be applicable to the pores formed by other AQP’s. Classical functional theory describes the pore as a simple cylinder which is long enough to traverse the thickness of the bilayer and narrow enough at one or more points along its course to require water molecules to pass single-file through it and to exclude small non-electrolyte solutes (Finkelstein, 1986; Stein, 1990). Under classical theory water flows through the pore as it does through a pipe, so that the conductance of the cylindrical pore is related to its radius by Poiseuille’s Law. Because the cylinder is narrow enough to force single-file flow of water molecules, the ratio of osmotic to diffusive water permeability (PF/PD) is used to determine the number of water molecules lined up within the single-file pore at any one time (Finkelstein, 1986; Stein, 1990). Although it has been unclear whether the classical formulation makes physical sense when applied to pores so narrow that their radii approach the size of water molecules, the theory has been applied successfully to gramicidin and nystatin (Finkelstein, 1986; Stein, 1990). In the case of gramicidin, the results obtained for radius of the pore and the length of its single-file portion appear to match the results of structural studies. Application of the classical formulation to AQPl also gives reasonable values for pore radius and length. However, the Molecular physiology of water transport
classical formulation may not apply well to other AQP’s and may be inadequate for AQPl as well. Alan Verkman’s group presented conductance data for AQP4 which, when put into the Poiseuille equation predict a radius of the pore which is so large as to permit water molecules to pass each other and solutes such as urea and glycerol to permeate the pore (Yang and Verkman, 1997). The Zeidel laboratory presented data on the plant symbiosome aquaporin NOD26 which also appear to contradict the classical formulation (Rivers et al, 1997). NOD26 permits permeation of glycerol and acetamide, but exhibits a 30-fold lower unit conductance than AQPl. If solutes permeate AQP’s through the water pore then we would predict that AQP’s which permit solute permeation should have the highest conductances for water, and not values markedly lower than AQP’s such as AQPl, which does not appear to mediate such solute fluxes in reconstitution experiments (Zeidel et al, 1992). Adrian Hill summarized his modification of the classical formulation, which involves considering both the diffusive and viscous aspects of the flux of water through the pore (Hill, 1994, 1995). An important aspect of his model is the prediction that small solutes such as glycerol and urea may be able to penetrate wider-diameter regions of the pore (socalled ‘atria’). This model has the advantage of considering directly the physical forces involved in water flow through pores only slightly larger than the molecule itself. The Hill formulation is consistent with the functional behavior observed in the limited available studies of gramicidin and AQPl, but has not been tested rigorously in any model system. Alan Verkman suggested that the best course might be to abandon all of the functional formulations until more detailed structural data are available, and then apply a mathematical modeling Zeidel
254
approach to defining the structure and function of the pore. This proposal was greeted with varying levels of enthusiasm. It appears highly likely that detailed functional data on individual AQP’s will be extremely important in developing a coherent interpretation of the structural data. Drs Mitra, Jap and Engel reviewed the structural data available from a variety of approaches including electron crystallography of glucose-embedded (Jap and Engel) or frozen-hydrated (Mitra) 2-D crystals, as well as electron microscopy of metalshadowed AQPl crystals (Engel) and surface topographs recorded with the atomic force microscope of crystals in buffer solution (Engel) (Walz et al, 1996; Cheng et aI, 1997). All methods have achieved a resolution of 6-7 angstroms, and the images obtained in the different laboratories appear to be consistent with one another. In assigning different portions of the protein molecule to the structural features observed at the current level of resolution, several features of AQPl obtained from structurefunction studies in oocytes have been agreed upon by all investigators: 1) the functional unit of the molecule is the monomer although AQPl forms tetramers (Preston et aI, 1993); 2) the molecule has six membrane-spanning domains, with ammo and carboxy termini inside the cell (Preston et al, 1994); 3) there are two domains which approach or touch the membrane, forming the ‘hourglass’ shape proposed by the Agre laboratory (Jung et al, 1994); 4) the NPA regions present in the two halves of the molecule are critical for AQP function (Preston et al, 1994); and 5) the AQPl structures being defined in the “imaging” studies represent functional forms of the molecule, so that inferences about pore structure can be obtained from them (Walz et al, 1994). The general models obtained by the different groups are relatively similar, and feature multiple tilted bilayer-spanning alpha helices. The Engel model proposes a right-handed orientation of the helices, while those of Mitra and Jap propose a lefthanded orientation. It was generally agreed that the resolution of the current structural information is inadequate to permit much definition of the structural properties of the pore, although some models can discern a vestibule of 8 angstroms in diameter between the helices and flanked by the NPA sequences in the two halves of the molecule (Cheng et al, 1997). In particular, the location and orientation of the amino acid side chains lining the pore cannot be defined. After vigorous entreaties to speculate on the structure of the pore, Dr Mitra proposed a version with atria at either end and a narrow cylindrical shape in the middle. It appears highly likely that further structural resolution combined with detailed functional stud-
Molecular physiology of water transport
Biology of the Cell (1997) 89, 253-254
ies will at last define the nature of the pore through AQPl. It will then become increasingly important to understand how this pore might be modified so as to permit some AQP’s to mediate the fluxes of small non-electrolytes. In addition, there is evidence that some plant AQP’s are gated, likely via phosphorylation of their carboxy terminal chains (Johansson et al, 1997; Rivers et al, 1997). Since the carboxy terminal regions of AQP’s exhibit the greatest variability and AQPl exhibits normal water channel function in the absence of its carboxy terminus (Zeidel et al, 1994), the mechanisms involved in gating via the carboxy terminus will be of great interest.
REFERENCES Cheng A, van Hoek AN, Yeager M, Verkman AS and Mitra AK (1997) Three-dimensional organization of a human water channel. Nature 387,627-630 Finkelstein A (1986) Water movement through lipid bilayers, pores and plasma membranes, theory and reality. Wiley and Sons, New York, New York Hill AE (1994) Osmotic flow in membrane pores of molecular size. J Membr Biol137,197-203 Hill AE (1995) Osmotic flow in membrane pores. Int Rev Cyfol 163,1-41 Johansson I, Larsson C, Ek B and Kjellbom P (1997) The major integral proteins of spinach leaf plasma membranes are putative aquaporins and are phosphorylated in response to calcium and apoplastic water potential. Plant Cell 8, 1181-1191 Jung JS, Preston GM, Smith BL, Guggino WB and Agre P (1994) Molecular structure of the water channel through aquaporin CHIP: The hourglass model. 1 Biol Chem 269, 14648-14654 Preston GM, Jung JS, Guggino WB and Agre P (1993) The mercurv-sensitive residue at cvsteine 189 in the CHIP28 water channel. J Biol Chem 268,17-20 Preston GM, Jung JS, Guggino WB and Agre P (1994) Membrane topology of aquaporin CHIP: Analysis of functional epitope-scanning mutants by vectorial proteolysis. J Biol Chem 269,1668-1673 Rivers RL, Dean RM, Chandy G, Hall JE, Roberts DM and Zeide1 ML (1997) Functional analysis of nodulin 26, an aquaporin in soybean root nodule symbiosomes. J Biol Chem 272, 16256-16261 Stein WD (1990) Channels, carriers, and pumps: An introduction to membrane transport. Academic Press, San Diego, CA Walz T, Smith BL, Zeidel ML, Engel A and Agre P (1994) Water transport by two-dimensional crystalline arrays of aquaporin CHIP. J Biol Chem 269,1583-1587 Walz T, Tittmann P, Fuchs KH, Muller DJ, Smith BL, Agre I?, Gross H and Engel A (1996) Surface topographise at subnanometer-resolution reveal asymmetry and sidedness of aquaporin-1. J Mel Biol264,907-918 Yang B and Verkman AS (1997) Water and glycerol permeabilities of aquaporins l-5 and MIP determined quantitatively by expression of epitope-tagged constructs in Xenopus oocytes. J Biol Chem 272,16140-16146 Zeidel ML, Ambudkar S, Smith B and Agre P (1992) Reconstitution of functional water channels in liposomes containing purified red cell CHIP28 protein. Biochemistry 31, 7436-7440 Zeidel ML, Nielsen S, Smith BL, Ambudkar SV, Maunsbach AB and Agre I’ (1994) Ultrastructure, pharmacologic inhibition, and transport selectivity of aquaporin CHIP in proteoliposomes. Biochemistry 33,1606-1615
Zeidel
Biology of the Cell (1997) 89, 253-254 o Elsevier, Paris
Proceedings
Molecular physiology of water transport: Molecular structure and function of MIP family members Mark L Zeidel Rena/-ElectrolyteDivision and Laboratory of Cell Biology, University of Pittsburgh Medical Center, A91 9 Sciafe Hall, 3550 Terrace Street, Pittsburgh, PA 15261, USA
The participants in the conference gathered to try to integrate the available functional and structural data into a model of the water pore running through the aquaporin molecule. Because the vast majority of the functional and all of the structural data currently available were obtained with AQPl, this protein served as the focal point of discussion. However, given the high degree of homology among AQP’s, especially in critical membranespanning domains, it appears likely that the major features of AQPl pore function will be applicable to the pores formed by other AQP’s. Classical functional theory describes the pore as a simple cylinder which is long enough to traverse the thickness of the bilayer and narrow enough at one or more points along its course to require water molecules to pass single-file through it and to exclude small non-electrolyte solutes (Finkelstein, 1986; Stein, 1990). Under classical theory water flows through the pore as it does through a pipe, so that the conductance of the cylindrical pore is related to its radius by Poiseuille’s Law. Because the cylinder is narrow enough to force single-file flow of water molecules, the ratio of osmotic to diffusive water permeability (PF/PD) is used to determine the number of water molecules lined up within the single-file pore at any one time (Finkelstein, 1986; Stein, 1990). Although it has been unclear whether the classical formulation makes physical sense when applied to pores so narrow that their radii approach the size of water molecules, the theory has been applied successfully to gramicidin and nystatin (Finkelstein, 1986; Stein, 1990). In the case of gramicidin, the results obtained for radius of the pore and the length of its single-file portion appear to match the results of structural studies. Application of the classical formulation to AQPl also gives reasonable values for pore radius and length. However, the Molecular physiology of water transport
classical formulation may not apply well to other AQP’s and may be inadequate for AQPl as well. Alan Verkman’s group presented conductance data for AQP4 which, when put into the Poiseuille equation predict a radius of the pore which is so large as to permit water molecules to pass each other and solutes such as urea and glycerol to permeate the pore (Yang and Verkman, 1997). The Zeidel laboratory presented data on the plant symbiosome aquaporin NOD26 which also appear to contradict the classical formulation (Rivers et al, 1997). NOD26 permits permeation of glycerol and acetamide, but exhibits a 30-fold lower unit conductance than AQPl. If solutes permeate AQP’s through the water pore then we would predict that AQP’s which permit solute permeation should have the highest conductances for water, and not values markedly lower than AQP’s such as AQPl, which does not appear to mediate such solute fluxes in reconstitution experiments (Zeidel et al, 1992). Adrian Hill summarized his modification of the classical formulation, which involves considering both the diffusive and viscous aspects of the flux of water through the pore (Hill, 1994, 1995). An important aspect of his model is the prediction that small solutes such as glycerol and urea may be able to penetrate wider-diameter regions of the pore (socalled ‘atria’). This model has the advantage of considering directly the physical forces involved in water flow through pores only slightly larger than the molecule itself. The Hill formulation is consistent with the functional behavior observed in the limited available studies of gramicidin and AQPl, but has not been tested rigorously in any model system. Alan Verkman suggested that the best course might be to abandon all of the functional formulations until more detailed structural data are available, and then apply a mathematical modeling Zeidel
254
approach to defining the structure and function of the pore. This proposal was greeted with varying levels of enthusiasm. It appears highly likely that detailed functional data on individual AQP’s will be extremely important in developing a coherent interpretation of the structural data. Drs Mitra, Jap and Engel reviewed the structural data available from a variety of approaches including electron crystallography of glucose-embedded (Jap and Engel) or frozen-hydrated (Mitra) 2-D crystals, as well as electron microscopy of metalshadowed AQPl crystals (Engel) and surface topographs recorded with the atomic force microscope of crystals in buffer solution (Engel) (Walz et al, 1996; Cheng et aI, 1997). All methods have achieved a resolution of 6-7 angstroms, and the images obtained in the different laboratories appear to be consistent with one another. In assigning different portions of the protein molecule to the structural features observed at the current level of resolution, several features of AQPl obtained from structurefunction studies in oocytes have been agreed upon by all investigators: 1) the functional unit of the molecule is the monomer although AQPl forms tetramers (Preston et aI, 1993); 2) the molecule has six membrane-spanning domains, with ammo and carboxy termini inside the cell (Preston et al, 1994); 3) there are two domains which approach or touch the membrane, forming the ‘hourglass’ shape proposed by the Agre laboratory (Jung et al, 1994); 4) the NPA regions present in the two halves of the molecule are critical for AQP function (Preston et al, 1994); and 5) the AQPl structures being defined in the “imaging” studies represent functional forms of the molecule, so that inferences about pore structure can be obtained from them (Walz et al, 1994). The general models obtained by the different groups are relatively similar, and feature multiple tilted bilayer-spanning alpha helices. The Engel model proposes a right-handed orientation of the helices, while those of Mitra and Jap propose a lefthanded orientation. It was generally agreed that the resolution of the current structural information is inadequate to permit much definition of the structural properties of the pore, although some models can discern a vestibule of 8 angstroms in diameter between the helices and flanked by the NPA sequences in the two halves of the molecule (Cheng et al, 1997). In particular, the location and orientation of the amino acid side chains lining the pore cannot be defined. After vigorous entreaties to speculate on the structure of the pore, Dr Mitra proposed a version with atria at either end and a narrow cylindrical shape in the middle. It appears highly likely that further structural resolution combined with detailed functional stud-
Molecular physiology of water transport
Biology of the Cell (1997) 89, 253-254
ies will at last define the nature of the pore through AQPl. It will then become increasingly important to understand how this pore might be modified so as to permit some AQP’s to mediate the fluxes of small non-electrolytes. In addition, there is evidence that some plant AQP’s are gated, likely via phosphorylation of their carboxy terminal chains (Johansson et al, 1997; Rivers et al, 1997). Since the carboxy terminal regions of AQP’s exhibit the greatest variability and AQPl exhibits normal water channel function in the absence of its carboxy terminus (Zeidel et al, 1994), the mechanisms involved in gating via the carboxy terminus will be of great interest.
REFERENCES Cheng A, van Hoek AN, Yeager M, Verkman AS and Mitra AK (1997) Three-dimensional organization of a human water channel. Nature 387,627-630 Finkelstein A (1986) Water movement through lipid bilayers, pores and plasma membranes, theory and reality. Wiley and Sons, New York, New York Hill AE (1994) Osmotic flow in membrane pores of molecular size. J Membr Biol137,197-203 Hill AE (1995) Osmotic flow in membrane pores. Int Rev Cyfol 163,1-41 Johansson I, Larsson C, Ek B and Kjellbom P (1997) The major integral proteins of spinach leaf plasma membranes are putative aquaporins and are phosphorylated in response to calcium and apoplastic water potential. Plant Cell 8, 1181-1191 Jung JS, Preston GM, Smith BL, Guggino WB and Agre P (1994) Molecular structure of the water channel through aquaporin CHIP: The hourglass model. 1 Biol Chem 269, 14648-14654 Preston GM, Jung JS, Guggino WB and Agre P (1993) The mercurv-sensitive residue at cvsteine 189 in the CHIP28 water channel. J Biol Chem 268,17-20 Preston GM, Jung JS, Guggino WB and Agre P (1994) Membrane topology of aquaporin CHIP: Analysis of functional epitope-scanning mutants by vectorial proteolysis. J Biol Chem 269,1668-1673 Rivers RL, Dean RM, Chandy G, Hall JE, Roberts DM and Zeide1 ML (1997) Functional analysis of nodulin 26, an aquaporin in soybean root nodule symbiosomes. J Biol Chem 272, 16256-16261 Stein WD (1990) Channels, carriers, and pumps: An introduction to membrane transport. Academic Press, San Diego, CA Walz T, Smith BL, Zeidel ML, Engel A and Agre P (1994) Water transport by two-dimensional crystalline arrays of aquaporin CHIP. J Biol Chem 269,1583-1587 Walz T, Tittmann P, Fuchs KH, Muller DJ, Smith BL, Agre I?, Gross H and Engel A (1996) Surface topographise at subnanometer-resolution reveal asymmetry and sidedness of aquaporin-1. J Mel Biol264,907-918 Yang B and Verkman AS (1997) Water and glycerol permeabilities of aquaporins l-5 and MIP determined quantitatively by expression of epitope-tagged constructs in Xenopus oocytes. J Biol Chem 272,16140-16146 Zeidel ML, Ambudkar S, Smith B and Agre P (1992) Reconstitution of functional water channels in liposomes containing purified red cell CHIP28 protein. Biochemistry 31, 7436-7440 Zeidel ML, Nielsen S, Smith BL, Ambudkar SV, Maunsbach AB and Agre I’ (1994) Ultrastructure, pharmacologic inhibition, and transport selectivity of aquaporin CHIP in proteoliposomes. Biochemistry 33,1606-1615
Zeidel