Molecular physiology of water transport: Current studies and future prospects

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Biology of the Cell (19971 89, 247-252 o Elsevier, Paris

Proceedings

Molecular physiology of water transport: Current studies and future prospects Richard M Hays Albert Einstein College of Medicine, 1300 Morris Park Ave Bronx, New York, NY 10461, USA

water transport / aquaporins / water channels

INTRODUCTION The following summary of the Symposium covers many contributions, some of which are included in this issue of Biology of the Cell. Additional contributions will be discussed as well, along with some references to the current literature. My emphasis will be on establishing where the problem of water transport is at the moment, and some of the questions that need to be answered for progress to take place. There is no doubt that with the discovery of the aquaporins (Preston et al, 1992) and the array of ideas and techniques that have been brought to bear on the problem, that progress will be rapid, and that the next International Symposium will provide many new answers.

A BRIEF HISTORY For decades, our picture of water transport rested on the data that biophysics could provide. The history of this period has been recently reviewed (Hays and L,eaf, 1997). Early measurements in vasopressin-treated amphibian skin and bladder gave diffusion rates for water that appeared to be far too slow to account for the observed water flow (Koefoed-Johnsen and Ussing, 1953) and it was suggested that bulk flow through relatively large pores was taking place. With more refined methods of measuring the movement of labeled water, the true rate of diffusion of water across the cell membrane proved to be far higher than originally thought (Hays and Franki, 1970) and the estimated pore diameter was reduced to the point that it might approximate the diameter of the water molecule. Molecular physiology of water transport

Ions, and solutes as small as urea would be excluded from these water channels, through which water might move in a single-file fashion. The freeze-fracture studies of Chevalier et al (1974) gave a biological reality to the water channels, showing that their appearance on the membrane required the presence of oxytocin, and Wade (1978), showed that the channels cycled between the apical membrane and subapical vesicles. With the molecular identification of proteins functioning as water channels by Agre and co-workers (Preston et al, 1992), the study of the aquaporins began. Many questions about aquaporin structure and function are still unanswered. Interestingly enough, biophysical measurements continue to provide important contributions, even at a time when our attention is centered on the models provided by electron crystallography, as the following review will show.

SOLUTE PERMEABILITY OF THE AQUAPORINS The preponderance of published studies @hang et aI, 1993; Preston et al, 1994; Mulders et al, 1995), and presentations at this symposium, show that AQPl and AQP2 are impermeable to solutes as small as Ht. Agre and co-workers, and Mulders and coworkers found no increase in ion conductance in AQPl oocytes compared to water-injected oocytes, and no effects of CAMP or forskolin on conductance. Other members of the aquaporin family do transport solutes; Zeidel has proposed a three-fold classification of the aquaporins, based on the extent to which they transport solutes. AQPl and AQP2 Hays

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are representative of aquaporins that have little to no solute conductance and high water permeability; NOD26 and possibly MIP show some solute permeability and intermediate water conductance; AQP3 and Fpslp of yeast have high solute conductance and low water permeability. The yeast Fpslp was discussed in detail by Hohmann, who described it as a glycerol channel, functioning as an osmoregulator in yeast cells. AQP7 (formerly AQP6), identified in rat testis by Ishibashi et al, facilitates both glycerol and urea transport. Delamarche et al made the interesting proposal that water-conducting aquaporins can be distinguished from glycerol facilitators on the basis of a very small number of amino acids. Sasaki et a2 suggested that hydrophilic loops C and E may be important in the functional differentiation of AQP3 and AQP2. While there is general agreement that AQPl is not a solute transporter, some questions remain about the extent to which solutes may influence water flow across the channel. Hill noted that even in the red cell, where AQPl is thought to account for almost all of the water transport, the reflection coefficients for a variety of solutes are significantly less than 1.0. Among the possible explanations are that: 1) there may be other types of water channels in parallel with AQPl that have high solute permeability; or 2) that AQPl does, in fact transport solutes at a rate higher than has been assumed. Hill has proposed an answer to this paradox, in which solutes can move into the relatively wider entry region of the channel, and thereby modify the flow of water through the entire channel. This would give rise to different reflection coefficients for different solutes without requiring that they traverse the entire channel. Reflection coefficients of several solutes were used by Whittembury et al to estimate the length and diameter of the narrowest region (‘selectivity filter’) of AQPl in the rabbit proximal straight tubule. Applying Hill’s bimodal theory, the diameter of the selectivity filter was estimated to be 4.5 A, the length 6-9 A, and the number of water molecules moving in single file through the filter to be 2-3. Henzler et al proposed that solutes can enter the single file water channel in Chara and that this ‘solute slippage’ can drag along considerable amounts of water.

OTHER PATHWAYS FOR WATER Several papers addressed the question of nonaquaporin pathways for water flow. Meinild and Zeuthen presented studies showing that solute cotransport can account for significant water flow in tissues such as human small intestine and bullfrog retinal pigment epithelium. In the small intesMolecular physiology of water transport

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tine, which is thought to have no aquaporins, the Nat/glucose cotransporter accounts for the uptake of 5 L of water a day, approximately half the total water reabsorption. Water molecules appear to be bound directly to the cotransport proteins, and are thought to be moved as protein conformation changes. In these systems, water can move ‘uphill’ against a concentration gradient. Parisi et al, using cultured cell lines which have no aquaporins, observed net water movements in the absence of an osmotic gradient, and attributed them to water-ion cotransport. Lipid membranes vary greatly in their water permeability. Studies by Zeide1 and co-workers have shown that ‘barrier membranes’ such as those of the medullary thick ascending limb and the mammalian urinary bladder have water permeability values at least lo-fold lower than those of other biological membranes. Among the factors that decrease water permeability are increased rigidity of the hydrocarbon layer in the membrane interior and increased cholesterol content. From the work discussed above, it is clear that the overall water and solute permeability of a given membrane is a function of the types of pathways open to each, and that the aquaporins themselves vary considerably in their relative conductances of water and solutes. Beyond this, the biophysical approach has left us with some unanswered questions. One is the dimensions and length of the narrowest segment of the AQPl channel. Does it truly approximate the diameter of the water molecule, and do water molecules move through it in singlefile fashion? Is there any room for solutes in this segment? These questions may be answerable by more refined biophysical experiments, but they will probably have to await the results of crystallographic studies.

ELECTRON CRYSTALLOGRAPHY Three groups reported their progress in determining the three-dimensional structure of AQPl at the Symposium, in separate papers and in a combined workshop. The principal technique was that of cryo-electron microscopy of 2D crystals. Mitra in La Jolla, Jap in Berkeley and Engel in Basel, with their co-workers, have arrived at an overall structure at 6-7 A resolution. (Cheng et al, 1997; Li et al, 1997; Walz et al, 1997). The AQPl complex consists of four independent channels. Each channel is formed by six tilted alpha-helices spanning the bilayer (figs 1,2). The helices surround a central density, which may represent, or be in close proximity to the narrowest segment of the channel. The structure of this density remains controversial. It has been proposed that the density is a ‘vestibule’ with a diameter of Hays

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are in position in the plasma membrane. The majority of studies were of AQP2, which, so far, is the major hormone-related aquaporin. Thus, one would anticipate that ADH levels, osmolality and related factors would influence AQP2 expression and function.

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Expression

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Fig 1. Current proposals for the structure of AQPl: the hourglass model joining helices 2-3 and 5-6, which have identical NPA motifs, become superimposed when the helices fold to form the outer barrel of the channel. The juxtaposed loops are thought to form the selective segment of the channel (From Jung et al, 1994, with permission).

Murillo and co-workers reported that water deprivation in rats increased mRNA for AQP’s 2, 3, and 4 in the cortical, but not the medullary collecting duct. Marples et al reported that long-term blockade of V2 receptors in mice significantly reduced AQP2 expression, suggesting that expression is regulated by CAMP. Signals via pathways other than V2 receptors may also be involved. Yasui et al, using transfected LLC-PKl cells, reached a similar conclusion, and showed in addition that AVP caused a time- and dose-dependent phosphorylation of AQP2. ‘In the developing rat kidney, Aperia et al showed that AQP2 mRNA levels increase progressively with age, V2 receptor stimulation and administration of glucocorticoids. In the developing lung, AQP4 mRNA is induced in an age-dependent manner by glucocorticoids and beta-adrenergic agents. AQPl expression in the sheep proximal tubule was found by Butkus et al to occur only after the mesonephros is completely regressed. The age-dependent expression and localization of AQP’s 1, 3, 4 and 5 in the rat upper respiratory tract was reported by King et al.

Trafficking approximately 8 A at its narrowest point, outlined by loops originating from the surrounding helices, the so-called ‘hourglass’ model (Jung et al, 1994). Jap and co-workers, on the other hand, have described the central density not in terms of the hourglass model, but as a branched rod-like structure that probably contains at least one alpha-helix (Li et al, 1997). In order to resolve these questions and to determine the structure of the water channel within the aquaporin molecule, studies at higher resolution (2 A or less) will be needed. In view of the recent progress in electron crystallographic resolution, we can expect that such studies will be available soon.

AQP EXPRESSION, TRAFFICKING AND FUNCTION A number of presentations dealt with the factors that control the expression of aquaporins, their trafficking with.in the cell, and their function once they Molecular physiology of water transport

Trafficking of AQP2-containing vesicles from cytoplasm to plasma membrane was the subject of several reports. Valenti et al (1996) reported studies with a cell line derived from human cortical collecting duct transfected with a coding sequence of rat kidney AQP2. These cells show a six-fold increase in osmotic water flow following vasopressin stimulation, with redistribution of AQP2 from an intracellular compartment to the apical membrane, the same site as in native AQP2 cells. A heterotrimeric G-protein was shown to be involved in vesicle redistribution; redistribution was inhibited by pertussis toxin. G-proteins colocalized with AQP2 on vesicles. Colocalization of AQP2 and G-proteins was also reported by Rosenthal et al in studies of purified vesicles from rat kidney. Brown and co-workers used green fluorescent protein (GFP) to tag the N- and C-termini of AQP2 in LLC-PKl cells transfected with AQP2 and treated with vasopressin. In this system, the AQP2 is mobilized from intracellular vesicles to the basolateral rather than the apical membrane. When GFP Hays

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Fig 2. Structure of AQPl from studies at 6-7 A resolution supports, the hourglass model. Six rod-like outer helices surround a central vestibular region thought to lead to the water-selective segment. a. From the work of Cheng et al (1997, with permission), a shows a bridging density with a central block (asterisk). The structure is linked to helices D and F, as in the hourglass model. b. From the work of Walz et al (1997, with permission), again shows the central density (XI, possibly linked to the side helices of the channel. Helices 5 and 6 are cut away in this view to show density X. c, d. From the studies of Li et al (1997, with permission), different views, of the structure of the selective segment. c. An end-on view of the aquaporin complex of four channels. The helices of each channel (l-6, upper left monomer) form a trapezoid. The trapezoid encloses a branched, rodlike structure (7/81, which is thought to be a short additional alpha-helix. A less hydrophobic component of this structure is pro posed to be related to the selective segment of the channel. d. Side view of the channel, again showing helix 7/8.

was present on the N-terminus, the GFP-Aqp2 construct moved to the basolateral membrane. When GFP was on the C-terminus, the construct remained Molecular physiology of water transport

predominantly on the plasma membrane. The authors concluded that the C-terminus of AQP2 contains one or more targeting elements required Hays

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for regulated recycling of AQP2. Additional studies suggested that phosphorylation of serine 256 on the AQP2 tail was necessary for movement to the plasma membrane, and that AQP2 is an actin-binding protein. Glycosylation is an important factor in trafficking and function of many proteins in mammalian cells. Baumgarten ef al presented studies in transfected MDCK cells suggesting that glycosylation of AQP2 is not essential for proper AQP2 routing and function. Cytoplasmic dynein appears to play a role as a microtubule motor in water channel delivery. Video microscopy studies by Ahrens et a2on material from rabbit and bovine renal medulla showed that renal dynein and kinesin induced microtubule gliding, and that kinesin-induced gliding was inhibited by vanadate and EHNA.

In situ regulation How may aquaporins be regulated once they are in place in the plasma membrane? A most interesting contribution was that of Johansson et al, who showed that PM 28A, a major intrinsic protein in the spinach plasma membrane, is an aquaporin, and that its activity is regulated by phosphorylation at its serine 274 site. Phosphorylation is turgordependent, and water transport is greater in the phosphorylated than in the non-phosphorylated state. Candia and co-workers presented studies in the frog bladder suggesting that serosal hypotonicity initiates a signaling mechanism that reduces basolateral water permeability, possibly due to regulation of basolateral water channels. The glyceroltransporting aquaporin in yeast, Fpslp, closes down in response to external hypertonic@, in studies reported by Hohmann. This permits the cell to accumulate glycerol, counterbalancing the external hypertonicity. External hypotonicity has the opposite effect, opening the channel and allowing glycerol release. These studies show a role for solutetransporting aquaporins in osmolyte transfer for cell volume control.

AQUAPORINS IN HUMAN DISEASE We are just beginning to understand the adjustments in aquaporin-mediated water flow that take place in human disease. Studies thus far have centered around AQP2. Here, changes in AQP2 expression are probably secondary to the vasopressin response in disease states. Nielsen and co-workers found an increase in AQP2 expression and enhanced plasma membrane targeting of AQP2 in rats with severe congestive heart failure and hyponatremia. The now well-established role of AQP2

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mutations in nephrogenic diabetes insipidus was not a major theme of the symposium, but certainly is an example of the importance of this area of investigation.

THE ROLE OF THE AQUAPORINS Finally, we may ask how important aquaporins are in the economy of the cell. This question was posed by Mark Knepper at the beginning of the Symposium, and the answer clearly lies in the system one is considering. At one end of the spectrum is the AQP2 system in the renal collecting duct. Here, the lipid membrane is a barrier membrane, to use Zeidel’s terminology. It is virtually impermeable to water, by virtue of its lipid structure and the absence of any parallel system for water transport. Any increase in its permeability is dependent on the incorporation of AQP2 channels. The proof is found in patients with severe congenital nephrogenie diabetes insipidus, who are almost completely unable to reabsorb water in their collecting ducts. At the opposite end of the spectrum one might place the renal proximal tubule, where parallel systems of water transport are probably present in the form of cotransport systems, as well as paracellular routes and the intrinsic permeability of the lipid membrane. Absence of AQPl, as in the Colton-null mutant (Preston et al, 1994), does not appear to have serious effects on renal function, at least under normal physiological circumstances. Knepper, in his analysis, proposed that the absence of aquaporins may significantly affect the speed of equilibration of water across the cell membrane, and may promote compensatory mechanisms. For example, significantly higher rates of sodium reabsorption in the proximal tubule may be required to transport the normal amount of water in the absence of AQP2. As we learn more about the aquaporins, we may encounter other compensatory mechanisms of this kind.

REFERENCES Cheng A, van Hoek AN, Yaeger M, Verkman AS and Mitra AK (1997) Three-dimensional organization of a human water channel. Nature 387,627-630 Chevalier J, Bourguet J and Hugon JS (1974) Membrane associated particles: Distribution in frog urinary bladder epithehum at rest and after oxytocin treatment. Cell Tissue Res 152,129-140 Hays RM and Franki N (1970) The rate of water diffusion in the action of vasopressin. J Membr Biol2,263-276 Hays RM and Leaf A (1997) Milestones in nephrology: Studies on the movement of water through the isolated toad bladder and its modification by vasopressin. J Am Sot Nephrol6, 1005-1015 Jung JS, Preston GM, Smith BL Guggino WB and Agre P (1994) Molecular structure of the water channel through aquaporin CHIP: The hourglass model. J Biol Chem 269, 1464% 14654

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Koefoed-Johnsen V and Ussing HH (1953) The contribution of diffusion and flow to the passage of D 0 through living membranes. Actu Physiol Scand Z&60-76 Li H, Lee S and Jap BK (1997) Molecular design of aquaporin-1 water channel as revealed by electron crystallography. Nature Struct Biol4,263-265 Mulders SM, Preston GM, Deen PMT, Guggino WB, van OS CH and Agre P (1995) Water channel properties of major intrinsic proteins of lens. J Biol Chem 270,9010-9016 Preston GM, Carroll TP, Guggino WB and Agre P (1992) Appearance of water channels in Xenopus oocytes expressing red cell CHIP28 protein. Science 256,385-387 Preston GM, Smith B, Zeidel ML, Moulds JJ and Agre P (1994) Mutations in aquaporin-1 in phenotypically normal humans without functional CHIP water channels. Science 265‘1585-1587

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Valenti G, Frigeri A, Ronco PM, D’Ettore C and Svelto M (1996) Expression and functional analysis of water channels in a stably AQPZ-transfected human collecting duct cell line. J Biol Chem 271,24365-24370 Wade JB (1978) Membrane structural specialization of the toad urinary bladder revealed by the freeze-fracture technique. III. Location, structure and vasopressin dependence of intramembranous particle array. ] Membr Biol 40, 281-296 Walz T, Hirai T, Murata K, Heymann JB, Mitsuoka K, Fujiyoshi Y, Smith BL, Agre P and Engel A (1997) The threedimensional structure of aquaporin-1. Nature 387,624-626 Zhang R, Skach W, Hasegawa H, van Hoek AN and Verkman AS (1993) Cloning, functional analysis and cell localization of a kidney proximal tubule water transporter homologous to CHIP28. J Cell Bioll20,359-369

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