Water Pumps 1

Water Transport Controversies Special Issue

Journal of Physiology (2002), 542.1, pp. 53–60 © The Physiological Society 2002

DOI: 10.1113/jphysiol.2002.018713 www.jphysiol.org

Topical Review Water pumps Donald D. F. Loo *, Ernest M. Wright * and Thomas Zeuthen † * Department of Physiology, UCLA School of Medicine, Los Angeles, CA 90095-1751, USA and † The Panum Institute, University of Copenhagen, DK-2200 Copenhagen N, Denmark

The transport of water across epithelia has remained an enigma ever since it was discovered over 100 years ago that water was transported across the isolated small intestine in the absence of osmotic and hydrostatic pressure gradients. While it is accepted that water transport is linked to solute transport, the actual mechanisms are not well understood. Current dogma holds that active ion transport sets up local osmotic gradients in the spaces between epithelial cells, the lateral intercellular spaces, and this in turn drives water transport by local osmosis. In the case of the small intestine, which in humans absorbs about 8 l of water a day, there is no direct evidence for either local osmosis or aquaporin gene expression in enterocytes. Intestinal water absorption is greatly enhanced by glucose, and this is the basis for oral rehydration therapy in patients with secretory diarrhoea. In our studies of the intestinal brush border Na+–glucose cotransporter we have obtained evidence that there is a direct link between the transport of Na+, glucose and water transport, i.e. there is cotransport of water along with Na+ and sugar, that will account for about 50 % of the total water transport across the human intestinal brush border membrane. In this short review we summarize the evidence for water cotransport and propose how this occurs during the enzymatic turnover of the transporter. This is a general property of cotransporters and so we expect that this may have wider implications in the transport of water and other small polar molecules across cell membranes in animals and plants. (Received 9 February 2002; accepted after revision 22 March 2002)

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Corresponding author D. Loo: Department of Physiology, UCLA School of Medicine, 10833 LeConte Avenue, Los Angeles, CA 90095-1751, USA. Email: [email protected]

The question of how water is transported across epithelial cells in the absence of external driving forces has intrigued physiologists for over a century, since Reid (1892) demonstrated that water is transported across the intestine in the absence of transepithelial osmotic and hydrostatic pressure differences. Much of the stimulus for investigations of the past 40 years originated with the hypothesis that active Na+ transport was responsible for the generation of local osmotic gradients within epithelial tissues, and that water is coupled to Na+ movement by osmosis. Peter Curran proposed a three-compartment model for intestinal fluid absorption (Curran & McIntosh, 1962), and Jared Diamond proposed a local osmosis model for the gall bladder (Diamond & Bossert, 1967). The local osmotic compartment was not defined in the Curran model, but Diamond proposed that the local osmotic gradient was generated in the lateral intercellular spaces. The latter hypothesis is consistent with the location of the Na+–K+-

pumps in the basolateral membrane of absorptive epithelia. Although considerable energy has been devoted to determine the osmolarity of the lateral spaces, especially in the gall bladder, there is little evidence for any significant hypertonicity (see Spring, 1998). One possible explanation is that the hypertonicity is small (< 3 %) and the water permeability of the basolateral membrane is high. With Peter Agre’s discovery of water channels (Preston et al. 1992), the aquaporins, there has been resurgence of interest in epithelial water transport, especially in the kidney. In the intestine there is little, if any, expression of aquaporins in enterocytes and this raises the possibility that the high water permeability of the brush border and basolateral membranes is either due to a high intrinsic water permeability of the lipid bilayer or the presence of other water channels. Yet another possibility is that local osmosis does not account for water transport across the intestine.

Presented at The Journal of Physiology Synthesium on Water Transport Controversies, Christchurch, New Zealand, 30 August, 2001.

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D. D. F. Loo, E. M. Wright and T. Zeuthen

There is ample evidence that intestinal water absorption, 8 l a day in man, is intimately linked to ‘active’ solute absorption and that glucose in the gut lumen greatly enhances salt and water absorption. Glucose stimulates sodium transport across the brush border membrane of the enterocytes by Na+–glucose cotransport, and the Na+ that enters the cell is pumped out into the lateral intercellular spaces by the basolateral Na+–K+ pump. It is commonly assumed that water follows sugar and salt transport by local osmosis. These links between glucose, salt and water transport form the basis of the therapy used to treat secretory diarrhoeas such as cholera, oral rehydration therapy (Hirschorn & Greenough, 1991). Our interest in tackling the enigma of the mechanism of intestinal water transport was rekindled by two independent observations. The first followed our successful cloning and expression of the brush border Na+–glucose cotransporter, SGLT1 (Hediger et al. 1987), when it was discovered that SGLT1 also behaves as a water channel (Zampighi et al. 1995). The second was that water transport across the apical membrane of the choroid plexus was found to be closely linked to the activity of the K+–Cl_ cotransporter (Zeuthen 1991, 1994). We then initiated a collaborative study in Los Angeles and Copenhagen to examine more closely the relationship between Na+, glucose and water transport using the cloned transporter (SGLT1) expressed in Xenopus laevis oocytes (Loo et al. 1996, 1999; Zeuthen et al. 1997, 2001; Meinild et al. 1998; Leung et al. 2000). The focus of this brief review is to summarize the evidence that SGLT1 behaves as a water channel and a Na+–glucose–water cotransporter, and to provide an alternative hypothesis for the mechanism of water transport across the intestine in the absence of external osmotic or hydrostatic forces (Reid, 1892).

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Experimental approach Our strategy has been to overexpress rabbit or human SGLT1s in Xenopus laevis oocytes, and to determine whether the cotransporter changes the water transport of the oocytes. This offers several unique advantages. (1) Oocytes are large cells, 1 mm in diameter. (2) Native oocytes have very low water permeabilities. (3) While native oocytes do not express SGLTs to any significant level, they do efficiently translate SGLT1 cRNAs injected into the cell and efficiently insert fully functional SGLTs into the plasma membrane, up to 1 w 1011 copies of SGLT1 are found in the plasma membrane (Zampighi et al. 1995). (4) Na+–glucose is electrogenic and so it is simple to measure the rate of Na+–glucose cotransport continuously using electrophysiological methods (Parent et al. 1992a; Loo et al. 1996). There is tight coupling between Na+ and glucose transport, two Na+ ions are transported along with one glucose molecule, and the glucose-activated inward current is a Na+ current (Mackenzie et al. 1998). The cotransporter currents are as high as 2–3 mA, whereas the

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currents in control oocytes are less than 0.001 mA. (5) It is possible to continuously measure the volume of voltageclamped oocytes with great precision (0.03 %) and time resolution (1 s) using optical methods (Loo et al. 1996; Zeuthen et al. 1997). Thus it is straightforward to continuously measure Na+–glucose cotransport and water transport in the same cell with high precision and time resolution. Water transport is measured by monitoring the changes in cross-sectional area of the oocyte, and Na+–glucose cotransport is measured using the two-electrode voltage clamp technique. Na+– glucose cotransport is recorded as a glucose-stimulated inward Na+ current, and cotransport is activated, or inactivated, by (1) the addition or withdrawal of sugar, or (2) changing the membrane potential (Na+–glucose cotransport is highly voltage dependent between 0 and _100 mV; Parent et al. 1992a). In our systems, we can accurately record both water transport and Na+–glucose cotransport in a single cell over a time scale of seconds to minutes.

SGLT1 is a water channel Expression of SGLT1 in oocytes revealed that the cotransporter behaves as a low conductance water channel (Zampighi et al. 1995; Loo et al. 1996, 1999; Loike et al. 1996; Meinild et al. 1998). The osmotic water permeability (Lp) of SGLT1-expressing oocytes is directly proportional to the number of cotransporters expressed in the membrane with a slope of 1.4 w 10_14 cm s_1, which is about 5 % of that for a single aquaporin (AQP1) molecule. The water permeability of SGLT1 is independent of the size and direction of the osmotic gradient (_100 to +300 mosmol), and for at least 50 s is independent of time of exposure to the osmotic gradient (Zeuthen et al. 2002). The SGLT1 Lp is also independent of the presence or absence of ligands (Na+ and sugar), but phlorizin inhibited water flow through SGLT1 with a Ki of ~5 mM in the presence of Na+. Phlorizin (100 mM) reduces the Lp of oocytes expressing SGLT1 to that of control oocytes. The activation energy for passive water flow through SGLT1 is 5 kcal mol_1, similar to that for AQP1 but much lower than that for water flow through the native oocyte membrane (14 kcal mol_1). We conclude that SGLT1 behaves as a water channel. There is also evidence that urea and ethylene glycol, but not mannitol, pass through this channel (Leung et al. 2000; Panayotova-Heiermann & Wright, 2001). However, the equivalent radius of the channel must be small as the reflection coefficient for urea is close to 1 (Zeuthen et al. 2001). The channel is formed by the five C-terminal transmembrane helices of the protein (PanayotovaHeiermann & Wright, 2001). Thus the high density of SGLT1 proteins in the brush border of enterocytes, 250 000 copies per cell, are expected

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to contribute to the passive fluxes of water and other small polar molecules in and out of the cell. The channel properties of SGLT1 are shared by other cotransporters such as the thyroid Na+–iodide, the brain Na+–Cl_–GABA, the renal Na+–dicarboxylate and plant H+–amino acid cotransporters (Loo et al. 1996; Meinild et al. 2000; Leung et al. 2000).

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(4) The coupling coefficient is the same when cotransport is driven by either Na+ or H+ gradients (D. D. F. Loo & A.-K. Meinild, unpublished observations). (5) The coupling coefficient is different for different cotransporters. For example, in one series of experiments

Cotransport of water When oocytes expressing SGLT1 are incubated in a sugarfree isotonic solution there is no change in the volume of the oocyte with time. However, when the cotransporter is activated by sugar there is an immediate increase in cell volume. Figure 1 shows one experiment where the solution superfusing an oocyte expressing hSGLT1 is abruptly changed to one containing sugar. Note that the osmolarity of the superfusate was maintained constant by replacing 10 mM mannitol with 10 mM a-methyl-D-glucopyranoside (aMDG, a model non-metabolized SGLT1 substrate). There was an immediate increase in the inward Na+ current through SGLT1 that reached a steady value of 1540 nA within 30 s, and returned towards the baseline when the specific blocker phlorizin was added (Fig. 1A). The volume of the oocyte also increased immediately on activation of the Na+–glucose cotransporter, and phlorizin blocked this volume increase (Fig. 1B). The initial increase in volume of the oocyte can be accounted for if we assume that the transport of two Na+ ions and one glucose molecule is coupled to the transport of 249 water molecules. In this series of experiments 203 ± 20 water molecules were transported for each turnover of the transporter (16 oocytes). In view of the rapid response, we hypothesized that the initial component of the sugar-coupled water flow is due to water cotransport.

What are the properties of sugar-coupled water flow?

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Our studies have revealed the following characteristics of the initial rate of sugar coupled-water transport. (1) There is a stoichiometric relationship between Na+– glucose cotransport and the rate of water transport. The coupling is independent of the level of SGLT1 expression and the rate of Na+–glucose cotransport. In these studies Na+–glucose cotransport was varied by changing the membrane potential, the external Na+ and sugar concentrations, and temperature (Loo et al. 1996; Meinild et al. 1998). (2) The activation energy for Na+–glucose cotransport is identical (25–30 kcal mol_1) to that for coupled-water transport (Loo et al. 1996; Meinild et al. 1998). (3) Coupled-water transport is independent of the osmotic gradient and even occurs against an osmotic gradient (Loo et al. 1996; Meinild et al. 1998).

Figure 1. Sugar-coupled water flow can be studied in very fast solution changes in the water by SGLT1 expressed in oocytes Water transport was measured optically and Na+–glucose cotransport electrically. At the time indicated, 10 mM sugar (a-methyl-D-glucopyranoside) replaced 10 mM mannitol in the saline superfusing the oocyte (mM: 90 NaCl, 20 mannitol, 2 KCl, 1 MgCl2, 1 CaCl2, 10 Hepes adjusted to pH 7.4). A, time course of the holding current. Membrane voltage was held at _50 mV. B, the noisy record is the oocyte volume and the smooth line is the Na+–glucose uptake reported as the charge influx (integral of the Na+–glucose inward current). The charge uptake superimposes on the volume uptake, assuming that 249 water molecules accompany two Na+ ions and one glucose molecule. C, the relationship between Na+ and water uptakes by an oocyte treated with 200 nM gramicidin. When the membrane potential was stepped from _45 to _100 mV by voltage clamping, a Na+ inward current of ~1900 nA was recorded. Note that in this case there was no correlation between the integrated current and volume change of the oocyte. Modified from Meinild et al. (1998).

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the amount of water transport per turnover of the cotransporter varied from 264 for human SGLT1 to 424 for rabbit SGLT1 (Zeuthen et al. 2001), and in another series the water coupling ranged from 50 for a plant H+–amino acid cotransporter (AAP5) to 200 for the rat thyroid Na+–iodide cotransporter (NIS) (Loo et al. 1996).

Osmosis and Na+–glucose cotransport The studies outlined above clearly demonstrate that there is a close link between Na+–glucose cotransport and the initial rate of water transport. Furthermore, the stoichiometry between Na+, glucose and water transport immediately after activation of hSGLT1 indicates that the transporter fluid is hypertonic to the saline bathing the oocyte, i.e. ~100 water molecules per transported solute through hSGLT1 compared with 258 water molecules per solute in the bathing solution. This means that the intracellular fluid will become hypertonic as transport proceeds, and this is expected to draw more water into the cell by osmosis. Figure 2 shows an experiment where

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Na+–glucose cotransport was maintained for over 5 min. When transport was switched on by the addition of sugar the volume of the cell increased within 2 s at an initial rate of 13 pl s_1 (D1, Fig. 2), equivalent to a coupling ratio of 269 ± 12 (n = 9) water molecule per turnover of the protein. As cotransport continued there was a slow increase in the rate of volume increase until a steady state is reached some 3–5 min after Na+–glucose cotransport was initiated. The steady-state rate was on average 3 times the initial rate and this remained constant for at least another 10 min. Note that in these experiments the sugar concentration was reduced from 10 to 1 mM and the lower transport rate, ~600 nA, declined by less than 15 % over the duration of the experiment. As expected, in the steady state, fluid transport was isotonic (775 water molecules per turnover). However, if cotransport is rapidly switched off, by removing sugar, or by depolarizing the membrane potential, there is an immediate (within 1 s) decrease in the rate of volume increase of the oocyte (D2, Fig. 2). In five hSGLT1 oocytes the initial increase in water transport with turning cotransport on by the addition of 1 mM sugar was 15 ± 2 pl s_1 (inward current 550 ± 74 nA), and after reaching a steady state (41 ± 6 pl s_1) the initial reduction of water transport on removing sugar was 14.5 ± 1 pl s_1, i.e. the initial rates of volume change of the oocyte were identical on switching the cotransport on and off by the addition and removal of sugar (Zeuthen et al. 2001). We estimate that in these five oocytes cotransport accounts for ~35 % of water transport in the steady state and that osmosis accounts for the remainder (~35 % by osmosis through SGLT1 and ~30 % by osmosis through the plasma membrane).

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Phlorizin cannot be used to specifically inhibit the water cotransport component of the steady-state water flow as it also blocks osmosis through SGLT1 (see above). In this series of experiments phlorizin initially reduced the steady-state flow by 70 %, i.e. twice as much as removing glucose. Figure 2. The two components of water transport by SGLT1 At the arrow labelled ‘ON’, sugar was abruptly added to the external solution. The data curve represented by Jvtotal is the time course of the oocyte volume. There was an initial linear component Jvini (continuous line, with slope 1.75 w 10_3 ± 3.85 w 10_19 cm s_1). Subtracting this component from the total Jvtotal yields the osmotic water flow Jvosm. In the steady state, the slope was 4.41 w 10_3 ± 2.29 w 10_5 cm s_1. The osmotic water flow occurred after a 40 s delay (the slope in the steady state was 2.66 w 10_3 ± 2.29 w 10_5 cm s_1). The delay seen is similar to that observed for osmotic water flow caused by Na+ fluxes through ion channels such as Connexin 50 and ionophores gramicidin and nystatin. When the Na+–glucose cotransporter was turned off with a step jump of the membrane voltage to 0 mV (see Fig. 3B), there was an immediate reduction in the rate of water transport to that observed for osmotic water flow. D1 and D2 are the changes in slope of the fluid transport vs. time curve (i.e. the differences in the rates of fluid transport), which would be predicted if Na+–glucose cotransport was turned on and off. Modified from Zeuthen et al. (2001).

Differentiation between water cotransport and osmosis We attempted to dissociate the glucose-stimulated water transport from osmosis by measuring the initial rate of water transport before Na+–glucose cotransport produces any significant change in intracellular osmolarity. The simplest way to do so is to change the rate of Na+–glucose cotransport by stepping the membrane potential within a few milliseconds using the two-electrode voltage clamp technique. Figure 3A illustrates one experiment with rbSGLT1 where the oocyte was superfused with saline containing 5 mM aMDG and the membrane potential was initially held at 0 mV. After stepping the membrane potential to _100 mV the transporter current increased immediately from 280 to 950 nA and the water transport rate increased within seconds from 9 to 40 pl s_1. Figure 3B illustrates another experiment where the membrane potential was initially held at _80 mV and then depolarized

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to 0 mV. The cotransporter current immediately decreased from 1100 to ~350 nA and the water flow decreased from 48 to 13 pl s_1 within 1.1 ± 0.7 s (n = 18). In these experiments the number of water molecules transported per turnover of the cotransporter was identical to that obtained by adding or withdrawing sugar from the perfusate (Zeuthen et al. 2001).

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access model accounts for all the known characteristics of SGLT1. Na+-coupled sugar transport is envisaged as a series of conformational changes induced by the binding of ligands (see Parent et al. 1992b; Loo et al. 1998; Meinild et al. 2002).

Our interpretation of these results is that there is cotransport of water through SGLT1, and that osmosis only becomes a contributing factor after sufficient time has lapsed to build up an intracellular osmotic gradient. Additional evidence in support of this hypothesis is as follows. (1) The rate of water cotransport is independent of the osmotic gradient across the membrane, and can even occur uphill against an osmotic gradient (Loo et al. 1996; Meinild et al. 1998). (2) No immediate water flow is observed on activation of inward Na+ currents through ion channels expressed in oocytes, e.g. Connexin 50 (Wright & Loo, 2000; Eskandari et al. 2002) or ionophores, e.g. gramidicin and nystatin (Zeuthen et al. 1997, 2001; Meinild et al. 1998). Even when the rates of sodium transport into the oocyte were comparable to the rates of Na+ transport through SGLT1, there was no increase in cell volume for 30–40 s (see Fig. 1C and Wright & Loo, 2000).

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(3) The stoichiometry for SGLT1 is constant when the rate of transport varies by over an order of magnitude but varies considerably from cotransporter to cotransporter. The coupling coefficients range from 50 for the plant H+–amino acid cotransporter, to 176 for the renal Na+–dicarboxylate cotransporter, to 210 for the human Na+–glucose cotransporter, and to as high as 424 for the rabbit Na+–glucose cotransporter. This is not expected if the coupling between water transport and solute cotransport is purely osmotic. (4) Finally, urea is transported by SGLT1 and other cotransporters such as the low affinity Na+–glucose, the Na+–iodide and the Na+–Cl_–GABA cotransporters (Leung et al. 2000). Although urea is transported through the cotransporter water channel (see above), the rate of urea transport increases when the cotransporter is activated by the addition of substrates. To a first approximation, the ratio of urea to water transport is in the same range as their concentrations in the saline bathing the oocytes. It is unlikely that the sugar-activated urea transport through SGLT1 is due to solvent drag as the reflection coefficient for urea is close to 1 (see Zeuthen et al. 2001).

How is water transport coupled to cotransport? We propose that water cotransport is simply a consequence of how sodium and glucose transport through SGLT1 are coupled. Our kinetic model for SGLT1 incorporating water cotransport is shown in Fig. 4. This six-state alternating

Figure 3. Sugar-coupled water flow can be turned rapidly on and off with voltage absence of an osmotic gradient The figure shows that the volume change of the oocyte can be accounted for by a stoichiometric coupling between sugar transport and water flow. In A, a SGLT1-expressing oocyte was superfused with a solution containing 5 mM a-MDG with an osmotic gradient of 15 mosmol l_1, and Na+–glucose cotransport was inactivated by clamping the membrane voltage to 0 mV. Na+–glucose cotransport was turned on by stepping membrane voltage to _100 mV. There was an immediate ∆4-fold increase in sugar-coupled current and water transport, from 280 to 950 nA and 9 to 40 pl s_1. Modified from Loo et al. (1996). B, Na+–glucose–water cotransport is turned off instantaneously by membrane voltage. Initially membrane voltage was held at _80 mV and the rate of Na+–glucose cotransport was 1100 nA. The oocyte swelled at a rate of 48 ± 3 pl s_1. When membrane voltage was jumped to 0 mV, the sugar-coupled Na+ current decreased to 350 nA, and the rate of water transport was reduced to 13 ± 1 pl s_1. Modified from Zeuthen et al. (2001).

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D. D. F. Loo, E. M. Wright and T. Zeuthen

SGLT1 is an integral protein with an apparent valence of _2. At a membrane potential of _50 mV and in the absence of Na+, all of the transporters are in the C6 form. Addition of saturating Na+ to the external solution results in two Na+ ions binding to the protein and a change in formation to the C2 form (80 %). In the absence of external sugar, Na+ is transported at a low rate (turnover 5 s_1, uniport mode). In the presence of external sugar the open C2 conformation permits sugar to bind to form C3, and the transport cycle proceeds to C4, where sugar dissociates from SGLT1 at the internal membrane surface, followed by Na+ dissociation due to the low intracellular Na+ concentration. The protein then returns to the original conformation C1 to complete the transport cycle. In the presence of saturating external Na+ and sugar, the turnover rate of human SGLT1 transport cycle is ~60 s_1 at 22 °C. This ordered reaction mechanism accounts for the strict coupling between Na+ and sugar transport and the kinetics of Na+ uniport in the absence of sugar.

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According to this scheme water and urea are cotransported along with Na+ and glucose simply as a consequence of the asymmetrical conformational changes that occur during the transport cycle. When external Na+ binds and produces the conformational change that permits high affinity sugar binding (C2 to C3), water and urea ‘bind’ to the open sugar binding pocket and pass to the cytosolic surface along with Na+ and glucose translocation (C3 to C4). Sugar and Na+ then dissociate from SGLT1, and the protein returns to the closed conformation (C6), extruding water and urea to the cytoplasm. Finally, SGLT1 in the closed conformation (C6) returns to the original state (C1) without any significant water movement. The ‘binding’ of more than 200 water molecules to the sugar-binding pocket is quite reasonable given that SGLT1 can transport glucosides with a molecular volume of 1700 Å3 (20 w 12 w 75 Å, Lostao et al. 1994; Hirayama et al. 2001). The volume occupied by 200–400 water molecules (6000–12 000 Å3) only amounts to 7 % of the volume of the cotransporter (100 000 Å3).

Figure 4. A kinetic model for Na+, glucose, water and urea transport by SGLT1 (see Parent et al. 1992b; Loo et al. 1998; Meinild et al. 2002) The six-state alternating access model shows ordered binding of substrates. Two external Na+ ions bind first and this promotes a conformational change in the cotransporter that allows sugar, water and urea to ‘bind’. The fully-loaded complex (C3) then undergoes an isomerization step to expose the substrates to the internal surface of the membrane (C4) where sugar and Na+ dissociate. Na+ dissociates because of the low intracellular concentration. The dissociation of Na+ results in a relaxation of the protein conformation to a closed state (C6) resulting in the extrusion of water and urea. The catalytic cycle is completed with the isomerization of the protein from C6 to C1. Glucose is transported across the membrane as a result of the sodium gradient across the membrane, the membrane potential, which drives the C6 to C1 conformational change as well as increasing the rate of external sodium binding to the transporter, and the strict coupling between sodium and sugar transport. It is our hypothesis that the asymmetrical conformation changes in the protein during the catalytic cycle results in the cotransport of water and urea along with Na+ and glucose. Note that to accommodate 200–400 water molecules (6000–12 000 Å3) in the sugar-binding pocket the volume of the pocket has to be only 6–12 % of the protein volume (100 000 Å3).

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The driving force for coupled transport of Na+, glucose and water across the membrane through SGLT1 is simply the sum of the electrochemical potential gradients across the membrane for these solutes. Normally it is the Na+ gradients that provide the energy for uphill sugar and water cotransport.

Physiological implications of water pumps These studies cast some light on the mechanism of water transport across the brush border membrane of enterocytes, and across the epithelium. The human intestine absorbs about 1 mol of D-glucose and 8 l of water per day and given that the stoichiometry of hSGLT1 is 2 Na+ : 1 glucose : 264 water, this indicates that cotransport alone can account for ~4 l of water transport. Since the cotransported fluid is hypertonic this will result in an additional component due to osmosis, but the exact amount is difficult to estimate due to uncertainties about the magnitude of the osmotic gradient and the water permeability of the membrane. These links between sodium, glucose and water transport across the brush border membrane rationalize the use of oral salt solutions containing glucose to treat patients with secretory diarrhoea, i.e. oral rehydration therapy. How is water transported across the basolateral membrane of the enterocyte in the absence of aquaporins and a hypertonic fluid in the lateral intercellular spaces? One possibility is that basolateral membrane transport proteins act as water pumps and channels. We have demonstrated that cotransporters in general behave as water channels and water cotransporters, which supports the notion that cotransporters such as the K+–Cl_ cotransporter play an important role in water exit from the cell (see also Zeuthen, 2000, 2002). The exact role will depend on both the type and density of cotransporters present in the basolateral membrane. Finally, it is possible that other transporters such as the exchangers and ATPases also transport water as a direct consequence of conformational changes.

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Concluding remarks The Na+–glucose cotransporter behaves as a low conductance water channel, and there is a sugar-coupled water flow that couples Na+, sugar and water stoichiometrically. In the steady state, direct sugar-coupled water flow constitutes one-third of the total water transport in isotonic water flow, the remaining being osmotic water flow. The sugar-coupled water flow or water cotransport, is distinguished from osmotic water flow by the instantaneous response to step jumps in the rate of Na+–glucose cotransport. Recently, Duquette et al. (2001) repeated our experiments on hSGLT1 and many of their results agree with ours. Unfortunately, these authors did not attempt to resolve the initial rates of water flow owing to technical difficulties. Under the assumption of extremely low intracellular mobilities (Zeuthen et al. 2002), and on the basis of steady-state flows alone, they concluded that water

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flow was linked to Na+–glucose cotransport by simple osmosis. As we have noted above, we concluded that 65 % of the steady-state water flow was osmotic. More definitive experiments are needed to confirm or reject our hypothesis that water cotransport accounts for 35 % of the steady-state flow. One obvious approach is to measure the thermodynamics of Na+, sugar and water transport. However this is difficult since the osmotic gradients required to influence Na+–sugar cotransport are large. Use of the Gibbs equation predicts that osmotic gradients of 100 mosmol l_1 will shift the reversal potential by only about 5 mV, a prediction supported by preliminary experiments (Zeuthen & MacAulay, 2002). Even with these experiments the interpretation will be clouded by the same ‘unstirred layer’ questions that bedeviled earlier studies of water transport. Ultimately the question will be resolved when the 3-D structure of SGLT1 is obtained. This may not be in the too distant future given current progress in isolating sufficient quantities of recombinant SGLTs for structural studies (Turk et al. 2000; Quick & Wright, 2002).

REFERENCES CURRAN, P. F. & MCINTOSH, J. R. (1962). A model system for biological water transport. Nature 193, 347–348. DIAMOND, J. M. & BOSSERT, W. H. (1967). Standing-gradient osmotic flow. A mechanism for coupling of water and solute transport in epithelia. Journal of General Physiology 50, 2061–2083. DUQUETTE, P.-P., BISSONNETTE, P. & LAPOINTE, J.-Y. (2001). Local osmotic gradients drive the water flux associated with Na+/glucose cotransport. Proceedings of the National Academy of Sciences of the USA 98, 3796–3801. ESKANDARI, S., ZAMPIGHI, G. A., LEUNG, D. W., WRIGHT, E. M. & LOO, D. D. F. (2002). Inhibition of gap junction hemichannels by chloride channel blockers. Journal of Membrane Biology 185, 93–102. HEDIGER, M. A.,COADY, M. J., IKEDA, T. S. & WRIGHT, E. M. (1987). Expression cloning and cDNA sequencing of the Na+/glucose cotransporter. Nature 330, 379–381. HIRAYAMA, B. A., DIEZ-SAMPEDRO, A. & WRIGHT, E. M. (2001). Common mechanisms of inhibition for the Na+/glucose (hSGLT1) and Na+/Cl-/GABA (hGAT1). cotransporters. British Journal of Pharmacology 134, 484–495. HIRSCHORN, N. & GREENOUGH, W. B. (1991). Progress in oral rehydration therapy. Scientific American 264, 50–56. LEUNG, D. W., LOO, D. D. F., HIRAYAMA, B. A., ZEUTHEN, T. & WRIGHT, E. M. (2000). Urea transport by cotransporters. Journal of Physiology 528, 251–257. LOIKE, J. D., HICKMAN, S., KUANG, K., XU, M., CAO, L., VERA, J. C., SILVERSTEIN, S. C. & FISCHBARG, J. (1996). Sodium-glucose cotransporters display sodium- and phlorizin-dependent water permeability. American Journal of Physiology 271, C1774–1779. LOO, D. D. F., HIRAYAMA, B. A., MEINILD, A.-K., CHANDY, G., ZEUTHEN, T. & WRIGHT, E. M. (1999). Passive water and ion transport by cotransporters. Journal of Physiology 518, 195–202. LOO, D. D. F., ZEUTHEN, T., CHANDY, G. & WRIGHT, E. M. (1996). Cotransport of water by the Na+/glucose cotransporter. Proceedings of the National Academy of Sciences of the USA 93, 13367–13370.

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D. D. F. Loo, E. M. Wright and T. Zeuthen

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Acknowledgements This work was supported by funds from the NIH (DK19567), USDA (99-35304-7975), the Danish Research Council, the Lundbeck Foundation, and the Novo-Nordisk Foundation. We gratefully acknowledge the contributions of colleagues to these studies on SGLT1: Drs G. Chandy, B. A. Hirayama, D. Klaerke, D. Leung, A.-K. Meinild, M. Panayotova-Heiermann and G. Zampighi.