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J Physiol Volume 542, Number 3, 817-828, August 1, 2002 DOI: 10.1113/jphysiol.2002.020586
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Journal of Physiology (2002), 542.3, pp. 817-828
© Copyright 2002 The Physiological Society
DOI: 10.1113/jphysiol.2002.020586

Passive water and urea permeability of a human Na+-glutamate cotransporter expressed in Xenopus oocytes

Nanna MacAulay, Ulrik Gether, Dan A. Klærke and Thomas Zeuthen

The Panum Institute, University of Copenhagen, Blegdamsvej 3C, DK-2200 Copenhagen, Denmark

  ABSTRACT
Top
Abstract
Introduction
 Methods
Results
Discussion
References

The human Na+-glutamate transporter (EAAT1) was expressed in Xenopus laevis oocytes. The passive water permeability, Lp, was derived from volume changes of the oocyte induced by changes in the external osmolarity. Oocytes were subjected to two-electrode voltage clamp. In the presence of Na+, the EAAT1-specific (defined in Discussion) Lp increased linearly with positive clamp potentials, the Lp being around 23 % larger at +50 mV than at -50 mV. L-Glutamate increased the EAAT1-specific Lp by up to 40 %. The K0.5 for the glutamate-dependent increase was 20 ± 6 µM, which is similar to the K0.5 value for glutamate activation of transport. The specific inhibitor DL-threo-beta-benzyloxyaspartate (TBOA) reduced the EAAT1-specific Lp to 72 %. EAAT1 supported passive fluxes of [14C]urea and [14C]glycerol. The [14C]urea flux was increased in the presence of glutamate. The data suggest that the permeability depends on the conformational equilibrium of the EAAT1. At positive potentials and in the presence of Na+ and glutamate, the pore is enlarged and water and urea penetrate more readily. The Lp was larger when measured with urea or glycerol as osmolytes as compared with mannitol. Apparently, the properties of the pore are not uniform along its length. The outer section may accommodate urea and glycerol in an osmotically active form, giving rise to larger water fluxes. The physiological role of EAAT1 for water homeostasis in the central nervous system is discussed.

(Resubmitted 14 March 2002; accepted after revision10 May 2002)
Corresponding author N. MacAulay: The Panum Institute, Blegdamsvej 3C, DK-2200 Copenhagen N, Denmark. Email: nmacaulay{at}mfi.ku.dk

  INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

The human glutamate transporter, EAAT1, is found in glial cells where it reabsorbs the glutamate released into the synaptic cleft (Rothstein et al. 1994; Lehre et al. 1995). The cloned human glutamate transporters (EAAT1-5) are members of the Na+- and K+-coupled neurotransmitter transporter family (Arriza et al. 1994, 1997; Fairman et al. 1995) and are made up of 8-10 transmembrane regions (Slotboom et al. 1996; Wahle & Stoffel, 1996; Grunewald et al. 1998). The glutamate uptake is electrogenic (Brew & Attwell, 1987) and associated with the cotransport of Na+ and H+ and countertransport of K+ (Kanner & Bendahan, 1982; Nelson et al. 1983; Barbour et al. 1988; Zerangue & Kavanaugh, 1996). The glutamate transporters have anion conductance in the presence of substrate (Fairman et al. 1995; Wadiche et al. 1995a) and are involved in water transport (MacAulay et al. 2001).

In an earlier study, we showed that EAAT1 has two modes of water transport, secondary active and passive. In the active mode, there is a translocation of around 400 water molecules per charge in association with the glutamate transport; this coupling is independent of external parameters, such as clamp potential and osmotic gradients (MacAulay et al. 2001). With regard to the passive transport, we showed that the EAAT1 acts as a water channel with a well-defined permeability (MacAulay et al. 2001). Similar results have been found for other cotransporters, i.e. the Na+-coupled cotransporters of sugar, SGLT1 (Zampighi et al. 1995; Loike et al. 1996; Loo et al. 1996; Meinild et al. 1998), of dicarboxylate, NaDC1 (Meinild et al. 2000), and of GABA, GAT1 (Loo et al. 1999).

The present paper deals with the passive water permeability, Lp, of the EAAT1. We wished to confirm and extend our previous finding that the Lp of the EAAT1 expressed in Xenopus oocytes increased in the presence of glutamate (MacAulay et al. 2001). This finding would suggest that the properties of the aqueous pore change with the conformational state of the protein. The aim of the present study was twofold: first, we wished to establish the link between conformational states and the Lp and second, to study the permeation properties of the water channel in more detail. To achieve this, the Lp was measured at different conformational states obtained by variation of external parameters, such as clamp potential, Na+, and glutamate concentrations. The physical- chemical properties of the aqueous pathway were studied using osmolytes of various molecular dimensions and properties and by studying the permeability properties of the pore for small hydrophilic molecules. Our main conclusions are that the permeability is indeed a function of the conformational states occupied by the protein and that the properties of the pore are not uniform along its length.

  METHODS
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Abstract
 Introduction
 Methods
Results
Discussion
 References

The human glutamate transporter (EAAT1) was subcloned into a vector optimized for oocyte expression (pNB1), in vitro transcribed and expressed in Xenopus laevis oocytes (50 ng RNA per oocyte) as previously described (Hediger et al. 1989; MacAulay et al. 2001). Xenopus oocytes were collected under anaesthesia from frogs that were humanely killed by decapitation after the final collection. The surgical procedures complied with Danish legislation and were approved by the controlling body under the Ministry of Justice. Oocytes were incubated in Kulori medium (mmol l-1: 90 NaCl, 1 KCl, 1 CaCl2, 1 MgCl2, 5 Hepes, pH 7.4) at 19°C for 3-7 days before experiments were performed. The experimental chamber was perfused by control solution (mmol l-1: 90 NaCl, 2 KCl, 1 CaCl2, 1 MgCl2, 10 Hepes, pH 7.4, 193 mosmol l-1). Hypertonic test solutions were obtained by adding 20 mosmol l-1 of mannitol, urea, glycerol, acetamide, or formamide to the control solution. Osmolarities of the test solutions (213 mosmol l-1) were determined with an accuracy of 1 mosmol l-1 by a cryoscopic osmometer from Gonotec, Berlin, Germany. NaCl was substituted with choline chloride (ChCl) in the Na+ affinity assays. L-Glutamate was obtained from RBI (Natick, MA, USA) and used in concentrations from 10-300 µmol l-1 [14C]urea (57 mCi mmol l-1), [14C]glycerol (149 mCi mmol l-1), and [14C]mannitol (56 mCi mmol l-1) were all obtained from Amersham, Little Chalfont, UK. The alcohol was removed from the isotopes by evaporation in a stream of N2 prior to use in order to avoid effects on the membrane permeability. DL-Threo-beta-benzyloxyaspartate (TBOA), DL-2-amino-5-phosphonovaleric acid (DL-AP5), and 6-cyano-7-nitroquinoxaline (CNQX) were obtained from Tocris Cookson Inc., USA. TBOA was solubilized to form a 10 mmol l-1 stock solution in water, DL-AP5 to form a 100 mmol l-1 stock solution in 1 N NaOH, and CNQX to form a 50 mmol l-1 stock solution in DMSO. For the CNQX experiment, DMSO was added to the control solution to obtain the same concentration of the solvent.

The isotope experiments were performed in 24 well plates with 50 µmol l-1 isotope added to the above control solution. Oocytes were incubated for 30 min at room temperature, during which time the uptake was linear with incubation time (data not shown), washed three times in ice-cold, Na+-free solution (mmol l-1: 90 ChCl, 2 KCl, 1 CaCl2, 1 MgCl2, 10 Hepes, pH 7.4), and dissolved in 200 µl 10 % SDS. Two millilitres of scintillation fluid (Opti-fluor from Packard, The Netherlands) was added and the samples counted.

The experimental chamber, the voltage clamp, and the optical system for the volume measurements have been described in detail previously (Parent et al. 1992a; Zeuthen et al. 1997; Mackenzie et al. 1998). In short, oocytes were impaled by two microelectrodes, one providing the clamp current and the other measuring the membrane potential. The microelectrodes were filled with 0.5 or 1 mol l-1 KCl. The experimental chamber had a volume of 30 µl and solution changes were 90 % complete in 5 s given a flow rate of 12 µl s-1. The solutions were fed into the chamber via a mechanical valve with a dead space of 5 µl. The oocytes were observed from below via a low magnification objective and focused upon at the circumference. The oocyte volume was recorded at a rate of one point per second with an accuracy of three in 10 000. The osmotic water permeabilities, Lp, are given per true membrane surface area. This is about nine times the apparent area due to membrane foldings and amounts to 0.44 cm2 (Zampighi et al. 1995). Lps are given in units of centimetres per second per osmole per litre (cm s-1 (osmol l-1)-1) . To obtain Lp in units of centimetres per second, the values should be divided by the partial molar volume of water, Vw, of 18 cm3 mol-1.

In order to obtain a measure of the oocyte plasma membrane area, the capacitance of the oocyte plasma membrane, Cm, was measured by means of two-electrode voltage clamp. The membrane potential was jumped from a holding potential of -50 mV, to either +50 or -150 mV, or by smaller jumps, to either -25 or -75 mV for 85 ms. The upper cut-off frequency was 50 kHz. Cm was obtained from the integrated pre-steady-state current (assuming exponential changes in the current) divided by the driving voltage. In order to minimize the influence from capacitive currents in the EAAT1 itself, the oocytes were bathed in Na+-free solution (mmol l-1: 90 ChCl, 2 KCl, 1 CaCl2, 1 MgCl2, 10 Hepes, pH 7.4, 193 mosmol l-1).

Values are given by means ± S.E.M. unless otherwise stated. The n is usually the number of oocytes or the number of batches; a batch is a collection of oocytes removed from the same frog on the same day and treated the same way. The Lps as a function of external parameters were analysed by linear and non-linear regression analysis using Prism 3.0 from GraphPad Software, San Diego, CA, USA.

  RESULTS
Top
Abstract
 Introduction
 Methods
Results
Discussion
 References

Passive water permeability Lp

Expression of the EAAT1 in Xenopus laevis oocytes increased the Lp of the oocyte membrane 4-fold which demonstrates an inherent passive water permeability in the EAAT1 protein (Fig. 1 and MacAulay et al. 2001). On average, the Lp increased from 2.6 ± 0.2 for non-injected oocytes (n = 12) to 10.7 ± 0.6 times 10-6 cm s-1 (osmol l-1)-1 for EAAT1-expressing oocytes (n = 21). We used oocytes with expression levels given by clamp currents in the range 200 to 600 nA at clamp potentials of -50 mV in the presence of 200 µmol l-1 of glutamate. These expression levels were similar to those achieved in a previous study (MacAulay et al. 2001). In most of the following experiments, Lp was derived from the addition of 20 mosmol l-1 mannitol to the bathing solution. The Lp was independent of the magnitude of the osmotic challenge for values up to 200 mosmol l-1 (n = 3), Fig. 2. This is a clear indication that the water fluxes, as such, do not affect the imposed osmotic gradient, i.e. there are no unstirred layer effects in this osmotic range (Finkelstein, 1987). For osmotic challenges of 400 mosmol l-1 a smaller Lp was measured (Fig. 2), which may be due to unstirred layers.

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Figure 1. Passive water permeability (Lp) of EAAT1-expressing oocytes

A, an example of the volume change of a non-injected oocyte during an hyperosmolar challenge of 20 mosmol l-1 of mannitol (black bar). B, an example of the response of an EAAT1-expressing oocyte to an hyperosmolar challenge of 20 mosmol l-1 of mannitol (black bar). The EAAT-specific Lp was determined as the difference between the initial rates of shrinkage observed between native (non-injected) oocytes and EAAT1-expressing oocytes from the same batch, see Discussion.

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Figure 2. Lp as a function of the osmotic gradient

EAAT1-expressing oocytes were exposed to hyperosmolar challenges of 20, 50, 100, 200, or 400 mosmol l-1 of urea ( filled circle) or mannitol (circle). Data are presented as the Lp relative to the Lp obtained with 20 mosmol l-1 mannitol, an average of three oocytes. The Lps derived (slope of the lines) were independent of the magnitude of osmotic gradients up to 200 mosmol l-1, whereas the Lps obtained with 400 mosmol l-1 were significantly lower than those obtained with the smaller osmolarities.

There was a small increase in oocyte membrane capacitance Cm associated with the expression of EAAT1. In native oocytes the capacitance was 143 ± 11 nF (n = 6 oocytes from three batches), while oocytes expressing EAAT1 had values of 208 ± 22 nF (n = 6 oocytes from three batches), 1.45 times larger (0.02 < P < 0.05). The values are similar to (Valentin et al. 2000) or about half (Hirsch et al. 1996) the values observed for SGLT1-expressing oocytes. Cm was the same whether it was obtained from positive or negative voltage jumps. The volumes of the oocytes were not affected by the expression of the EAAT1, for the native oocytes it was 1.1 ± 0.04 mm3 (n = 5), for the EAAT1-expressing oocytes 0.97 ± 0.04 mm3 (n = 5).

The Lp of native oocytes was determined for each batch of oocytes. The S.E.M. for each individual batch was usually within 5 %. The Lp of the native oocyte membrane was independent of external parameters such as clamp potential, Na+ concentration (data not shown), and by the presence of glutamate and various specific inhibitors, see below. In the following, the properties of EAAT1-expressing oocytes are presented, i.e. the data include both the effects of the EAAT1 and those of the native oocyte membrane. To obtain the EAAT1-specific effects the properties of the native oocyte membrane has to be corrected for, see Discussion.

The effects of membrane potential. The Lp of EAAT1-expressing oocytes was measured at various clamp potentials; oocytes were bathed in control solutions (90 mmol l-1 Na+) with no glutamate present (Fig. 3A). The Lp increased linearly with the clamp potential by 1.3 ± 0.1 times 10-8 cm s-1 (osmol l-1)-1 mV-1 (n = 4), values normalized to an expression level of 500 nA at -50 mV. On average, the Lp was 16 ± 2 % larger at clamp potentials of +50 mV than at -50 mV (n = 4). If Na+ was replaced by Ch+ the change in Lp as a function of clamp potential was abolished. In one representative example out of three oocytes, the Lp at -100 mV was 12.0 ± 0.4 and at +50 mV it was 12.5 ± 0.1 times 10-6 cm s-1 (osmol l-1)-1.

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Figure 3. Lp as a function of external parameters

A, Lp obtained at different clamp potentials. The Lp was a linear function of the clamp potential. For the typical oocyte shown, the regression line had the slope: 2. 1 ± 0. 2 times 10-8 cm s-1 (osmol l-1)-1 mV-1 (r = 0. 88). B, Lp as a function of the glutamate concentration (Glu). The oocyte was clamped to -30 mV (filled circle) or +30 mV (circle). Glutamate was present in both control and test solutions. In both cases, Lp was an increasing and saturable function of the glutamate concentration. The data were fitted to a Michaelis-Menten equation, see text. C, the effect of glutamate on Lp was abolished if Ch+ completely replaced Na+ in the bathing solution; oocytes were clamped to -30 mV. D, there was no effect of the Na+ concentration on the Lp. Different Na+ concentrations were obtained by Ch+ replacement. The membrane potential of the oocytes was clamped to -40 mV and no glutamate was present. Data in each panel are from one oocyte representing at least three oocytes.

The effects of glutamate. The Lp increased 1.26 ± 0.10-fold (n = 4) in the presence of saturating amounts of glutamate (100-300 µmol l-1) both with the oocytes clamped to -30 and +30 mV (this study) and in unclamped oocytes (MacAulay et al. 2001). Glutamate was present in both the control solution as well as in the hyperosmolar test solution. The presence of glutamate itself induces a relatively slow swelling in the oocyte due to the cotransport of water; this appears in the recording as a slow drift in the baseline but does not affect the measurement of the passive water permeability (MacAulay et al. 2001). The osmotically induced shrinkage induced by the osmotic challenge of 20 mosmol l-1 of mannitol was 4.3 ± 0.9 (n = 4) times larger than the glutamate-induced background swelling (at -30 mV, 300 µmol l-1 glutamate).

The Lp of the non-injected oocytes was not significantly affected by the addition of glutamate. Triple determinations in three oocytes clamped to -30 mV in the presence (+) or absence (-) of 100 µmol l-1 glutamate gave (+)/(-) 2.37 ± 0.13/ 2.23 ± 0.08, 3.37 ± 0.20/3.42 ± 0.10, 2.45 ± 0.21/ 2.33 ± 0.23 (10-6 cm s-1 (osmol l-1)-1) ± S.D. The membrane potential of the non-injected oocytes changed <= 2 mV at the application of glutamate. The Lp was also not affected by the glutamate-gated channel blockers DL-AP5 and CNQX (50 µmol l-1) either with or without glutamate present (data not shown).

With no clamp potential applied, the membrane potential approach the reversal potential of EAAT1 when glutamate is added (+24 ± 5 mV, n = 16) (MacAulay et al. 2001). This may mask the specific effects of glutamate, since the membrane potential itself changes the Lp, Fig. 3A. To circumvent the problem, we measured Lp in oocytes clamped to -30 mV as well as to +30 mV, Fig. 3B. The data show that the Lp is an increasing function of the concentration of glutamate, and that the Lp exhibits saturation. The results could be fitted to the Michaelis-Menten equation with a K0.5 of 22 ± 7 µmol l-1 at -30 mV (n = 4 oocytes) and 20 ± 6 µmol l-1 at +30 mV (n = 4 oocytes); these two values are not significantly different, suggesting that the K0.5 is independent of the membrane potential (Klockner et al. 1994; Otis & Kavanaugh, 2000). The K0.5 for the glutamate-induced increase in Lp was analysed by assigning the Lp in the absence of glutamate to zero and calculating the half-maximal effect of the increase in the Lp as a function of the glutamate concentration. The glutamate-dependent increase in Lp was abolished in the absence of Na+ ions. This was tested in three oocytes clamped to -30 mV, data in Fig. 3C.

The effects of Na+. Na+ in the glutamate-free bathing solution was substituted with Ch+ to obtain Na+ concentrations in the range 0 to 90 mmol l-1. With a K0.5 for Na+ on glutamate transport of around 45 mmol l-1 (Arriza et al. 1994) this range should be sufficient to see a possible effect of changes in the Na+ concentration. Oocytes were clamped to -40 mV. However, no Na+-dependence on the Lp was observed (n = 4), Fig. 3D.

The effects of osmolyte size. We observed distinct Lps of the EAAT1 depending on the size of the test osmolyte; 20 mosmol l-1 of mannitol (molecular weight, MW 182), urea (MW 60), glycerol (MW 92), acetamide (MW 63), or formamide (MW 45) were tested. The experiments were performed under unclamped conditions in the absence of glutamate. The membrane potential of the oocyte was not affected (<= 1 mV) by the addition of 20 mosmol l-1 of these osmolytes. The Lps are given relative to the Lp obtained with mannitol, Fig. 4. Mannitol served as a reference since we considered this molecule too large to enter into the putative pore of the EAAT1 as was supported by the uptake experiments shown in Fig. 5. The Lps obtained with urea and glycerol were significantly higher than that obtained with mannitol, for urea by a factor of 1.18 ± 0.02 (n = 8 oocytes), for glycerol by 1.12 ± 0.04 (n = 8). The Lps obtained with formamide and acetamide were significantly lower than that obtained with mannitol, by a factor of 0.70 ± 0.04 in the case of acetamide (n = 7) and by 0.44 ± 0.06 in the case of formamide (n = 7).

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Figure 4. Lp with different osmolytes

The Lps were obtained with 20 mosmol l-1 mannitol, urea, glycerol, acetamide, or formamide as osmolyte. Data are presented as the average Lp obtained with the osmolyte relative to that obtained with mannitol, Lp, man. Man, mannitol; Urea, urea; Gly, glycerol; Acet, acetamide; Form, formamide (n = 7-8 oocytes). The t test represents the significance of the difference between the Lp obtained with the osmolyte and that of mannitol, *** P < 0.001; ** 0.001 < P < 0.01.

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Figure 5. Uptake of [14C]mannitol, urea or glycerol

The oocytes were incubated for 30 min with 50 µmol l-1 [14C]mannitol, urea, or glycerol in the absence of glutamate. The data are from one representative batch (out of four, comprising five oocytes for each experimental condition). The open bars represent the uptake into native oocytes, and the black bars represent the uptake into EAAT1-expressing oocytes. The t test represents the significance of the isotope uptake into the EAAT1-expressing oocyte as compared with the native oocytes. *** P < 0.001.

Permeability for urea and glycerol

The increased Lp obtained with urea and glycerol as osmolytes indicated that these osmolytes gained access to the aqueous pore of the EAAT1. This was supported by the uptake of [14C]urea and glycerol into EAAT1-expressing oocytes, no glutamate present. A representative example from one batch is shown in Fig. 5. The uptake of urea was 20.7 ± 1.9 pmol [14C]urea oocyte-1 (30 min)-1 (n = 5) and that of glycerol was 10.2 ± 0.7 pmol [14C]glycerol oocyte-1 (30 min)-1 (n = 5). Both values were significantly higher than that of mannitol, 1.6 ± 0.2 pmol [14C]mannitol oocyte-1 (30 min)-1 (n = 5). The isotope uptake by the native oocytes was obtained in each batch (see Fig. 5). If these values are subtracted from the values obtained with the EAAT1-expressing oocytes a measure of the EAAT1-specific uptake is obtained, see Discussion. The average EAAT1-specific uptake of experiments from four batches of oocytes gave 16.1 ± 2.7 pmol [14C]urea oocyte-1 (30 min)-1, 7.6 ± 2.5 pmol [14C]glycerol oocyte-1 (30 min)-1, and 0.4 ± 0.3 pmol [14C]mannitol oocyte-1 (30 min)-1.

Temperature-dependence. The uptake of [14C]urea was compared at 7 and 22 °C in unclamped oocytes. The Q10 value in the range 10 to 20 °C (assuming a linear relationship between urea flux and temperature, as seen for the SGLT1(Leung et al. 2000)) was 1.32 ± 0.09 in the absence of glutamate and 1.45 ± 0.02 in the presence of glutamate. The low Q10 suggests that urea is present in the aqueous pore in an unbound form and that urea transport takes place by simple diffusion both in the absence and presence of glutamate. It should be noted that the presence of glutamate depolarizes these (unclamped) oocytes to around +25 mV, which makes the data comparable to those performed under voltage clamp (+30 mV, Fig. 6).

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Figure 6. Uptake of [14C]urea under voltage clamp

The oocytes were voltage clamped to -50 or +30 mV and exposed for 10 min to [14C]urea in the presence (+) or absence (-) of 100 µmol l-1 glutamate. The data are from one representative batch (out of three, comprising four oocytes for each experimental condition). The black bars represent the uptake into the EAAT1-expressing oocytes and the open bar represents the uptake into non-injected oocytes from the same batch. The t test represents the significance of the increase of the [14C]urea uptake induced by glutamate at a given clamp potential. *** P < 0.001.

Urea uptake under voltage clamp. The presence of glutamate leads to an activation of the EAAT1-mediated cotransport and to an enhanced uptake of urea. The increased uptake could result from two effects: (i) an enlargement of the aqueous pore diameter induced by the binding of glutamate and the associated depolarization of the membrane potential (Fig. 3A and B) or (ii) from the glutamate-induced secondary active water transport of EAAT1 (MacAulay et al. 2001). The latter mechanism was suggested for the Na+-activated glucose transporter SGLT1 (Leung et al. 2000). To test between these possibilities, the uptake of [14C]urea was measured in EAAT1-expressing oocytes clamped to -50 or to +30 mV, with and without glutamate present. At the negative potential, the cotransport of glutamate is active. At the positive potential, which is approaching the equilibrium potential for the EAAT1 (Wadiche et al. 1995a; Mitrovic et al. 1998), cotransport is reduced or abolished.

In the absence of glutamate, the [14C]urea uptake in a representative batch was 10.7 ± 0.4 pmol oocyte-1 (10 min)-1 (n = 4) at clamp -50 mV, slightly smaller (although only significant in one out of three experiments) than that obtained at clamp potentials of +30 mV, 13.4 ± 0.8 pmol oocyte-1 (10 min)-1 (n = 4), Fig. 6. In the presence of 100 µmol l-1 of glutamate in the bathing solution, the uptake of [14C]urea was increased to 40.2 ± 2.4 pmol oocyte-1 (10 min)-1 (n = 4) at clamp potentials of -50 mV. This value was similar to that obtained at clamp potentials of +30 mV, 45.1 ± 3.7 pmol oocyte-1 (10 min)-1 (n = 4). For analysis of significance, see legend of Fig. 6. The same (significant) glutamate-induced increase in urea uptake was found in all three batches of oocytes. The average of three different batches (with the contribution from the native oocytes membrane subtracted, see Discussion) gave (in pmol [14C]urea oocyte-1 (10 min)-1): 11.9 ± 4.8 at -50 mV, 13.7 ± 4.7 at +30 mV, 34.6 ± 3.4 at -50 mV, + glutamate, and 39.9 ± 1.8 at +30 mV, + glutamate. The [14C]urea uptake of the non-injected oocytes were not affected by the addition of 100 µmol l-1 glutamate (-glutamate/+glutamate): 6.2 ± 0.3/ 5.8 ± 0.4 pmol oocyte-1 (30 min)-1 for [14C]urea (data from four batches, five oocytes from each). Please note that these experiments were run for 30 min as in Fig. 5.

The effects of the specific inhibitor TBOA

Under unclamped conditions and with no glutamate present, the competitive, non-transported inhibitor of EAAT1, TBOA (Shimamoto et al. 1998) reduced the Lp of the EAAT1-expressing oocyte to 82 ± 3 % of control values (n = 3). TBOA was present in the bathing solutions at a concentration of 200 µmol l-1, Fig. 7A. The Lp of the non-injected oocytes was not significantly affected by the addition of TBOA. Triple determinations for two oocytes with (+) or without (-) 200 µmol l-1 TBOA gave (+)/(-) 2.06 ± 0.13/2.31 ± 0.18 and 2.41 ± 0.05/ 2.82 ± 0.39 (10-6 cm s-1 (osmol l-1)-1) ± S.D. The membrane potential of the non-injected oocytes changed <= 2 mV by the application of TBOA. It follows that TBOA reduces the EAAT1-specific Lp by up to 72 ± 4 % (n = 3), see Discussion.

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Figure 7. [14C]Urea uptake as a function of TBOA

A, the Lp of the EAAT1-expressing oocytes was assessed in the absence of glutamate by the application of 20 mosmol l-1 mannitol. The data are presented as the Lp obtained with 200 µmol l-1 TBOA relative to that obtained under control conditions (n = 3 oocytes). B, oocytes were incubated in test solution for 30 min with 50 µmol l-1 [14C] urea in the absence (-) or presence (+) of 200 µmol l-1 TBOA, glutamate absent. The data are the average of five oocytes from a representative batch; in total three batches were tested. The open bars represent the uptake into non-injected oocytes, and the black bars represent the uptake into EAAT1-expressing oocytes. The t tests represent the significance of the change in the Lp and [14C]urea uptake in EAAT1-expressing oocytes induced by TBOA. *** P < 0.001.

Under the same conditions of no clamp and no glutamate, the [14C]urea uptake in a representative batch of EAAT1-expressing oocytes was significantly reduced from 28.2 ± 1.3 pmol [14C]urea oocyte-1 (30 min)-1 to 14.1 ± 1.2 pmol [14C]urea oocyte-1 (30 min)-1 in the presence of TBOA (n = 5), Fig. 7B. The average from three batches showed a reduction of the uptake to 57 ± 7 % of control. The uptake in native oocytes was not affected by TBOA: three independent batches with five oocytes in each gave (+TBOA/-TBOA): 5.7 ± 0.6/ 5.9 ± 0.1, 7.5 ± 1.8/ 6.2 ± 0.6, and 7.1 ± 1.0/ 5.6 ± 0.4 pmol [14C]urea oocyte-1 (30 min)-1. The membrane potential of the EAAT1-expressing oocytes changed <= 3 mV by the application of the inhibitor.

  DISCUSSION
Top
Abstract
 Introduction
 Methods
Results
Discussion
References

Hydrophilic pores or cavities have been found in a variety of membrane-spanning proteins. This applies to cotransporters (Golovanevsky & Kanner, 1999; Slotboom et al. 1999, 2001a), channels (Chang et al. 1998; Doyle et al. 1998), and receptors (Nayeem et al. 1994; Sankararamakrishnan & Sansom, 1995; Miyazawa et al. 1999). Indeed, significant passive water permeabilities have been demonstrated in the sodium-glucose cotransporter, SGLT1 (Loike et al. 1996; Loo et al. 1996; Meinild et al. 1998), the GABA cotransporter, GAT1 (Loo et al. 1999), the dicarboxylate transporter, NaDC1 (Meinild et al. 2000), the glutamate transporter, EAAT1 (MacAulay et al. 2001), as well as in the electroneutral cotransporters for KCl (Zeuthen, 1991, 1994) and H+-lactate, MCT-1 (Zeuthen et al. 1996). Passive water permeability has also been found in the uniports for glucose, GLUT (Fischbarg et al. 1990) and for urea, UT3 (Yang & Verkman, 1998). This raises two questions: what are the mechanisms behind the water permeability of these proteins, not usually associated with water transport, and does the phenomenon have a physiological role as compared with that of the model water channels, the aquaporins (AQPs)? For a review of AQPs, see (Borgnia et al. 1999).

Quantitative evaluation of the data

Our data on the effects on the Lp by changes in membrane potential, Na+, glutamate, and TBOA concentrations (Fig. 3 and Fig. 7) and those on urea and glycerol permeation (Figs 4-7) are presented for the EAAT1-expressing oocyte, i.e. they include the properties of both the expressed EAAT1 and the native oocyte membrane. It seems fair, however, to conclude that the effects are specific to the EAAT1 since no such effects were found in the native oocyte. A quantitative evaluation of the EAAT1-specific values of Lp and Pu (urea permeability) will rest on a number of assumptions. The expression of EAAT1 is associated with an increase in the capacitance of the oocyte membrane, Cm, by a factor of about 1.45. This could result from three mechanisms. (i) Capacitive currents in the inactivated cotransporter, ad modum the SGLT1 where the transition from C1 to C6 contribute to the capacitive current, also under Na+-free conditions (Loo et al. 1993). This mechanism is supported by the finding that the Lp of SGLT1- and GAT-expressing oocytes poisoned by phlorizin and SKF89976A resembles that of native oocytes (Meinild et al. 1998; Loo et al. 1999). (ii) An increase in the oocyte membrane area due to fusion of the membrane vesicles carrying the EAAT1 to be inserted. This mechanism is under debate; an increase in Cm was observed for the rSGLT1 (Hirsch et al. 1996), while Valentin et al. (2000) report no change associated with expression of human SGLT1. A positive effect would imply that the increase in Lp and Pu associated with EAAT1 expression would partly arise from the increased plasma membrane area. (iii) We cannot rule out that heterologous expression of EAAT1 may lead to upregulation of endogenous membrane proteins (Tzounopoulos et al. 1995; Sha et al. 2001) and that these proteins could affect our results. However, we find this unlikely as no apparent effect was observed with SGLT1- and GAT-expressing oocytes, see above (Meinild et al. 1998; Loo et al. 1999).

The quantitative evaluation of the EAAT1-specific effects will depend on which background values are subtracted. If we, as a minimum, subtract those of the native oocyte (non-injected) membrane, the effect of clamp potential on the EAAT1-specific Lp will calculate as a 23 ± 3 % increase going from -50 to +50 mV, instead of 16 ± 2 %. Likewise, the EAAT1-specific glutamate-induced increase in Lp would be (1.40 ± 0.10)-fold instead of (1.26 ± 0.10)-fold. The EAAT1-specific Lp measured with urea as test osmolyte would be 1.25 ± 0.04 times that measured by mannitol; the factor would be 1.15 ± 0.05 in case of glycerol as test osmolyte. If it was assumed that the plasma membrane surface area increased with expression, a larger background would have to be subtracted and the estimated effects would be larger. However, the 4-fold increase in Lp seen on expressing EAAT1 would become only a 2.8-fold increase if an increased surface area were taken into account.

Molecular properties of the pore

The capacity of EAAT1 as a water channel is significant. A rough estimate of the unit water permeability of EAAT1, 2 times 10-15 cm3 s-1 (see below), gives a value somewhat smaller than that of the highly permeable aquaporin AQP1, (1.2 to 6 times 10-14 cm3 s-1) (Chandy et al. 1997; Yang & Verkman, 1997) but higher than that of the less conductive AQP0, where 2.8 times 10-16 cm3 s-1 has been suggested (Chandy et al. 1997). In an EAAT1-expressing oocyte, the number of transporters per oocyte calculates as 12 times 1010, given a clamp current of 400 nA at a clamp potential of -30 mV in the presence of glutamate and a turnover of two unit charges at a rate of 10.5 s-1 (Wadiche & Kavanaugh, 1998). With a surface area of the oocyte of 0.44 cm2 (see Methods) this is equivalent to 27 times 1010 transporters cm-2. At this expression level, the contribution of the EAAT1 to the Lp of the oocyte would be around 9 times 10-6 cm s-1 (osmol l-1)-1 or 5.4 times 10-4 cm s-1 (MacAulay et al. 2001). Accordingly, the unit water permeability of the EAAT1 is calculated as 5.4 times 10-4/ 27 times 1010 = 2 times 10-15 cm3 s-1. The unit permeability for EAAT1 is of the same order of magnitude as that determined for the SGLT1 (Zampighi et al. 1995; Loo et al. 1999). The unit urea permeability (Pu) of the EAAT1 can be estimated by a similar calculation. With the data from Fig. 6, it becomes 1.7 times 10-17 cm3 s-1, two orders of magnitude smaller than the unit water permeability. Pu for the EAAT1 is about twice that calculated for SGLT1 (Leung et al. 2000). It should be stressed that the calculations above are crude and rest on a number of assumptions, particularly in regard to turnover rates, expression levels, and surface areas. In the calculation, the permeability of the native oocyte has been subtracted. If it is assumed that the background Lp and Pu increased by, say, a factor of 1.45 above that of the native oocyte (see discussion above), our estimates of the unit Lp and Pu of the EAAT1 would be about 25 % smaller.

The Lp of the EAAT1 obtained with the smaller osmolytes, urea and glycerol, was larger than that recorded by the larger osmolyte, mannitol (Fig. 4). The simplest interpretation is that the part of the aqueous pore facing the outside solution can accommodate the smaller osmolytes in an osmotically active form, but that the larger osmolyte will be excluded, Fig. 8A. This effect could result from two mechanisms: simple steric exclusion due to the physical dimensions of the mouth region of the pore, or exclusion of the larger osmolyte from the layer of surface water at the protein by physical-chemical forces (Parsegian, 2001). Urea and glycerol are small, hydrophilic, and polar molecules that have access to these inner layers of water (Collins & Washabaugh, 1985; Courtenay et al. 2000) while mannitol has not. In either case, the resistance of the aqueous pathway across which the osmotic force acts would be smaller in case of urea and glycerol than in case of mannitol. Consequently the resulting Lp would be larger when measured with the smaller osmolytes.

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Figure 8. Molecular determinants of the Lp of EAAT1

A, molecular properties of the pore. The data suggest that the outer end of the pore can accommodate urea and glycerol in an osmotic active form, while excluding larger osmolytes such as mannitol. During transport, the profile of water chemical potential (Cw) will differ in the two cases. In case of urea (continuous line), the transmembrane difference in Cw will be present across a shorter portion of the pore. In case of mannitol (dashed line), it will be present across the entire length of the pore. Accordingly, a larger Lp will be measured with urea as osmolyte. As discussed, the exclusion of mannitol could result from steric hindrance or from the properties of the water-protein interphase. B, simplified reaction scheme for EAAT1. The glutamate molecule (filled square) and the Na+ ions (circle with positive charge) are shown to be bound from the outside and transported to the inside of the membrane. The filled up triangle is the inhibitor TBOA. State C3, with glutamate and Na+ bound, exhibited an increased Lp, which is symbolized by a large pore size. State C* with bound TBOA stops the protein in its reaction cycle and is therefore set outside the cycle. The Lp of this state was reduced, indicated by the small pore size. The pathways for K+, H+, and Cl- ions have been left out for simplicity. See also Discussion.

Access of urea and glycerol into the pore was confirmed by the observation that [14C] urea and, to a lesser extent, [14C]glycerol both crossed the membrane of the EAAT1-expressing oocyte, as opposed to [14C]mannitol (Fig. 5). The low Q10 supports the notion that the transport of the ions takes place by diffusion in an aqueous milieu. It should be emphasized that there is no discrepancy between the ability of urea and glycerol on the one hand to give larger Lps and on the other hand to permeate the entire channel. As long as the permeabilities of urea and glycerol are below certain limits, irreversible thermodynamics of serial elements confirm that the Lp obtained with more permeable osmolytes can be larger than that obtained with less permeable osmolytes (Kedem & Katchalsky, 1964, their eqn (35)). Small Lp values were obtained with the smallest osmolytes acetamide and formamide (Fig. 4). Acetamide and formamide must be expected to have high permeability values and therefore small reflection coefficients. Consequently, only small osmotic driving forces and Lp s were obtained with these osmolytes (Kedem & Katchalsky, 1964).

We have presently no definite knowledge of the location of the aqueous pore through the protein. A proteinaceous pathway for water would follow from the suggestion of Slotboom and coworkers (Slotboom et al. 2001b) who have drawn attention to the topological resemblance between EAAT1, AQPs, and channels: they all have re-entrant loops. Permeability of anions as wide as 5 Å in the EAAT1 (similar to the diameter of glycerol and urea) has been demonstrated (Wadiche & Kavanaugh, 1998), suggesting a mutual pathway through the protein. However, we cannot rule out the possibility that water and urea permeate through the central pore of a pentameric glutamate transporter complex, a structure found for EAAT3 (Eskandari et al. 2000).

A recent study of the SGLT1 reported an uptake of [14C]urea in the absence of sugar, and an increase in uptake induced by sugar. The latter was suggested to take place by a mechanism strictly coupled to the cotransport (Leung et al. 2000). As outlined above, this seems to conflict with the finding for EAAT1, where the uptake of [14C]urea, both in the absence and presence of glutamate, took place mainly by diffusion, as shown by the low Q10 value and the independence of the polarity of the clamp potential and hence the level of transport activity. The difference may, at least in part, result from the fact that the EAAT1 increases its Lp in the presence of the organic substrate, and thereby also the permeability for urea. Such an increase was not observed for the SGLT1 (Meinild et al. 1998). Considering the much larger urea fluxes observed for the EAAT1-expressing oocytes compared with the SGLT1-expressing oocytes (about a factor of ten) we cannot exclude the possibility that the increase in permeability in the EAAT1 masked a smaller cotransport-mediated component of [14C]urea transport.

Passive water permeability and conformational states

We found that in the presence of saturating amounts of glutamate, the EAAT1-specific Lp increased by a factor of about 1.4. The increase required the presence of Na+, but was independent of the rate of the cotransport (low at clamp potential +30 mV and higher at clamp potential -30 mV), Fig. 3C and B. It is debateable which step(s) in the glutamate transport cycle is voltage dependent and which is rate limiting (Klockner et al. 1994; Kanai et al. 1995; Otis & Jahr, 1998; Wadiche & Kavanaugh, 1998; Auger & Attwell, 2000). Under our experimental conditions and with an assumption of the glutamate translocation (C3 right C4 in Fig. 8B) being the rate-limiting step, it follows that the glutamate-bound state (C3) has a larger water permeability than the glutamate-free states (C1-C2). Glutamate increased the Lp with K0.5 of 20 µmol l-1. The value for K0.5 agrees with studies of glutamate activation in EAAT1 which gave K0.5s in the range 15-21 µmol l-1 (Arriza et al. 1994; Conradt & Stoffel, 1995; Vandenberg et al. 1998). This indicates that it is the conformational changes directly associated with glutamate binding that are responsible for the change in the Lp.

In the absence of glutamate there was a Na+-dependent linear relation between the Lp and the clamp potential with the largest Lps observed at positive potentials, Fig. 3A. This finding suggests a change in conformation following shifts in the membrane potential. This agrees with the observation of Na+-dependent transient currents elicited by jumps in the clamp potential, which represents Na+ binding and associated conformational changes of the glutamate transporter (Wadiche et al. 1995a,b). Accordingly, in the absence of Na+, the effect of clamp potential on the Lp was abolished. It is possible that the occupancy of the inwardly facing conformation (C6) increases with positive clamp potentials at the cost of the outward facing C1 and C2, as has been suggested for the SGLT1 (Parent et al. 1992b). It would follow that the conformation C6 has a larger Lp than the outward facing conformations C1 and C2. The independence of the Lp on Na+ concentrations, Fig. 3D, would suggest that the states C1 and C2 have the same Lps. However, a detailed analysis of the Lp as a function of clamp potential must await further studies of conformational occupancy of EAAT1 at different membrane potentials.

Binding of the competitive inhibitor TBOA brings the transporter into a new state, C* in Fig. 8B, with a low Lp and Pu. The conformational states occupied by the protein subsequent to binding of TBOA or glutamate are different, as judged by the reduction of Lp and Pu in the presence of TBOA and the increase of these parameters in case of glutamate binding. We cannot rule out that the larger TBOA molecule (MW 257 vs. 147 for glutamate) simply acts as a plug in the water pore, although conformational differences upon binding of glutamate and the inhibitor kainate have been suggested in a study of a rat glutamate transporter, GLT-1 (Zarbiv et al. 1998).

Physiological relevance

A problem under neural activity is the large and rapid transport of osmotic active solvents between the intra- and extracellular compartments. Unless mechanisms of cellular water homeostasis are activated, deleterious volume changes might occur. It has been shown that glial cells swell in response to neuronal activity with an accompanying release of glutamate into the extracellular space. This swelling has been proposed to result from water uptake via AQP4 (Jung et al. 1994; Manley et al. 2000; Niermann et al. 2001), driven by the osmotic gradient built up from K+ uptake (Dietzel et al. 1980; Holthoff & Witte, 2000) and Na+-dependent glutamate uptake (Schneider et al. 1992; Izumi et al. 1999; Chen et al. 2000; Koyama et al. 2000). The AQP4-knockout mice, however, have no general motor, sensory, or co-ordination deficits (Ma et al. 1997; Manley et al. 2000), indicating that there are other aqueous pathways across the glial cell membrane. We therefore suggest that the glutamate transporter plays a role in the water homeostasis of the glial cells. On our model, water is transported into the cell along with re-absorption of glutamate (MacAulay et al. 2001), a process which would be aided by the depolarization/ glutamate-induced increase in passive water permeability of the transporter demonstrated in the present paper.

In addition, the EAAT3 isoform of the glutamate transporter, which is expressed in the small intestine and in the kidney (Kanai & Hediger, 1992), might contribute to the water absorbance of these epithelia along the same lines as the SGLT1, given that the EAAT3 transports water in a similar manner as the homologous EAAT1.

  REFERENCES
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Abstract
 Introduction
 Methods
Results
Discussion
References

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Acknowledgements

The EAAT1 clone was a kind gift from Dr Susan Amara. We are grateful for the technical assistance of S. Christoffersen, B. Lynderup, and T. Soland. This study was supported by the Lundbeck Foundation and the Danish Research Council.


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