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J. Biol. Chem., Vol. 277, Issue 34, 30991-30997, August 23, 2002

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Changes in Glucose Transport and Water Permeability Resulting from the T310I Pathogenic Mutation in Glut1 Are Consistent with Two Transport Channels per Monomer^*

Pavel Iserovich^§, Dong Wang^§¶, Li Ma, Hong Yang^¶, Felipe A. Zuniga, Juan M. Pascual^¶, Kunyan Kuang, Darryl C. De Vivo^¶, and Jorge Fischbarg^**

From the Departments of  Ophthalmology, ^** Physiology and Cellular Biophysics, and ^¶ Neurology, Columbia University, New York, New York, 10032

Received for publication, March 22, 2002, and in revised form, May 16, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION CONCLUSION REFERENCES

We studied glucose and water passage across wild type (WT) glucose transporter Glut1 and its T310I pathogenic mutant, expressing them in Xenopus laevis oocytes. We found that the T310I mutation produced a 8-fold decrease in glucose transport (zero-trans influx, 13 ± 2% compared with WT), accompanied by a 2.8-fold increase in the osmotic water permeability (P_f 280 ± 40% compared with WT), and no change in the diffusional water permeability (P_d). The dependence of glucose and water transports on the amounts of mutant cRNA injected was identical exponential buildups (k = 19.7 ng), suggesting that they depend similarly on the quaternary structure. The E_a values for P_f were 16 ± 0.4 (WT) and 11 ± 1 kcal mol¹ (T310I). We report for the first time that 10 mM D-glucose and L-glucose inhibit P_f by ~45% in the WT but not in the T310I mutant. In addition, 10 mM maltose reduces P_f (15-20%) in both cases. However, 5 mM L-glucose increased the P_f of T310I, consistent with a cooperative effect. These experimental observations and an analysis of our three-dimensional model strongly suggest the presence of two channels per Glut1 monomer, one of which can be blocked by the mutation T310I.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION CONCLUSION REFERENCES

Glucose transporters (Gluts)¹ are members of the major facilitator superfamily of membrane proteins (1) and facilitate the transport of several monosaccharides. Among them, Glut1 is probably the one most extensively studied partly because of its relative abundance within the erythrocyte cell membrane. An extensive kinetic analysis of Glut1-mediated transport in human erythrocytes is consistent with a simple alternating conformational model for facilitative transport (2), although alternate mechanistic models have been proposed previously (3, 4). In addition, it has been suggested that the transport of substrates would be accompanied by desolvation-solvation cycles that would store and then recover the associated energies as Glut1 conformation changes (5). Based upon hydropathy analysis of the primary sequence, a 12-transmembrane-spanning -helical model for the structure of the Glut1 protein was first proposed by Mueckler et al. (6). They also recognized that several helices were amphipathic and therefore capable of forming a water-accessible pore. This idea received experimental support from demonstrations of moderate water permeability through Gluts (7, 8). A later analysis led to the hypothesis that perhaps five transmembrane helices can form a channel that is able to admit hexoses but too small for disaccharides (9, 10). A water-filled transport channel is highlighted in a recent review (11) in which it is asked of MF proteins: "has nature hijacked a common structure for an aqueous pore?" An exploration of the hypothesis of a water-filled pore in Glut1 led to a series of experimental papers (12-15) in which the Mueckler group used cysteine-scanning mutagenesis to describe the possible roles of helices II, V, VII, and XI in solvent accessibility and glucose transport. This information was used in suggesting for Glut1 a scheme for helical packing (6, 13, 14) similar to that of Lac permease (16, 17). Additional models have been proposed for Gluts. Based on the similarity with an ion channel and general hints from aquaporin 1, Dwyer (18) has described a three-dimensional spatial arrangement for Glut3, and we have recently modeled a three-dimensional structure of Glut1 (19) based on the packing schemes above and have communicated its coordinates (Protein Data Bank code 1JA5 (19)).

Additional information on Glut1 structure can be deduced from Glut1 pathogenic mutations found in patients with the Glut1 deficiency syndrome discovered by Dr. D. C. De Vivo and colleagues (20-25). In this disease, a Glut1 deficiency disrupts the glucose transport through the blood-brain barrier, which in turn can cause serious clinical complications. The presence of the mutated Glut1 results in reduced cerebrospinal fluid glucose concentrations (hypoglycorrhachia) and reduced erythrocyte glucose transporter activities in the patients (20). The genetic abnormalities include hemizygosity, splice site mutations, deletions, insertions, and nonsense and missense mutations, as well as genetic transmission as the autosomal dominant trait (20-25). There are eight known missense Glut1 mutations: S66F (21), G91D (25), R126L (21), R126H (23), E146K (21), K256V (21), T310I (24), and R333W (21).

In one of the missense mutants, a polar amino acid threonine at position 310 is replaced by a non-polar one, isoleucine (24), which has also a larger side chain. The residue Thr-310 in helix 8 is conserved in Glut1 through Glut5 and also across species (26), suggesting that Thr-310 may play an important role for glucose permeation. In this paper, we concentrate on the functional and structural changes that the mutation induces.

The effects of mutations of Glut1 on its P_f have not been reported in the literature. We find that in the mutant T310I glucose transport activity is decreased, and paradoxically, P_f is increased. We also report that in the C-less mutant the P_f is essentially unchanged. In addition, we find that D-glucose competes for water passage in the wild type but not the T310I mutant. Analyzing the data and our Glut1 structure, we offer an interpretation of these effects based on the existence of two channels per monomer.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION CONCLUSION REFERENCES

Mutagenesis and cRNA Preparation from cDNA Templates-- A 1913-bp Eco47III and HindIII hGLUT1 cDNA fragment derived from pcDNA3 (Invitrogen) was subcloned into a custom plasmid (pM) containing fragments of 5'- and 3'-untranslated regions of Xenopus -globin cDNA. This newly constructed plasmid was named pM-GLUT1 and was confirmed by direct DNA sequencing with the appropriate primers. The mutation T310I was introduced by PCR. Mutagenic primers were: 5'-AGGGCGCAATTGCGGTACCCAGCTTGCTG-3' (forward 1); 5'-CCTGTGTATGCCATCATTGGCTCAGGTATCGTCAACACGGCC-3' (forward 2); 5'-GAAGGGCCGTGTTGACGATACCTGAGCCAATGATGGCATACAC-AGG-3' (reverse 1); and 5'-GCCTGCAACGGCAATGGCAGCTGGACGTGG-3' (reverse 2). PCR was conducted on the pM-GLUT1 plasmid DNA using the above primers for 35 cycles with denaturation at 95 C for 30s, annealing at 58 C for 1 min, and elongation at 72 C for 2 min. The right-sized PCR fragment was purified from the gel and cut with StuI and MunI. The gel-purified StuI and MunI fragment was then directionally ligated into the StuI and MunI sites of pM-GLUT1. The presence of the mutation T310I was confirmed by DNA sequence analysis. Capped, the runoff cRNA transcripts of both the wild type and the mutant T310I GLUT1 were synthesized from the pM-GLUT1 constructs after linearization at a unique NotI site using the mMESSAGE mMACHINE^TM kit and T7 RNA polymerase (Ambion, Austin, TX). The C-less mutant construct we used where all of the six cysteines existing in the wild type were mutated (133, 207, 347, and 429 into serines and 201 and 421 into glycines (27)) was a gift from Dr. M. Mueckler. This plasmid was linearized at the XbaI site, and the cRNA was synthesized using the SP6 RNA polymerase.

Xenopus laevis Oocytes Injection-- Oocytes were prepared as described previously (28). Oocytes were removed from the ovaries of X. laevis (NASCO, Fort Atkinson, WI) and were defolliculated and separated (29). The largest undamaged oocytes (stages V and VI) were transferred to Barth's medium ((in mM) 88 NaCl, 2.4 NaHCO₃, 1 KCl, 0.33 Ca(NO₃)₂, 0.41 CaCl₂, 0.82 MgSO₄, 10 Hepes; osmolarity: 178 mosM, pH 7.4). The oocytes were manually microinjected with 50 nl of either water or water plus cRNA (1 µg/µl) and were incubated at 18 °C for 3 days.

Preparation of Purified Oocyte Membranes and Western Blot Analysis-- Purified oocyte membranes were prepared according to the procedure of Garcia et al. (30) with minor modifications. After disruption of oocytes with a Pipetman, the oocyte ghosts were washed free of yolk as described previously (30). The ghosts were treated with 50 µl/oocyte ice-cold homogenization buffer ((in mM) 83 NaCl, 1 MgCl₂, 10 Hepes, 5 EDTA, 5 EGTA, 0.5 phenylmethylsulfonyl fluoride, 5 g/ml each of aprotinin, pepstatin, and soybean trypsin inhibitor, pH 7.8) in a glass homogenizer. The total cell homogenate was centrifuged three times at 3000 rpm for 10 min at 4 C in a microcentrifuge to pellet the yolk granules and melanosomes. The final supernatant was loaded in 4 ml of 15% sucrose and was spun at 165,000 × g for 80 min to generate the total membrane fraction. The membrane pellet was suspended in homogenate buffer at 5 µl/oocyte and stored at 80 °C.

Ten µg of purified plasma membrane samples from oocytes injected with wild type, T310I, and H₂O were fractionated in 4-20% SDS-polyacrylamide gradient gel (Bio-Rad) and transferred to a nitrocellulose membrane. Western blot analysis was performed using Glut1-specific primary antibodies and horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) as previously described (31). Glut1-specific signals were visualized and quantified by electrochemiluminescence (ECL) reactions and then transformed into digital values using a scanner and SigmaScan Pro software (SPSS Inc., Chicago, IL).

Transporter Activity Assays, Uptake of 2-deoxy-D-[³H]Glucose (Deoxyglucose, DOG)-- Groups of six oocytes were placed into 0.6 ml of Barth's solution containing 10 µCi of [³H]DOG (5 Ci/mmol, PerkinElmer Life Sciences) and 2 mM unlabeled DOG in the wells of a 24-well plate. After incubation for 10 min, the oocytes were washed four times with ice-cold Barth's solution containing 0.2 mM phloretin. Individual oocytes were then placed into vials, and 0.2 ml of 0.2 N NaOH plus 0.2% SDS was added. After the oocytes were dissolved, 5 ml of scintillation fluid was placed into each vial for counting. The results were expressed in picomoles of DOG uptake/10 min/oocyte.

Osmotic Water Permeability (P_f) Measurement-- To measure oocyte volumes as a function of time, we used the method we first outlined in 1990 (7). The oocytes were placed in a 0.3-ml glass-bottom chamber containing Barth's medium at room temperature. The temperature of the perfusing liquid could be set to a range of 5-37 C. The temperature setting was considered the working temperature given the low thermal capacity of the plastic chamber. The oocyte was loosely attached to the chamber bottom using a small amount of Cell-Tak^® glue (Collaborative Research, Bedford, MA (29)). The oocyte equatorial cross-section was viewed with a Nikon^TM inverted microscope equipped with a ×4 objective and a video camera (model NC-65, Dage-MTI, Michigan City, IN). A computer with frame grabber digitized the oocyte image and calculated the oocyte area and volume every 6 s. For P_f measurements, the oocytes were superfused with isotonic Barth's solution (178 mosM) for a period of 60 s and then with hypotonic solution (15 mosM, obtained by omitting NaCl) for another 100 s. The P_f values were calculated from the induced change in oocyte volume as shown in Equation 1

(Eq. 1)

where A is the area (assumed spherical) of the oocyte, dV/dt is the rate of volume change, C is the osmolarity gradient (all three at zero time), and V_w is the water molar volume (29).

Diffusional Water Permeability (P_d) Measurement-- We calculated P_d from the changes in oocyte volume resulting from H₂O-D₂O exchange. Initially, the oocytes were equilibrated for 10 min in Barth's solution prepared with D₂O or "heavy" water (Sigma) as a solvent (heavy water-based Barth's solution). After such incubation, the bathing solution was replaced by Barth's solution for which the solvent was H₂O (water-based Barth's solution). In response, the oocytes swelled because of the 10% faster self-diffusion coefficient for H₂O compared with D₂O. In other words, under these conditions, H₂O diffuses into the oocyte faster than D₂O can leave it. Fig. 3 shows a family of curves obtained from such experiments.

To determine oocyte membrane diffusional permeability to H₂O (P_d), we note that the extent of the cell volume change observed and the intracellular H₂O concentration are linearly related (32). Therefore, monitoring the oocyte volume transient yields information on the oocyte membrane diffusional exchange of D₂O for H₂O governed in turn by the oocyte membrane P_d. In mathematical terms, because the oocyte volume is much smaller than that of the external solution, the intracellular concentration of water (C_i) at time t during the D₂O for H₂O exchange can be taken as Equation 2 (8, 33-35)

(Eq. 2)

where C_e is the extracellular concentration.

If the initial oocyte volume and surface area are Vo and A from Equation 1, the diffusional permeability P_d is as follows in Equation 3

(Eq. 3)

We found the values of Vo and A as described above, and  was obtained by fitting the oocyte volume changes during H₂O-D₂O substitution (Fig. 3) to an exponential buildup curve (Equation 2) using Origin^TM software.

P_f, P_d Determination in the Same Oocyte-- A sizable advantage of our method is that, if desired, it allows one to obtain the values for both P_d and P_f in the same experiment. This yields an accurate determination of the P_f/P_d measurements, because both can be obtained from the same oocyte. An oocyte was incubated in heavy water-based Barth's solution for ~5 min and then was transferred to the chamber described above, which also contained heavy water-based Barth's solution. The oocyte was challenged first with isotonic water-based Barth's solution to determine the P_d and after that with hypotonic (15 mosM) water-based Barth's solution to determine the P_f. Typical curves are shown in Fig. 3.

Modeling-- For molecular modeling, we used a Silicon Graphics octane work station with InsightII software (Accelrys, Inc.) as described previously (19). To explore the cavities in Glut1, we had previously used the program HOLE (36, 37), developed to explore the pore of ion channels. For the current purpose, as the internal structure of Glut1 appeared more complex than anticipated, we decided to use a combination of several algorithms, among them the program Swiss Pdb viewer (38).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION CONCLUSION REFERENCES

It has been shown previously (22) that glucose uptake by erythrocytes is only 35% of normal in a Glut1 deficiency syndrome patient affected with the T310I mutation in one of the alleles. However, work with erythrocytes does not allow one to quantify precisely the function of the mutant. We used instead the X. laevis oocytes expression system.

Sugar Uptake by Wild Type, T310I Mutant, and C-less-- Fig. 1 shows the results of zero-trans DOG influx by oocytes expressing one of the proteins above. The unlabeled DOG concentration in the medium was 2 mM. As can be seen, the T310I mutant transports DOG at a rate of only ~13 ± 2% of that of wild type Glut1. As mentioned below, the expression of the proteins referenced was assessed with the control experiments shown in Fig. 2.

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Effect of the pathogenic T310I and the C-less Glut1 mutants on glucose and water transports. The left ordinate corresponds to zero-trans influx uptake of 2 mM unlabeled DOG and 10 µCi of ]3H]DOG. The right ordinate corresponds to the Pf. The x axis legends denote whether cRNA encoding wild type, T310I, and C-less mutant Glut1 or only water (Control) were injected into oocytes. Values represent mean ± S.E. Results for the T310I mutant were normalized using the intensity of the Glut1 protein band.

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Western blot analysis of the expression of wild type and T310I mutant Glut1 in X. laevis oocytes membranes. Groups of three oocytes were incubated for 3 days after injection of 50 ng of cRNA or water (as a control). Positive controls were purified Glut1 and red blood cell membranes. The expression rate for the T310I mutant was 85% that of wild type (WT) and was used for normalization for all T310I data. RBC, red blood cells.

We also utilized the C-less Glut1 mutant. In accordance with the data from Mueckler and Makepeace (27), the uptake of DOG by the C-less Glut1 mutant (Fig. 1) was 80 ± 9% of that by wild-type Glut1. The uptake of DOG by water-injected oocytes was <1% than in oocytes expressing wild type Glut1.

Water Permeability of Wild Type and T310I and C-less Mutants-- Fig. 1 shows the P_f values determined for oocytes expressing the proteins described. Interestingly, the P_f for the pathogenic T310I mutant was approximately three times (280 ± 40%) larger than that for the wild type (after subtraction of the background P_f for water-injected oocytes). As for the C-less, the P_f determined (Fig. 1) was 69 ± 13% of that of the wild type (also after the subtraction of the background P_f for water-injected oocytes). Clearly this mutant, even if all six of the original cysteines in wild type Glut1 have been replaced, can transport glucose and water at rates near those obtained with the wild type (Fig. 3).

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Typical curves for determinations of diffusional and osmotic water permeabilities on same given oocytes. For these experiments, oocytes expressing Glut1 were preincubated for 10 min in isotonic Barth's medium prepared with D2O. We calculated the Pd from the kinetics of the fast increase in oocyte volume after challenging oocytes with isotonic Barth's medium prepared with H2O. After steady state, Pf was determined from the oocyte volume in response to osmotic challenge (15 mosM).

Fig. 4 shows the P_f and P_d data we obtained. The P_f values we determined here are comparable with those we reported earlier (39) for the P_f of oocytes injected with GLUT1 cRNA (28 ± 2 here versus 23 ± 5 µm s¹) and water (control: 15.8 ± 1 here versus 13 ± 1 µm s¹). As for P_d values in oocytes, from what we know this is the first such report. We found that in contrast to the increase seen in P_f for the T310I mutant (Fig. 4B), the P_d for this mutant was quite similar to that of the wild type (Fig. 4B). Consequently, the P_f/P_d ratio (Fig. 4C) was approximately three times higher for the T310I mutant than for Glut1. The significance of these findings is considered under "Discussion." The P_f/P_d ratio for water-injected oocytes presumably means that endogenous transporters or channels contribute to water passage. There is in fact evidence for the presence of some endogenous membrane proteins in oocytes (28). From our data, they could have some water permeability.

View larger version (24K): [in this window] [in a new window]   Fig. 4.   Water passage across Glut1 and the T310I mutant. A and B, values of Pf and Pd obtained from oocytes expressing either wild type Glut1 or mutant T310I or just injected with water as a control. To obtain the Pf/Pd ratios for wild type and T310I mutant shown in C, we subtracted the corresponding Pf and Pd of water-injected (control) oocytes. The data in A are the same as those in Fig. 1. Values represent mean ± S.E. Results were normalized using the intensity of the Glut1 protein band.

Assessments of Expression-- We also determined the expression rate of the wild type and mutant Glut1 in oocyte membranes using Western blot analysis with Glut1-specific antibodies. We found that the expressions of wild type Glut1 and T310I mutant in X. laevis oocytes are very close (Fig. 2); the calculated expression ratio was used to normalize the P_f, P_d, and glucose uptake data obtained with oocytes injected with T310I mutant RNA. It has also been reported that the C-less expression by oocytes is comparable with that of the wild type (40).

It has been shown previously (28) that in oocytes expressing wild type Glut1, the rate of DOG uptake characteristically depends on the amount of cRNA injected. The dependence of P_f on the amount of Glut1 cRNA injected has been also demonstrated (8). In our present case, the relatively high P_f of the mutant appeared useful for us to try to determine the dependencies of glucose transport and water permeability on the amount of T310I cRNA injected. Fig. 5A shows that they change in concert as the amount of the cRNA injected increases and that their dependence can be approximated to a single exponential buildup y = y₀ + A [1  exp(x/x_c)]. In Fig. 5B, the values were normalized by subtracting y₀ and dividing by the asymptotic maximal A for each set. The exponential constant x_c was 19.7 ng. After normalization, the same Fig. 5B shows that DOG uptake and P_f data practically overlap with each other, displaying the same exponential buildup dependence.

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Dependence of DOG uptake and osmotic water permeability on the amount of T310I Glut1 mutant cRNA injected. A, the actual experimental values plus curves fitted to the data using exponential buildup functions: y = y0 + A [1exp(x/xc)]. Values represent mean ± S.E. in five oocytes. B, the values were normalized by subtracting y0 and dividing by the asymptotic maximum for each set. The exponential constant xc was 19.7 ng.

Activation Energy-- Fig. 6 shows the temperature dependence of the P_f for the T310I mutant. The data were fit to the standard expression: P_f = A exp(E/RT), where E is the activation energy, R is the gas constant, and T = 310 K. The activation energies for water transport through the wild type and the T310I mutant were 17.0 ± 2.0 and 13.0 ± 0.5 kcal mol¹, respectively.

View larger version (15K): [in this window] [in a new window]   Fig. 6.   Temperature dependence of the osmotic permeability. Background oocyte Pf values were subtracted in each case. Values represent mean ± S.E. (five oocytes).

Effects of Saccharides on Glut1 P_f-- Fig. 7, A and B, shows the effects of external saccharides on the osmotic water flow across membranes of oocytes expressing either wild type Glut1 or the T310I mutant. The compounds used were (a) the normal substrate of Gluts, D-glucose, (b) the non-transported enantiomer, L-glucose, and (c) a disaccharide that inhibits glucose transport, maltose. Fig. 7A exemplifies the original data obtained. Importantly, after taking into account the base-line P_f of water-injected oocytes, 10 mM D-glucose inhibits osmotic flow by ~45% in oocytes expressing Glut1. On the other hand, D-glucose (up to 30 mM) does not significantly affect the P_f of oocytes expressing the T310I mutant. Fig. 7B further shows that, interestingly, the inhibitory effects (40-45%) on water permeability in oocytes expressing wild type Glut1 were also observed with 10 mM (but not with 5 mM) L-glucose and maltose. Crucially, for oocytes expressing the mutant T310I, 10 mM maltose decreased P_f by 20%, 5 mM L-glucose increased P_f by 35%, and 10 mM D- and L-glucose did not significantly affect water permeation.

View larger version (24K): [in this window] [in a new window]   Fig. 7.   Effect of saccharides on the Pf of oocytes expressing either wild type or the pathogenic mutant T310I. Oocytes were preincubated for 10 min with the saccharides indicated. A, effects of D-glucose. Oocytes were injected with cRNA encoding wild type Glut1 or T310I mutant and with water only as controls. The numbers inside the bars denote the D-glucose concentration (mM) used in each case. B, permeabilities for both wild type and T310I mutant. Base-line permeabilities (for water-injected controls) were subtracted, and data were normalized with respect to the corresponding permeabilities in the absence of saccharides. Asterisks, averages significantly different from controls at a level of 0.05 (one-way ANOVA).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION CONCLUSION REFERENCES

Movements of Glucose and Water Across Wild Type and Pathogenic T310I Mutant-- Our finding of reduced glucose transport (down to 13%, Fig. 1) by the pathogenic mutant was in line with our expectations. Still, the mutation reduces but does not abolish glucose transport. Moreover, the P_f of the mutant was paradoxically increased (Fig. 1) over that of the wild type. These results lead us to consider what might be the shape of the putative water pathway(s) in Glut1; whether glucose and water may share the same pathway(s), and in which manner could these have changed because of the T310I mutation.

There is already evidence for moderate water passage through oocyte membranes in connection with Glut expression as first shown by our group (7) and quickly confirmed by Verkman's laboratory (8), and that the rate of water passage in Glut1 rises in proportion to the amount of Glut1 cRNA injected in oocytes (8). Here we have ascertained the simultaneous behavior of glucose and water passage upon the expression of T310I. As shown in Fig. 5, we determined that the rates of glucose and water passage rose together exactly in parallel with the amount of copies of T310I expected to be expressed (Fig. 5B). This reaffirms the idea that both glucose and water traverse the same transporter. Furthermore, the dependence shown in Fig. 5B suggests that both glucose and water transports depend similarly on the oligomeric structure of the functional transporter.

The P_f of the C-less and the Function of the Monomer-- DOG glucose uptake and osmotic water permeability of the C-less are 80 and 69% of those of the wild type, respectively. In other words, the function of this mutant is similar to that of the wild type. From a previous report (41), the formation of Glut1 oligomers would depend on the formation of disulfide bonds between residues 347 and 421. Accordingly, when Glut1 and/or its mutants are expressed in the X. laevis system, C-less Glut1 mutants would be expressed by oocytes as functional monomers capable of transporting both glucose and water. In addition, the co-expression of Glut isoforms differing in kinetic properties suggested a functional monomeric form and do not support oligomerization as being a prerequisite for functional activity (42). However, to be noted from our Glut1 structure, cysteines 347 and 421 are in the periphery of the monomer and face outwards (data not shown), so clearly S-S-bonded dimers could still form in the wild type and T310I mutant. In addition, the ability of Glut1 to be functionally active as a monomer does not exclude the significant cooperativity among subunits as proposed by Carruthers and colleagues (41).

Number of Channels per Monomer-- We consider first the one-channel alternative. Is a channel wide enough to be described by classical hydrodynamics, or should we use a single file paradigm? In the first case, an increase in P_f/P_d can be explained by an increase in the channel radius (43). However, that would also have led to an increase in P_d, which did not happen in our case (Fig. 4B). As for single-file water channels, the P_f/P_d ratio allows one to draw inferences on the number of water molecules occupying the pore and, hence, on the length of the pore (35, 44). In this context, a larger P_f/P_d ratio for the T310I mutant would entail higher occupancy, a longer pore, and lower P_f. However, this possibility contradicts the experimental finding of a higher P_f in the T310I mutant (Fig. 4A). In other words, assuming the presence of only one channel leads to difficulties in explaining the present evidence.

Therefore, we turn to the two-channel alternative. Although it is not known with certainty how many transport channels exist across Glut1, there is evidence consistent with the presence of two channels. A channel for glucose was theorized (6) and was located between helices 7 and 5 using mutagenesis data from Mueckler's laboratory (12, 40, 45). As we detail below, the locations of the mutagenized residues fit very well in the boundaries of what we term "main channel" in our Glut1 structure (Ref. 19 and this study). As for a second channel through Glut1, Brasseur and colleagues (46) presented theoretical arguments in favor of this idea, and Keller and colleagues (15) detected a solvent-accessible exofacial cleft between helices 2 and 7 and suggested that it could lead to a possible alternative pathway for transported substrates. This finding is consistent with the presence of the auxiliary channel we have described in our structure (19) located in that part of the protein.

Evidence for Two Channels from Water Permeation Across Wild Type and Pathogenic T310I Mutant Inhibitory Effects of Saccharides-- Interestingly, externally added 10 mM (but not 5 mM) D-glucose, L-glucose, and maltose cause a 40-45% inhibition in the P_f of the wild type (Fig. 7). This observation is apparently the first documented instance of inhibition of water passage through Glut1 by its substrate, D-glucose and suggests that glucose and water share the same path(s) through Glut1. The fact that both a transported sugar (D-glucose) and its non-transported enantiomer (L-glucose) inhibit water passage suggests that such inhibition takes place in a channel externally to a selectivity filter.

On the other hand, in the case of the mutant, D-glucose (up to 30 mM) or L-glucose (10 mM) do not affect the P_f value. This finding is consistent with the existence of a second channel that would become a preferential pathway for water passage. The access of the sugars to the main channel might be greatly reduced or blocked at the 310 site (Fig. 8, right panel), which would partially account for the lack of sugar-induced inhibition of P_f. Furthermore, the mutant transports only ~10% glucose that the wild type does. This would be consistent with reduced sugar penetration into the auxiliary channel and the virtual lack of water flow inhibition observed.

View larger version (56K): [in this window] [in a new window]   Fig. 8.   Effects of the pathogenic mutation T310I on the Glut1 structure. The main channel is colored in green, and the auxiliary channel in blue, both in space-filling representation. The yellow structure consists of segments of both channels plus a narrow connection. The left panel represents wild type Glut1, and the right panel represents the pathogenic T310I Glut1 mutant. The channel coordinates were generated with the program HOLE (36, 37). Channels are cut at both ends to depict only the interhelical segments. The residue 310 is in surface rendering. Thr-310 is in light green, and the mutated residue Ile-310 in blue. Only helix 7 is shown (white ribbon).

The effects of 10 mM maltose are revealing. The decrease in P_f for the case of the wild type is in line with its known effect of glucose transport blocker and seems to implicate the main channel. It also decreases the P_f of the mutant by a similar amount. Maltose would not be expected to enter the transport channel(s) and would inhibit P_f at its own binding sites at the openings of both channels. In this connection, there is strong recent kinetic evidence from Carruthers laboratory for the existence of two sugar import sites (and two maltose binding sites) for the human erythrocyte Glut1 (47). Remarkably, the study (47) argues convincingly for two channels per functional glucose transport unit. Our evidence here leads us to very similar conclusions with the exception that they are inclined to equate the functional unit to a dimer, whereas from previous (42, 48) and present studies given experimental evidence and a structural analysis of our three-dimensional model, we think that a monomer can be a functional transport unit and have the two channels in question when using the X. laevis expression system.

Lastly, there is a trend toward the stimulatory effects on P_f by 5 mM saccharides, significant for L-glucose. This finding is consistent with cooperative effects that might develop given the presence of two channels.

Activation Energy-- As mentioned under "Results", the activation energies for water transport through wild type and T310I mutant were 17.0 ± 2.0 and 13.0 ± 0.5 kcal mol¹, respectively. These numbers are of the order of the activation energies reported for glucose transport into human erythrocytes (14.1 kcal mol¹ (49)), for water flow into large unilamellar vesicles with reconstituted purified human erythrocyte Glut1 (10-13 kcal mol¹ (50)), and for the passage of water into oocytes expressing Glut1 (13 kcal mol¹ (8)). Such relatively large activation energies contrast with the low values associated with water channels (3-4 kcal mol¹ (51, 52)) and indicate that water passage across Glut1 depends on the conformational changes of the protein.

As shown in Fig. 6, the activation energy for water transport in T310I is approximately 5 kcal mol¹ smaller than that for wild type Glut1. This may correspond to an increase in the rigidity of the mutant with consequent increase in the number of open states.

Evidence from Structural Analysis: Update on the Glut1 Channels-- As mentioned above, we have recently modeled a three-dimensional structure for Glut1 (19), which accounts for existing data. We have also drawn from it new insights on transports pathways through the protein and possible effects of pathogenic mutations. Our work on the structure continues as we incorporate new experimental evidence. In this regard, in a recent paper by Mueckler and colleagues (54), the authors mention that two residues in helix 5 (Gln-161 and Val-165) of our structure do not seem to fit well with the experimental data observed (40) as pCMBS blocked the single Cys mutants at those sites, suggesting that they lie next to the transport pathway. We have examined this issue. It would seem that the program HOLE (36, 37) that we had been using to determine the coordinates of cavities in the Glut1 structure is optimal for gramicidin-type simple pores, but the more complex cavities in Glut1 would require additional procedures. Therefore, we are now using the Swiss Pdb viewer (38) to provide the initial coordinates to be explored further with HOLE. We were pleased to ascertain that the main transport channel that we had located near the exofacial end of helix 7 actually extended all the way through the protein with an opening on the endofacial side. With this extension, helix 5 is in the vicinity of the main channel (data not shown), which immediately gives a structural basis for the inhibitory effects on Q161C and V165C crucially pointed out (54). We also reexamined which side of helix 5 faces the main channel using the mutational data available for this amphipathic helix (40). We verified that with the angular position we chose for helix 5 (19), it has most of its pCMBS-accessible residues facing that channel (data not shown) from which our coordinates seem to require no change.

We originally described two channels termed main and auxiliary (19). However, as anticipated above, we have found that the main channel extends in the endofacial direction among helices 7, 5, 8, and 10 (Fig. 9, green color) all through the protein, including the endofacial loop region. This finding is consistent with this channel serving to transport glucose. As for the second or auxiliary channel, we have found a prolongation that traverses the region of endofacial loops. Together with a short connecting piece described previously, these two channels are now seen forming a H-like structure (Fig. 9). They have separate ends on both the exofacial and endofacial sides and are connected together in the middle around helix 7 (Fig. 9, yellow color). In the protein conformational state represented by our model, the main channel is open on both sides, whereas the auxiliary channel is open endofacially but closed exofacially (Fig. 9, blue color). Importantly, as explained below, it seems that the mutations in the vicinity of the auxiliary channel disable glucose transport (Fig. 9).

View larger version (51K): [in this window] [in a new window]   Fig. 9.   Transport channels in the Glut1 structure and the positions of seven known pathogenic mutants. For clarity, we only show the mutated residues in red surface rendering. The channels are shown in space-filling representation as in Fig. 8. Only helix 7 is shown (white ribbon).

Evidence from Structural Analysis: Changes Attributed to the T310I Pathogenic Mutation-- In our model, the threonine residue at position 310 lines the main channel (Fig. 8, left). Hence, the replacement of Thr (hydrophilic) by Ile (hydrophobic) plus the fact that the Ile side chain is ~2.3 Å longer than that of Thr could provide a straightforward explanation for the drastic decrease in glucose transport function shown experimentally. To investigate these possible effects, we substituted Ile for Thr-310 in our structure and ran the molecular dynamics for 50 ps. We examined the main and auxiliary channels of the mutant at the end of the simulation. The main channel was clearly blocked by the isoleucine residue, which protrudes to the middle of the putative glucose pathway (Fig. 9, right), consistent with the deficit in glucose transport observed. In addition, the auxiliary channel happened to be open on its exofacial end (Fig. 9), which supports its possible role for water passage, and is consistent with the elevated water permeation through the mutant.

This information on the arrangement of the channels after molecular dynamic simulation gives insight into the consequences of the mutation. It seems that the simple introduction of Ile-310 results in the mutant reaching a stable conformation accompanied by a lower mobility (data not shown) plus an important steric blockage of a glucose pathway. The mobility loss in itself would make it difficult for the mutant to undergo the conformation changes necessary for glucose transport, and the steric blockage would make the transport deficit even more pronounced. In fact, it may be surprising that any transport at all does remain under the circumstances.

Structural Analysis: the Locations of Pathogenic Mutants-- Interestingly, these mutants are not distributed randomly in the structure but are very close to well defined regions (Fig. 9). Three of them are on the exofacial side: S66F (in the loop between helices 1 and 2), T310I (in helix 8), and R126L or R126H (in helix 4). Of these, R126L or R126H is on the auxiliary channel, S66F is close to it, and T310I is on the main channel. The other pathogenic mutants are located on the endofacial side: G91D (loop between helices 2 and 3), E146K (loop between helices 4 and 5), K256V (loop between helices 6 and 7), and R333W (loop between helices 8 and 9). They seem to be distributed in pairs. G91D and E146K are close to each other and to the auxiliary channel near the endofacial side of the protein. Interestingly, this is close to the cytochalasin B binding site (55). It is possible that these mutations could act on Glut1 flexibility, perhaps mimicking in some way the inhibitory effect that cytochalasin B has. Finally, K256V and R333W are also located close to each other and to the putative main channel exit where they could obviously affect glucose transport.

	CONCLUSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION CONCLUSION REFERENCES

It seems possible to offer consistent explanations for the complex effects that the mutation T310I causes in the functional properties of Glut1 with the help of our three-dimensional model for Glut1. An examination of water passage gives useful clues for the study of transport of substrates through this protein. Both our experimental evidence and an analysis of our Glut1 structure agree in suggesting the presence of two channels per monomer. They also indicate that the obstruction of glucose flux and increased rigidity of the T310I mutant are probably responsible for its pathogenicity. The changes that this mutation causes in the dynamic behavior of Glut1 will be detailed separately.

	ACKNOWLEDGEMENT

We thank Dr. M. Mueckler for the generous gifts of purified Glut1 and C-less plasmid.

	FOOTNOTES

^* This work was supported in part by National Institutes of Health, U. S. Public Health Service Grants EY08918 (to J. F.), NS01698, NS37949, and RR00645 (to D. C. D.), the Will and the Colleen Giblin Foundations (to D. C. D.), and Research to Prevent Blindness, Inc. (to J. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ Both authors contributed equally to this work.

Recipient of a Fight for Sight Fellowship.

To whom correspondence should be addressed: Dept. of Physiology, College of Physicians & Surgeons, Columbia University, 630 W. 168th St., New York, NY 10032. Tel.: 212-305-9092; Fax: 212-305-2461; E-mail: jf20@columbia.edu.

Published, JBC Papers in Press, May 24, 2002, DOI 10.1074/jbc.M202763200

	ABBREVIATIONS

The abbreviations used are: Glut, glucose transporter; DOG, deoxyglucose; WT, wild type; P_f, water osmotic permeability; P_d, water diffusional permeability; pM, plasmid; pCMBS, para-chloro-mercuri- benzenesulfonate.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION CONCLUSION REFERENCES