Neutralization of a Conserved Amino Acid Residue in the Human Na+/Glucose Transporter (hSGLT1) Generates a Glucose-gated H+ Channel*

Matthias QuickDagger, Donald D. F. Loo, and Ernest M. Wright

From the Department of Physiology, UCLA School of Medicine, Los Angeles, California 90095-1751

Received for publication, June 23, 2000, and in revised form, August 31, 2000



    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The role of conserved Asp204 in the human high affinity Na+/glucose cotransporter (hSGLT1) was investigated by site-directed mutagenesis combined with functional assays exploiting the Xenopus oocyte expression system. Substitution of H+ for Na+ reduces the apparent affinity of hSGLT1 for glucose from 0.3 to 6 mM. The apparent affinity for H+ (7 µM) is about three orders of magnitude higher than for Na+ (6 mM). Cation/glucose cotransport exhibits a coupling ratio of 2 Na+ (or 2 H+):1. Pre-steady-state kinetics indicate that similar Na+- or H+-induced conformational changes are the basis for coupled transport. Replacing Asp204 with Glu increases the apparent affinity for H+ by >20-fold with little impact on the apparent Na+ affinity. This implies that the length of the carboxylate side chain is critical for cation selectivity. Neutralization of Asp204 (Asp right-arrow Asn or Cys) reveals glucose-evoked H+ currents that were one order of magnitude greater than Na+ currents. These phlorizin-sensitive H+ currents reverse and are enhanced by internal acidification of oocytes. Together with a H+ to sugar stoichiometry as high as 145:1, these results favor a glucose-gated H+ channel activity of the mutant. Our observations support the idea that cotransporters and channels share common features.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The human high affinity Na+/glucose cotransporter (hSGLT1)1 is a member of a family of secondary transporter proteins encompassing more than 55 homologues from archaea, bacteria, yeast, insects, and mammals (1, 2). This family uses electrochemical Na+ gradients to drive the coupled uphill transport of a variety of substrates (sugars, amino acids, vitamins, osmolytes, ions, myo-inositol, urea, and water). The expression of hSGLT1 in Xenopus laevis oocytes has resulted in a comprehensive study of both steady-state and pre-steady-state kinetics (3-5). A six-state ordered binding model has been proposed in which transport results from ligand-induced conformational changes (6, 7). In this model Na+ binds before sugar, with a coupling ratio of 2 Na+:1 glucose, and voltage influences both Na+ binding and the conformational states of the unloaded transporter. Functional analysis of SGLT chimeras and truncated proteins strongly suggests that the sugar pathway is located in the C-terminal domain of the protein (8, 9). Site-directed thiol labeling of a residue in the proposed sugar pathway indicates that conformational changes are responsible for the coupling of Na+ and sugar transport (10). We suggest that these conformational alterations are induced by cation binding in the N-terminal domain of the protein.

Although the functional importance of the N terminus in cation binding/translocation was shown for another SGLT family member (the Na+/proline transporter (PutP) of Escherichia coli) (11-13), there is little information on the role of the N-terminal domain in hSGLT1. We have initiated a study to explore the role of N-terminal residues in hSGLT1 in cotransport. In the present study, we have targeted a conserved residue, Asp204, located in a short cytoplasmic loop of hSGLT1 connecting transmembrane domains V and VI, which has been implicated in cation selectivity in PutP (12). Replacing Asp204 in hSGLT1 with Asn, Cys, or Glu dramatically modulated the steady-state and pre-steady-state kinetics of the transporter. Remarkably, although a transporter with a negative amino acid (Asp or Glu) exhibited cation/glucose cotransport with a stoichiometry of 2 (Na+ or H+) to 1, neutralization of Asp204 (by Asn or Cys) resulted in the activation of a glucose-activated H+ channel.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
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Molecular Biology-- A plasmid containing human SGLT1 (hSGLT1) cDNA was used as template for site-directed mutagenesis. Replacement of Asp204 with Asn, Cys, and Glu was performed using a two-step polymerase chain reaction protocol (14). For each pair of mutagenic oligonucleotides the sequence of the sense primer is presented with the altered nucleotide(s) underlined: D204C, 5'-GATTTACACGTGCACCTTGC-3'; D204E, 5'-GATTTACACGGAAACCTTGC-3'; D204N, 5'-GATTTACACGAACACCTTGC-3'. Polymerase chain reaction products were digested with BglII and Eco47III, and the resulting 428-bp fragments were ligated into a similarly treated wild-type hSGLT1-containing plasmid. The fidelity of the inserted DNA fragments was confirmed by sequencing double-stranded DNA (Sequenase version 2.0, DNA sequencing kit, United States Biochemical, Cleveland, OH) after alkaline denaturation (15). Each mutagenized DNA template was linearized with XbaI, transcribed, and capped in vitro using the T3 RNA promoter (MEGAscript kit, Ambion, Austin, TX). X. laevis oocytes were injected with 50 ng of mRNA and were incubated in Barth medium containing gentamicin (5 mg/ml) at 18 °C for 3-7 days (4).

Transport Assays and Electrophysiological Techniques-- For transport and electrophysiological experiments, oocytes were bathed in an assay buffer composed of 2 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 10 mM HEPES-Tris, pH 7.5, and a combination of Na+ or choline+ chloride salts to give a final concentration of 100 mM. For H+ activation experiments the pH of 100 mM choline buffer was varied between 8.0 and 4.5 by titration with Tris or Mes. Uptake of methyl-alpha -D-[U-14C]glucopyranoside (293 Ci/mol, Amersham Pharmacia Biotech) and electrophysiological measurements using the two-microelectrode voltage clamp technique were performed as described (16, 17). The stoichiometry of cation-coupled D-[U-14C]glucose (316 Ci/mol, ICN Radiochemicals) uptake was determined under voltage clamp conditions (18).

Data Analysis-- Sugar-evoked steady-state currents were fitted to Eq. 1,


I<SUP><UP>Glc</UP></SUP>=<FR><NU>I<SUP><UP>Glc</UP></SUP><SUB><UP>max</UP></SUB>×[<UP>Glc</UP>]</NU><DE>K<SUP><UP>Glc</UP></SUP><SUB>0.5</SUB>+[<UP>Glc</UP>]</DE></FR>, (Eq. 1)
where IGlc and ImaxGlc represent D-glucose-induced current and maximal D-glucose-induced current, respectively, at saturating [cation], [Glc] is the concentration of D-glucose, and K0.5Glc is [Glc] at 0.5 ImaxGlc. Kinetic parameters for the phlorizin-inhibited cation leak or cation-activated glucose transport were determined by using Eq. 2,
I<SUP><UP>cation</UP></SUP>=<FR><NU>I<SUP><UP>cation</UP></SUP><SUB><UP>max</UP></SUB>×[C]<SUP>n</SUP></NU><DE>(K<SUP><UP>cation</UP></SUP><SUB>0.5</SUB>)<SUP>n</SUP>+[C]<SUP>n</SUP></DE></FR>, (Eq. 2)
where Ication and Imaxcation is the cation-evoked (leak) current and the maximal cation (leak) current at saturating cation concentration [C], respectively, K0.5cation is [C] at 0.5 Imaxcation, and n represents the Hill coefficient. Charge-voltage (Q-V) relations for each membrane voltage (Vm) were obtained by integrating pre-steady-state current transitions (after subtracting the capacitive and the steady-state currents from the total currents) with time and were fitted to Eq. 3 (17, 19),
<FR><NU>Q−Q<SUB><UP>hyp</UP></SUB></NU><DE>Q<SUB><UP>max</UP></SUB></DE></FR>=<FR><NU>1</NU><DE>1+<UP>exp</UP>[z(V<SUB><UP>m</UP></SUB>−V<SUB>0.5</SUB>)F/RT]</DE></FR>, (Eq. 3)
with Qhyp and Qdep for Q at hyperpolarizing and depolarizing limits, respectively. V0.5 represents Vm at which 50% of the total charge in the membrane electric field has moved, z is the apparent valence of the moveable charge, and F, R, and T have their usual meanings. All experiments were repeated at least three times with oocytes from different donor frogs. Data fits were performed using the non-linear regression algorithm in SigmaPlot (version 5.0, SPSS Inc., Chicago, IL). Unless otherwise noted, figures are based on data obtained from a typical experiment on a single oocyte, and errors represent S.E. of the fit.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In these studies all comparisons between Na+ and H+ kinetics were performed in the same oocyte expressing WT, D204C, D204E, or D204N cRNA. For comparison of WT kinetics and the kinetics of a transporter with a substitution at position 204, oocytes of the same batch were analyzed on the same day within 10 h.

Uptake Experiments-- The most conservative substitution Asp204 right-arrow Glu resulted in a reduction of Na+-dependent alpha MDG uptake by 87%, whereas the uptake rates of D204C and D204N were reduced by ~50% (Fig. 1). In the absence of Na+ ions, the rate of alpha MDG uptake in oocytes expressing D204E was about 1 pmol/h, a value comparable to oocytes with the native transporter (1.7 pmol × h-1 × oocyte-1). However, replacing Asp204 with Cys or Asn increased the uptake rate by 7- and 16-fold. This increase may reflect the enhanced apparent affinity for sugar (see Table I).



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Fig. 1.   Uptake of [14C]alpha MDG by oocytes 5 days after injection with hSGLT1, D204C, D204E, or D204N cRNA. Transport of 50 µM [14C]alpha MDG (293 µCi/µmol) was assayed in the presence of 100 mM NaCl (Nac) or 100 mM choline chloride buffer, pH 7.5, for 60 min at 22 °C. H2O-injected oocytes (NI) served as a control. Data are from the same batch of oocytes, and errors represent the S.E. (n = 7).


                              
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Table I
Summary of kinetic parameters in presence of Na+ and H+
Steady-state kinetic parameter and turnover rates for cation-coupled sugar transport (kcatcation) are shown at Vm = -110 mV. ImaxGlc, K0.5Glc, and Qmax were determined at 100 mM Na+ and 3.2 µM H+.

Steady-State Currents-- Fig. 2 shows representative sugar-induced steady-state current-voltage (I-V) relationships for WT, D204C, and D204E in the presence of Nac and Hc at saturating [Glc]. For WT both curves were sigmoidal and saturated at hyperpolarizing voltages. A similar I-V relation was observed for D204E in Nac, but in Hc the sugar-induced currents exhibited a supralinear increase with hyperpolarization. D204C and D204N (data not shown) exhibited essentially identical I-V relations with no saturation of the sugar-evoked currents at the most negative potential (-150 mV) in either Nac or in Hc. The glucose-evoked currents in Hc for these transporters were much greater than the Na+ currents (see Table I). In Hc for the latter transporters, glucose activated outward currents at potentials more positive than ~+20 mV (~+100 nA at +50 mV). A comparison of the sugar-induced currents at Vm = -150 mV revealed similar values in the presence of Hc for WT (-1220 nA), D204C (-1120 nA), and D204N (-870 nA), and a reduction of about 75% for D204E (-300 nA). In Nac, WT and D204E showed comparable currents generated by 100 mM D-glucose at -150 mV (-1480 and -930 nA). Under the same test conditions, D204C and D204N exhibited less than 10% of the current observed for WT.



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Fig. 2.   Voltage dependence of sugar-induced steady-state currents. Net currents induced by 100 mM D-glucose (Inet = Isubstrate - Ino substrate) were measured in the presence of Nac (open symbols) or Hc (solid symbols) and are plotted as a function of the membrane potential (Vm) for WT (,black-square), D204C (open circle ,), and D204E (triangle ,black-triangle). Experiments were carried out in single oocytes from one batch.

We measured the sugar kinetics of each transporter by varying external [Glc] from 0.05 to 100 mM in the presence of Nac or Hc. The apparent affinity constants for D-glucose (K0.5Glc) at Vm = -110 mV are shown in Table I. As described earlier (20), hSGLT1 exhibited a K0.5Glc of 0.3 mM in Nac. When Hc substituted for Nac the apparent affinity for Glc was reduced by 20-fold (K0.5Glc = 6 mM). A similar reduction of the apparent Glc affinity in Hc was also observed for each transporter with a replacement of Asp204. A closer examination of K0.5Glc revealed two classes of mutations. First, Glu in place of Asp204 reduced the apparent affinity for Glc in either Nac or Hc about 4-fold. The second class encompasses transporters with a neutral side chain at position 204 (Asp204 right-arrow Asn or Cys), which showed increased apparent affinities for Glc. Although the K0.5Glc of D204C in Hc was only slightly decreased (3 mM), a ~5-fold reduction of K0.5Glc was observed in Nac (0.07 mM). D204N exhibited a more dramatic increase of the apparent affinity for Glc with a 10- and 15-fold reduction of K0.5Glc in Hc (0.6 mM) and in Nac (0.02 mM), respectively. Over a range from -150 to -30 mV, the K0.5Glc determined in Nac was essentially voltage-insensitive for all transporters. From -150 to -50 mV, WT exhibited an approximate 4-fold increased K0.5Glc in Hc, but no voltage dependence of the K0.5Glc was observed for the other transporters.

The ImaxGlc values for each transporter increased (more negative) with hyperpolarizing potentials and were comparable to the currents generated by 100 mM D-glucose (see Fig. 2). The cation had no impact on the magnitude of ImaxGlc for WT (Table I). WT and D204E exhibited a sigmoidal ImaxGlc-V relation in Nac and thus, the similar ImaxGlc values of these two transporters shown at -110 mV (Table I) were not significantly changed by more hyperpolarizing potentials. With the exception of WT, no saturation of the ImaxGlc-V relationship was observed for each transporter at hyperpolarizing potentials in Hc.

To elucidate whether the substitution of Asp204 in hSGLT1 changed the sugar specificity of the transporter, oocytes were held at -50 mV and currents generated by 10 mM sugar were monitored in the presence of Nac or Hc. Under either test condition the selectivity pattern for each transporter was in the order Glc >=  alpha MDG >=  D-galactose > 3-O-methyl-D-glucose (data not shown). Consistent with the effect of the cation on the apparent Glc affinity for WT and D204E, each sugar induced ~10-fold lower currents in the presence of Hc than in Nac. The effect on the apparent Glc affinity of D204E was reflected by ~4-fold lower sugar-induced currents in Nac and Hc than with WT. In Hc, D204C and D204N exhibited sugar-induced currents similar to WT, but the currents generated by each sugar in Nac were ~50-fold lower than the sugar-evoked currents observed for WT.

We determined the apparent half-maximal cation concentration for Na+- and H+-activated glucose transport (K0.5cation). Table I summarizes K0.5cation for all transporters at Vm = -110 mV. For WT the K0.5H+ (7 µM, pH 5.2) was about three orders of magnitude smaller than the K0.5Na+ (6 mM). Again, the nature of the substitution of Asp204 grouped transporters with a neutral amino acid side chain (Asp204 right-arrow Asn or Cys) in one group. These transporters exhibited a 10-fold reduced K0.5Na+, but the K0.5H+ was not significantly altered. On the other hand, Glu in place of Asp204 reduced K0.5Na+ by about 3-fold (2 mM). However, this conservative substitution reduced K0.5H+ of this transporter by a factor of >20, shifting the pH of the apparent half-maximum concentration for H+-activated glucose transport from pH 5.2 to ~6.5. In general, for each transporter K0.5Na+ was slightly voltage-dependent and increased about 4-fold from -150 to -50 mV, while K0.5H+ over the same voltage range increased by a factor of about 2.

The Hill coefficient (n) of WT for cation-activated glucose transport was larger than unity for both Na+ (nNa+ > 1.5) and H+ (nH+ > 1.2) and was not affected by voltage (-50 to -150 mV). nNa+ of D204E was >1.3 but for H+/sugar cotransport nH+ was <1. Independent on the cation, D204C and D204N exhibited a nNa+ or nH+ of < 1.

Whereas at -110 mV ImaxNa+ for each transporter was essentially indistinguishable from ImaxGlc in Na+, this similarity in Hc was only observed for WT and D204E (note that the ImaxNa+-V relation of D204C and D204N and the ImaxH+-V relation of D204E did not saturate at hyperpolarizing potentials). On the other hand, at Vm = -110 mV, a transporter with a neutral amino acid side chain at position 204 exhibited a ~3-fold larger ImaxH+ (D204C, -1630 nA; D204N, -2450 nA) than ImaxGlc in Hc (D204C, -675 nA; D204N, -769 nA) and ImaxH+ increased supralinearly with more negative potentials.

To determine the net Na+ and H+ leaks through each transporter, [Na+] or [H+] was varied between 0.1 and 100 mM Na+ or between 0.03 and 32 µM H+ and phlorizin-sensitive currents and choline currents at pH 8.0 were subtracted from the total currents. The kinetics of the cation leak were calculated at Vm = -110 mV by fitting the data to Eq. 2. In general, for each transporter K0.5Na+ for glucose cotransport and leak were essentially identical, but the K0.5H+ value for the leak was ~10-fold smaller than K0.5H+. WT exhibited a leak ImaxNa+ of -97 ± 4 nA and replacement of Asp204 with Asn (54 ± 5 nA), Cys (18 ± 2 nA), or Glu (47 ± 3 nA) reduced the leak ImaxNa+ ~2- to 5-fold. The leak ImaxH+ for WT (430 ± 4 nA) was 2.5 to 3 times larger than for D204C (-172 ± 16 nA) and D204N (-142 ± 7 nA), but there was apparently no H+ leak through D204E. Although the Hill coefficient (n) for the Na+ or the H+ leak pathways was >1.2 only for WT, the value of n for each modified transporter was <= 1.

Pre-Steady-State Charge Movement-- Fig. 3A shows representative current records of oocytes injected with WT or D204E cRNA in the presence of Nac or Hc after stepping the membrane potential from the holding potential (-50 mV) to the test potential (Vm = -150 to +50 mV in 20-mV decrements). After the initial fast membrane capacitive transients (tau < 1 ms) each transporter exhibited currents that relaxed to a steady-state with a single time constant (see Fig. 4). These relaxations were abolished after addition of saturating [sugar] and/or [phlorizin] (data not shown). The removal of a negative amino acid side chain at position 204 caused a dramatic reduction of the current transients (compare Qmax in Table I).



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Fig. 3.   Pre-steady-state kinetics associated with WT and D204E. A, current traces in Nac and Hc were obtained by a pulse protocol in 20-mV decrements (Vm = +50 to -150 mV from a holding potential of -50 mV). To determine the relaxation time constant of D204E (see Fig. 4), the pulse duration was increased from 100 to 200 ms. B, Q-V relationship for WT and D204E at various [Na+] or [H+] in the absence of substrate or inhibitor. Smooth lines represent fits to Eq. 3 (see "Experimental Procedures"). For comparison, curves were normalized (Qnorm) and aligned vertically with respect to Qdep in Nac or Hc. Symbols represent [Na+] (in mM) of 10 (open circle ), 50 (triangle ), and 100 (), and [H+] (in µM) of 0.032 (black-diamond ), 0.316 (black-down-triangle ), and 10 (). C, V0.5 of WT (rectangle) and D204E (triangle) as a function of [cation].



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Fig. 4.   Voltage dependence of relaxation time constant. Time constants (tau ON and tau OFF) were determined from the charge transfer in Nac (open symbols) and Hc (solid symbols) shown in Fig. 3A. tau ON of WT (rectangle) and D204E (triangle) was plotted as a function of membrane potential (Vm). Smooth lines represent a Gaussian fit to tau ON. tau OFF for both transporters was voltage-independent and shown as the average over the test range (+50 to -150 mV) for WT (square with black diamond, in Nac; square with white diamond, in Hc) and D204E (triangle with black diamond, in Nac; triangle with white diamond, in Hc).

The charge movement was obtained by integration of the transporter-mediated relaxation. At each Vm, a plot of the charge (Q) versus the membrane voltage (Vm) yielded a sigmoidal charge-voltage (Q-V) curve (Fig. 3B). Data were fitted to Eq. 3 to obtain the Boltzmann parameters Qmax (maximal charge), V0.5 (Vm at which 50% of the charge has moved in the membrane electric field), and z (the apparent valence of the charge). Qmax of WT and D204E were comparable in Nac and Hc (~23 nanocoulombs (nC)), and Qmax of D204C and D204N were reduced about 5-fold (Table I). The substitution of Nac by Hc shifted V0.5 of WT from -36 ± 1 mV to -74 ± 4 mV. Glu in place of Asp204 exhibited a V0.5 in Nac of -50 ± 2 mV. However, the V0.5 of this transporter in Hc was shifted to more positive potentials (V0.5 ~+40 ± 5 mV).

In Fig. 3B the effect of varying [cation] on the charge-voltage (Q-V) relationship is shown for WT and D204E. At [Na+] or [H+] below 25 mM or 3.2 µM, respectively, the Q-V curve for WT didn't become saturated at negative potentials. D204E exhibited a sigmoidal Q-V curve over the entire [Na+] range tested. At [H+] higher than 3.2 µM, the Q-V relationship of the latter transporter did not become saturated at +50 mV.

Fig. 3C summarizes V0.5 as a function of [cation]. Reducing [Na+] from 100 to 25 mM shifted V0.5 of WT from -36 ± 1 mV to -91 ± 2 mV. Plotting V0.5 as a function of log[Na+] revealed a linear relation with a slope of 98 ± 3 mV per 10-fold change in [Na+] (Delta V0.510). V0.5 for D204E was apparently not dependent on [Na+] (Delta V0.510 of 8.7 ± 0.2 mV). In H+ WT (Delta V0.510 = 92 ± 7 mV), and D204E (Delta V0.510 = 87 ± 11 mV) exhibited a similar slope of Delta V0.510. However, V0.5 of D204E was shifted by about 110 mV toward positive potentials. Over the concentration range shown in Fig. 3C, WT and D204E exhibited a z value of ~1.

Relaxation Time Constants-- Substraction of capacitive and steady-state currents from the total currents (see Fig. 3A) revealed a monoexponential time constant (tau ) of the pre-steady-state relaxation currents. tau  was voltage-dependent in the ON response but not dependent on voltage in the OFF response (Fig. 4). In Nac the shapes of the tau ON-V curves for the transporters tested were bell-shaped and described a Gaussian fit. The maximum (tau max) for WT and D204E was ~20 ms and ~27 ms at ~-100 mV. In contrast, in Hc the tau -V curve of WT didn't reach tau max over the applied Vm (+50 mV to -150 mV) but the bell-shaped tau -V relation of D204E was dramatically shifted to the right (tau max of ~32 ms at ~-22 mV). As reported above, D204C and D204N exhibited very small pre-steady-state currents that were at the border of resolution and precluded us from determining reliable tau max values over the entire voltage range.

Stoichiometry-- The stoichiometry of ion/sugar cotransport was determined by simultaneously measuring the unidirectional flux of D-[14C]glucose into oocytes expressing WT or D204N and monitoring the sugar-evoked inward current. Integration of the inward current with time revealed the net positive charges that entered the oocyte. A plot of the net charge against glucose uptake by oocytes expressing WT revealed a linear relation in both Nac and Hc with a slope of ~2 inward charges per glucose molecule transported (2.1 ± 0.1 in Nac; 2 ± 0.3 in Hc) (Fig. 5). In the presence of Nac the slope of the inward charge/glucose relation of D204N was 2.1 ± 0.4. Replacing Nac with Hc changed the slope for D204N to 39 ± 3. By reducing the final D-[14C]glucose concentration in Hc from 5 to 0.1 mM, the slope for D204N was 144 ± 7 (Fig. 5, inset), but no significant effect on the H+/glucose stoichiometry was observed for WT (data not shown). No Nac- or Hc-dependent glucose uptake or sugar-evoked current was observed in control (H2O-injected) oocytes.



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Fig. 5.   Stoichiometry of cation/sugar cotransport of WT and D204N. Glucose-dependent charge (Qglc) and D-[14C]glucose uptake were simultaneously determined at Vm = -90 mV in the presence of Nac (, WT; down-triangle, D204N) or Hc (black-square, WT; black-down-triangle , D204N) for time periods ranging from 5 to 15 min. The final concentration of D-[14C]glucose was 1 mM (1 µCi/µmol) in Nac and 5 mM (1 µCi/µmol) or 0.1 mM (100 µCi/µmol) (inset) in Hc, respectively. Integration of the glucose-evoked inward current with time was used to calculate the net cation influx by converting nanocoulombs to nanomoles using the Faraday constant. Uptake of D-[14C]glucose by oocytes expressing given cRNA was corrected for basal uptake by control (H2O-injected) oocytes.

Outward Currents-- We next studied the effect of intracellular acidification on glucose-induced H+ currents. Acidification of the intracellular compartment was produced by rapid replacement of external choline chloride with potassium acetate (21). Fig. 6 shows representative I-V curves on the effect of internal acidification of oocytes expressing D204N or WT in the presence of 31.6 µM H+ (pH 4.5). In the absence of acetate, the glucose-induced currents for D204N increased with hyperpolarization and reversed at +23 mV (Fig. 6A). Internal acidification increased the magnitude of outward currents with depolarization (+800 nA at +50 mV) but had only minor effects on currents at potentials more negative than -70 mV. No outward currents were observed after internal acidification of oocytes expressing WT under either condition (Fig. 6B), despite the fact that the amount of WT in the plasma membrane was about four times that of D204N (Qmax for WT = 14 nC, for D204N = 4 nC). Addition of 0.2 mM phlorizin reversibly blocked the inward and outward glucose-induced currents for D204N completely (Fig. 6C).



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Fig. 6.   Effect of intracellular acidification on sugar-induced steady-state currents. A and B, net currents induced by 10 mM D-glucose in the presence or absence of 50 mM potassium acetate (equimolar replacement for choline chloride) at pH 4.5 are plotted as functions of the membrane potential (Vm) for (A) D204N (in the presence (inverted triangle with black diamond) or absence (black-down-triangle ) of acetate) and (B) WT (in the presence (square with black diamond) or absence (black-square) of acetate) as described in the legend of Fig. 2. C, phlorizin inhibition of glucose-evoked inward and outward currents for D204N in the presence of 50 mM potassium acetate at pH 4.5. Net currents were presented as the difference of total currents induced by 10 mM glucose in the absence (black-down-triangle ) or presence (open circle ) of 0.2 mM phlorizin minus H+ currents in the presence of 0.2 mM phlorizin. Data for D204N (A and C) are from the same oocyte.



    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The present study confirms and extends our previous reports on the kinetics of SGLT1 (4, 5): First, the steady-state and pre-steady-state kinetics of the transporter in Na+ are in close agreement with previous observations on hSGLT1 (10, 17, 20, 22-24). Second, we confirm and extend our findings on rabbit SGLT1 (25, 26) that H+ can drive sugar cotransport through hSGLT1. The kinetics of H+ and Na+ sugar cotransport are quite similar, even though the apparent affinity of hSGLT1 for H+ is ~1000-fold greater than for Na+, and the apparent affinity for sugar is about 10-fold lower in Hc than in Nac (see Table I). In fact, the maximum rates of cotransport, the cation-to-sugar stoichiometry (2:1), the Hill coefficients (>1), turnover numbers (~50 s-1), and voltage dependence of transport are virtually identical for Na+ and H+ cotransport when measured in the same oocyte. Furthermore, the pre-steady-state kinetic parameters Qmax, V0.5, and tau max are comparable in Nac and Hc. The only quantitative differences are that the turnover number for the leak pathway (uniport) (27) is about 5-fold higher in Hc (22 s-1) than in Nac (4.4 s-1) and that K0.5H+ is about one order of magnitude smaller for the leak than for the cotransport. The latter observation is consistent with the results for hSGLT1 reported by Chen et al. (24). In contrast to their work (they reported a Hill coefficient for Na+ uniport = 1 and for H+ uniport > 1), the present study clearly emphasizes that the Hill coefficients for both Na+ and H+ uniport are >1. Together with the concentration dependence of V0.5 (95 mV per 10-fold change in [Na+] or [H+]), this finding supports our hypothesis that two cations, Na+ or H+, bind to SGLT1 before sugar. This is in direct contradiction to the Na+ (or H+)-sugar-Na+ binding sequence model proposed by Chen et al. (24) based on their experiments. However, Chen's model is inconsistent with their reported results, because it does not take into account that the Hill coefficient for the H+ leak current was >1 and that the currents for the Na+ leak were no greater than 15 nA (compared with 100 nA in the present study). Furthermore, their conclusion that Na+ is the last substrate bound to the transporter cannot account for the steady-state kinetics, because the maximum rate of Na+/glucose cotransport is independent of [Na+] (4, 5).

To explore the role of the N terminus of hSGLT1 in cation/sugar cotransport, we have analyzed the effect of replacing a conserved amino acid residue, Asp204. This residue is aligned with Asp187 in the Na+/proline cotransporter (PutP), which is functionally important for cation selectivity and Na+-dependent proline binding and transport (12). Replacing Asp204 revealed two functional classes of mutations, depending on the presence or absence of a negative-charged amino acid side chain at position 204. This stands in contrast to PutP, where a polar rather than a charged residue at position 187 is essential for function.

The first class of mutations consists only of a transporter with a Glu in place of Asp204. Independent of the coupling cation, the apparent affinity for glucose is increased by ~4-fold, indicating a cation species-independent effect on the glucose translocation through D204E. The apparent affinity of the transporter for Na+ is increased by 3-fold, whereas the I-V relation and ImaxGlc in Nac+, ImaxNa+, and the leak ImaxNa+ are all comparable to WT. These minor kinetic differences are reflected by similar turnover numbers by these transporters. On the other hand, D204E exhibits an increased apparent H+ affinity. This implies that lengthening the side chain at position 204 by only one methylene group (about 1.5 Å) dramatically influences, directly or indirectly, the geometry of the cation site(s). A similar observation was reported for the glutamate transporter (GLT-1 or EAAT-2) (28). The increased apparent H+ affinity of D204E is mirrored by the alkaline shift in the Q-V relation (Fig. 3B), suggesting that, even at very low [H+], binding of H+ induces conformational changes that are the basis for coupled transport (10). This fact is most likely the reason for the apparent independence of the Q-V relation of D204E on [Na+], because H+ is the preferred cation species. At [H+] larger than 3 µM no saturation at depolarizing potentials is detectable, precluding reliable calculations in this [H+] range. However, between 0.1 and 3 µM H+ Delta V0.510of D204E is similar to Delta V0.510for WT from 1 µM to 32 µM H+. The ~20-fold increase in apparent affinity is also reflected by a ~110-mV shift of V0.5 toward positive potentials and a tau max of D204E at -25 mV (no tau max is detectable for WT in Hc from -150 mV to +50 mV). According to computer simulations of the 6-state kinetic model for SGLT1 (5), this right shift of tau max and the generally higher tau  values are due to higher binding and lower dissociation rate constants for the cation. This prediction is consistent with the observation that no H+ leak through D204E is observed and may explain why the glucose-evoked H+ currents (Fig. 2) were about ~5-fold smaller for D204E than for WT. Because the I-V relation of D204E in H+ does not saturate at hyperpolarizing potentials, the turnover number for H+/glucose cotransport for D204E at -110 mV must be greater than 9 s-1.

The second class of mutations encompasses transporters with a neutral side chain at position 204 (Asp right-arrow Asn or Cys). By using thiol labeling, the predicted cytoplasmic location of Asp204 (1, 29) is confirmed, because Cys204 is only accessible from the cell inside (data not shown). Furthermore, introduction of a positive charge (by 2-aminoethyl methanethiosulfonate) doesn't have a significant effect on the kinetics of the transporter. Based on this observation, we may exclude a possible role of Asp204 in salt-bridge formation with a positive amino acid as proposed for Asp residues in the lactose permease (LacY) (30-32). However, as indicated by the 5-fold reduction of Qmax for D204C and D204N, removal of the negative charge at position 204 reduces the number of transporters in the plasma membrane. These data imply a trafficking defect of the protein to the plasma membrane: a common problem observed with the expression of a membrane protein in a eukaryotic expression system (33, 34). This trafficking problem precludes us from a detailed analysis of the pre-steady-state kinetics of D204C or D204N.

Analysis of steady-state kinetics of D204C and D204N shows a >10-fold reduction of K0.5Glc in Nac and Hc. The finding that this single mutation also produces a 10-fold reduction in K0.5Na+ raises the idea that cation and sugar sites are in close proximity or may overlap. A similar hypothesis is proposed for the melibiose permease of E. coli, where an N-terminal domain of the transporter plays a fundamental role in connecting cation- and sugar-binding sites in terms of coupling (35, 36). Although the apparent affinity parameters of the (co)substrates for D204C and D204N were altered and the steady-state activation of the currents revealed a Hill coefficient < 1, neither the coupling ratio of Na+ to glucose transport (Fig. 5) nor the turnover number for Na+/glucose cotransport (ImaxNa+/Qmax, see Table I) was significantly altered.

Unexpectedly, all experimental evidence indicates that neutralization of Asp204 (D204C and D204N) in hSGLT1 results in the activation of a glucose-activated H+ channel. This conclusion is based on the observations that: 1) The glucose-evoked H+ currents are an order of magnitude greater than the Na+ currents (Fig. 2). Because the H+ currents do not become saturated within the applied voltage range, this would imply that the turnover number (ImaxH+/Qmax) increases by more than one order of magnitude over that in Na+ (Table I). 2) The H+ currents reverse at positive voltages, unlike either the Na+ currents for D204C or D204N or the H+ and Na+ currents for WT or D204E (Fig. 2). These outward H+ currents are enhanced by internal acidification of the oocyte (Fig. 6A) and are blocked by the addition of phlorizin (Fig. 6C). 3) The H+ currents greatly exceed those expected for the strict coupling of H+/sugar cotransport. The H+/sugar stoichiometry increases from the expected value of 2 to as high as 144 (Fig. 5). The Na+/glucose stoichiometry for both WT and D204N was 2:1. 4) There is no change in the H+ leak currents for these mutants. The turnover numbers for the H+ leak currents for D204C and D204N (-172 nA/5 nC = 34.5 s-1; -142 nA/4.4 nC = 32 s-1) are comparable to that for the wild-type protein (22 s-1).

These results imply that minor structural changes in hSGLT1, i.e. replacing a carboxyl side chain with an amine or sulfhydryl group, are sufficient to open a glucose-activated H+ channel. This channel activity is intimately associated with sugar transport, because the competitive blocker phlorizin is unable to activate the H+ channel but is only able to block the effect of glucose. Clearly, based on our results we cannot distinguish whether neutralization of this conserved acidic residue generates a new "artificial" H+ pore or affects interactions with the coupling cation and/or other parts of the protein, thereby modifying the "original" H+ pathway. Because Asp204 is located in a cytoplasmic loop, it seems unlikely that this residue is part of the membrane-spanning H+ pathway. However, with the lack of high resolution structural data of this transporter, this question will remain an enigma.

Our observations lend support to the suggestion that cotransporters and channels share features in common. For example, the glutamate cotransporters also behave as ligand-induced chloride channels (37, 38, see also Ref. 39 for review). However, this report shows that hSGLT1 appears to be unique in that: 1) ion channel activity is only observed in the mutagenized protein; 2) the ligand (sugar) opens a channel for the driving cation (H+) for cotransport, and 3) SGLT1 functions as a monomer (40). The glutamate cotransporter EAAT3 occurs as a homopentamer in oocyte plasma membranes, and it has been suggested that the chloride channel is formed by the oligomer (41).


    ACKNOWLEDGEMENTS

We are indebted to Daisy W. Leung and Mary Lai Bing for excellent technical assistance with the Xenopus oocytes and our colleagues for critical discussions.


    FOOTNOTES

* This work was supported by National Institutes of Health Grant DK19567 (to E. M. W.).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.

Dagger A postdoctoral fellow of the Deutsche Forschungsgemeinschaft. To whom correspondence should be addressed: Tel.: 310-825-6905; Fax: 310-206-5886; E-mail: mquick@mednet.ucla.edu.

Published, JBC Papers in Press, October 6, 2000, DOI 10.1074/jbc.M005521200


    ABBREVIATIONS

The abbreviations used are: hSGLT1, human high affinity Na+/glucose transporter; alpha MDG, alpha -methyl-D-glucopyranoside; Glc, D-glucose; Hc, 3.2 µM H+ pH 5.5; Nac, 100 mM Na+; WT, hSGLT1-wild-type; PutP, Na+/proline transporter; Mes, 4-morpholinoethanesulfonic acid; nC, nanocoulomb(s).


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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