©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Kinetics of Steady-state Currents and Charge Movements Associated with the Rat Na/Glucose Cotransporter (*)

(Received for publication, May 31, 1995; and in revised form, August 9, 1995)

Mariana Panayotova-Heiermann (§) Donald D. F. Loo Ernest M. Wright

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

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The rat Na/glucose cotransporter (SGLT1) was expressed in Xenopus oocytes and steady-state and transient currents were measured using a two-electrode voltage clamp. The maximal glucose induced Na-dependent inward current was 300-500 nA. The apparent affinity constants for sugar (alpha-methyl-D-glucopyranoside; alphaMDG) (K) and sodium (K) at a membrane potential of -150 mV were 0.2 mM and 4 mM. The K increased continuously with depolarizing potentials reaching 40 mM at -30 mV. K was steeply voltage dependent, 0.46 mM at -30 mV and 1 mM at -10 mV. From all tested monovalent cations only Li could substitute for Na, but with lower affinity. The relative substrate specificity was D-glucose > alphaMDG approx D-galactose > 3-O-Me-Glc beta-naphthyl-D-glucoside uridine. Phlorizin (Pz), the specific blocker of sugar transport, showed an extremely high affinity for the rat cotransporter with an inhibitor constant (K) of 12 nM. SGLT1 charge movements in the absence of sugar were fitted by the Boltzmann equation with an apparent valence of the movable charge of 1, a potential for 50% maximal charge transfer (V) of -43 mV, and a maximal charge (Q) of 9 nanocoulombs. The apparent turnover number for the rat SGLT1 was 30 s. Model simulations showed that the kinetics of the rat SGLT1 are described by a six-state ordered nonrapid equilibrium model, and comparison of the kinetics of the rat, rabbit and human cotransporters indicate that they differ mainly in their presteady-state kinetic parameters.


INTRODUCTION

Cotransporters are membrane proteins that use the electrochemical potential gradient for ions to accumulate sugars, amino acids and osmolytes into cells. Using the electrochemical potential gradient for Na, the Na/glucose cotransporter (SGLT) (^1)accumulates glucose across the brush border membrane of the epithelial cells of the intestine and the proximal tubule of the kidney.

Several members of the SGLT family have been cloned, and these include the high affinity glucose cotransporters (SGLT1, K 0.2 mM) from rabbit small intestine (1) and kidney (2) , pig(3) , and rat kidney(4) , human intestine(5) , as well as the low affinity glucose cotransporter (pSGLT2, K 2 mM) from pig kidney(6) . Mapping the genomic arrangement of the human SGLT1 gene, Turk et al.(7) showed that SGLT1 is a single-copy gene, so that the amino acid sequences from various tissues of a given species are identical. Comparison of the amino acid sequences from the rat, human and rabbit clones reveal 86-87% identity and 93-94% similarity. How does this high degree of homology between the three clones affect their kinetic properties? In this study, we characterized the presteady-state and steady-state kinetics of the rat SGLT1 clone with a view to understand the relationship between structure and function of members of the SGLT1 family.


MATERIALS AND METHODS

The pBluescript II SK plasmid containing the coding sequence for rat SGLT1 (4) was linearized with SalI and transcribed in vitro with T3 RNA polymerase(11) . The cRNA was overexpressed in Xenopus oocytes and protein function studied 5-10 days after injection using the two-microelectrode voltage clamp(8, 11) . To obtain a current-voltage (I-V) relationship, the membrane voltage was stepped for 100 ms to various test values (V) between 50 and -150 mV in 20-mV decrements and returned to the holding potential (-50 mV). Averaged currents from three sweeps were low-pass filtered at 500 Hz by an 8-pole Bessel filter and digitized at 100 µs/point. In experiments to study the substrate and cation specificity (see Fig. 3and Fig. 4) the currents were continuously monitored on a chart recorder.


Figure 3: Substrate specificity of the rat SGLT1. A, sugar specificity. Continuous current record from a single rat SGLT1 cRNA-injected oocyte showing the sugar-induced inward currents as the membrane potential was held at -50 mV. The dashed line represents the base-line in 100 mM NaCl. At the time, indicated by the arrows 20 mM of each of the potential sugar substrates were added. The sugars were: D-glucose, L-glucose, 3-O-methyl-D-glucopyranose, alpha-methyl-D-glucopyranoside, D-galactose, myo-inositol, and beta-naphthyl-beta-D-glucopyranoside. Prior to the addition of the sugar the oocyte was equilibrated in 100 mM NaCl. After the sugar exposure it was rinsed in 100 mM choline chloride. B, nucleoside specificity. Uridine was tested at 50 mM alone, or in the presence of 400 µM alpha-methyl-D-glucopyranoside. Formycin B was tested at 10 mM.




Figure 4: Cation specificity of the sugar transport. Continuous current record showing the effects of Na and Li in alphaMDG on the inward currents mediated by the same rat SGLT1 cRNA-injected oocyte, described in Fig. 1. V was -50 mV and the oocyte was first perfused with choline (base line is indicated as a dashed line), followed by a 1-min equilibration in 100 mM solution of the appropriate cation (large arrows). At the time indicated by the small arrows, 25 mM alphaMDG was added. The substrate was continuously washed out until currents returned to the base line. The record for V = -50 mV is shown. The sugar-induced currents in the presence of lithium and sodium were -25 nA and -130 nA, respectively, and the leak currents were -8 and -20 nA.




Figure 1: Steady-states kinetics of the alphaMDG induced transport. A, membrane current records obtained before and after the addition of 400 µM alphaMDG to a cRNA rat SGLT1 injected oocyte. The oocyte was clamped at -50 mV. Presented traces are the response to voltage steps (30, -10, -50, -90, and -150 mV) applied for 100 ms. The traces were averaged from three sweeps. B, current-voltage (I-V) relationships of the steady-state currents induced by alphaMDG. The external Na concentration [Na] was fixed at 100 mM while the [alphaMDG] was varied (in mM: 0.015, 0.031, 0.062, 0.125, 0.250, 0.5, 1, 5, and 20). The I-V relationships were obtained as the difference of the steady-state currents in the presence and absence of alphaMDG. The box symbols represent the calculated maximal current (I). C, voltage dependence of the apparent affinity for alphaMDG (K). At each membrane potential (V) the alphaMDG-induced inward currents (I) were fitted to the equation: I = I [alphaMDG]/((K) + [alphaMDG]) where I is the apparent maximal current at saturating alphaMDG concentrations and K is the sugar concentration at 50% I. K was 0.2 mM at -150 mV, increased to 0.46 mM at -30 mV and 1 mM at -10 mV. I was -265 nA at -150 mV. Similar results were obtained in two other experiments.



Nonlinear regression analyses were performed using the software ENZFITTER (Elsevier-Biosoft, Cambridge, UK), and the fitting routines in Sigmaplot (Jandel Scientific, San Rafael, CA). The Marquardt-Levenberg algorithm was used by both programs.

Data presented in Fig. 3, Fig. 4, and Fig. 5A were carried out on the same oocyte. Similar results were obtained by repeating the experiments 2-4 times on oocytes from different donors.


Figure 5: Inhibition of the sugar induced currents by phlorizin. A, continuous current record illustrating the inhibition of the alphaMDG-induced currents by two different phlorizin concentrations. The dashed line represents the base line in 100 mM NaCl, the arrows show the time when the substrate or inhibitor were added. V was -50 mV. Steady-state currents were measured in 100 mM NaCl containing 5 mM alphaMDG. Addition of 5 µM phlorizin inhibited the maximal sugar-induced current 84% (-170 to -27 nA), whereas 50 µM phlorizin completely blocked the sugar-induced current. B, voltage independence of K. Shown are the calculated K in the presence of two concentrations of alphaMDG. For the inhibition at 5 mM alphaMDG the following concentration of inhibitor were added (in µM): 0.05; 0.1, 0.5, 1, 5, 10, and 20, and for the inhibition at 250 µM (in µM): 0.025, 0.05, 0.1, 0.5, 1, and 5. The current differences between the measured steady-state currents in 5 mM alphaMDG and those measured in each concentration of the inhibitor in 5 mM alphaMDG at each V were fitted to the equation described in Fig. 1C. Same calculations were repeated for the currents measured at 250 µM alphaMDG. Note that on reducing the sugar concentration from 5 mM to 250 µM, K decreased from 0.9 to 0.09 µM. For both curves error bars are mean of three oocytes. C, Dixon plots of the phlorizin inhibition of alphaMDG induced currents as function of different phlorizin concentrations. The obtained reciprocal currents (1/I) at each V for 0.4 mM or 5 mM alphaMDG at the following phlorizin concentrations (in µM: 0.01, 0.02, 0.05, 0.1, 0.25, and 0.5) were plotted against these phlorizin concentrations [Pz]. The straight lines fit the equation: 1/I = K[Pz]/I[alphaMDG]K+ 1/I(1+K/[alphaMDG]), where K is the apparent binding constant for substrate, I is the maximal current at the applied substrate concentration [alphaMDG], [Pz] is the applied concentration of the inhibitor. Lines obtained for different fixed concentrations of substrate have a different positive slope and cross at the point equivalent to the K for a competitive inhibitor. As an example we show the plot at V = -150 mV where the K was 12 nM.




RESULTS

Steady-state Kinetics

Fig. 1A (left panel) shows the current records from a rat SGLT1 cRNA-injected oocyte bathed in the 100 mM NaCl buffer in absence of sugar. The membrane potential was held at -50 mV and then stepped for 100 ms to test potentials (V(t)) of 30, -10, -50, -90, and -150 mV. The current relaxation during both the ON and OFF current responses consisted of the capacitive transient followed by a slower decay to the steady state. The slow decay is the presteady-state current of SGLT1 and has been observed in the human and rabbit intestinal Na/glucose cotransporters(8, 9, 10, 11) . Addition of alphaMDG (400 µM) to the bath solution generated an inward Na-current, and abolished the presteady-state current (Fig. 1A, right panel). The sugar-induced steady-state current and the presteady-state currents were not observed in noninjected oocytes.

The steady-state current-voltage (I-V) relationship of the sugar-induced current is the difference in steady-state current in the absence, and in the presence of alphaMDG. Fig. 1B shows a family of sigmoidal I-V curves obtained as [alphaMDG](o) increased from 31 µM to 20 mM. At each test potential (V(t)), increasing [alphaMDG](o) increased the sugar-induced current until saturation was reached at 5 mM. For each alphaMDG concentration, as the test potential was made more negative, the current increased for V(t) between 0 and -100 mV, and then became independent of membrane voltage.

The voltage dependence of the apparent K(0.5) for sugar (K) is shown in Fig. 1C. Between -150 and -50 mV, K was relatively insensitive to membrane voltage. However, between -30 and -10 mV, K increased steeply with depolarizing potentials, from 0.46 ± 0.03 at -30 mV to 1.0 ± 0.2 mM at -10 mV. The calculated maximal current I at -150 mV for this oocyte was -265 nA.

To determine the Na-dependence of the sugar-evoked currents, the steady-state inward currents were measured as [Na](o) was varied from 0 to 100 mM while [alphaMDG](o) was maintained at 5 mM. Fig. 2A shows the Na-dependent sugar-evoked current at V(t) = -30, -50, and -70 mV. At each V(t) the current was described by the Hill equation (see legend to Fig. 2B). There was a steep voltage dependence of the apparent affinity for sodium (K). K increased from 4 ± 0.6 mM at -150 mV to 40 ± 2 mM at -30 mV. The Hill coefficient (1.8 ± 0.3) was independent of voltage for V between -150 to -50 mV, and the I was -318 nA at -150 mV.


Figure 2: Na-activation of the steady-state sugar induced currents. A, dependence of the steady-state currents on the Na concentration. [Na] was varied between 0 and 100 mM (0, 5, 10, 20, 50, 70, and 100), [alphaMDG] was 5 mM. The alphaMDG-induced steady-state currents were measured as a function of [Na]. The measured inward currents corresponding to three different membrane voltages are shown. Curves were drawn according to the Hill equation (see B). B, for each test potential (V), the currents shown in A were fitted to the equation: I = I [Na]/((K) + [Na]). I is the maximal current at saturating Na concentrations, K is the value of [Na] at 50% I, and n is the apparent coupling coefficient for Na. K was more voltage-dependent at depolarizing membrane potentials. The values for K were 4 ± 0.6 mM at -150 mV and 40 ± 2 mM at -30 mV, but n remained independent on voltage. As an example, at V = -50 mV all three parameters were: I = -221 ± 2 nA; K = 26 ± 2 mM; n = 1.8 ± 0.3. The errors are errors of the fit. Almost identical findings were obtained from two additional oocytes.



Substrate and Cation Specificity

Fig. 3A shows the currents induced by various substrates with the oocyte membrane potential held at -50 mV. Since the experiment was performed on the same oocyte, the magnitude of the current induced by the different substrates indicates the relative affinity of the cotransporter for the substrates. The current induced byD-glucose, alphaMDG, and D-galactose were the largest (approx200 nA). The L-isomer of glucose is a poor substrate (5 nA). 3-O-Methyl-D-glucopyranose acts as a substrate with moderate activity (-160 nA). The Na/myo-inositol cotransporter shares high amino acid sequence homology (46% identity) to SGLT1(12) , and myo-inositol has been shown to be a substrate of rabbit SGLT1(13) . Fig. 3A shows that myo-inositol is not transported by the rat SGLT1. The Na/glucose cotransporter also shows high amino acid sequence homology to the Na/nucleoside cotransporter(14) . Competition experiments using radioactive tracers showed that uridine inhibited the uptake of alphaMDG, suggesting that the nucleoside may be a substrate (4) . We tested whether uridine and formycin B, substrates of the nucleoside transporter, are transported by rat SGLT1. Fig. 3B shows that both compounds are transported by rat SGLT1, but with very low affinity since the current generated by 50 mM uridine or 10 mM formycin B was only -7 and -11 nA, respectively. The low affinity for uridine transport by rat SGLT1 is also indicated by the observation that uridine caused a weak inhibition of the current induced by 400 µM alphaMDG (Fig. 3B, left panel).

Consistent with reports about the rat intestinal absorption of glucose-conjugated compounds (15) we observed beta-naphthyl beta-D-glucopyranoside induced Na inward currents. Addition of 20 mM of this compound induced about 18-20% (-50 nA at -150 mV) of the recorded currents induced by D-glucose (Fig. 3A).

The ability of the monovalent cations Li, K, Rb, and Cs to substitute for Na was also examined. NaCl in the 100 mM NaCl buffer was replaced isoosmotically by LiCl, KCl, RbCl, and CsCl and the currents induced by addition of 25 mM alphaMDG was measured. In the presence of 100 mM KCl, RbCl or CsCl, no detectable inward currents were generated by 25 mM alphaMDG, indicating that they cannot support sugar transport by rat SGLT1. Li was found to be able to support sugar transport (Fig. 4). There was an inward current upon substitution of LiCl for choline. Similar to the Na leak, this current was also blocked by 50 µM phlorizin (not shown), and indicates that there is a leak of Li by rat SGLT1 in the absence of sugar. This Li leak was approx50% of the Na leak. The current carried by Li in 25 mM alphaMDG was about 25% (-25 versus -130 nA, V(t) = -50 mV) of the current carried by Na, suggesting lower affinity for sugar in LiCl, as detected for rabbit SGLT1(16) . At V(t) = -150 mV, the alphaMDG-induced currents were -150 nA in 100 mM Li and -250 nA in 100 mM Na.

Phlorizin Sensitivity

Phlorizin is a high affinity competitive inhibitor of Na-dependent glucose transport in renal and intestinal epithelia (K approx 10 µM, 17). Fig. 5A shows that addition of 5 µM phlorizin into the bath medium inhibited the currents induced by a saturating concentration of alphaMDG (5 mM) by 85%. The inhibition was complete at 10 µM phlorizin (data not shown). Fig. 5B shows the voltage dependence of the K for the inhibition by phlorizin (K) studied at external alphaMDG concentrations of 5 mM and 0.25 mM. K was 0.9 µM at 5 mM sugar and decreased to 0.09 µM when the alphaMDG concentration was 250 µM.

To determine the inhibitor constant K(i) for phlorizin inhibition (K) we performed a series of Dixon plots. We plotted the reciprocal of the currents (1/I) against the phlorizin concentration. The lines in Fig. 5C were obtained by linear regression on phlorizin inhibition of the steady-state currents (V = -150 mV) generated by 1 mM and 0.4 mM alphaMDG. The lines intersect at a phlorizin concentration of -0.012 µM. Thus the inhibitory constant K is 0.012 µM (at -150 mV). It remained slightly voltage dependent in the range -150 mV to -50 mV, increasing to 0.053 ± 0.003 µM and 0.030 ± 0.010 µM at -70 mV and -50 mV. The errors are S.E. from three oocytes.

Presteady-state Charge Movements

In absence of sugar the Na/glucose cotransporter exhibits a presteady-state current after step changes in membrane voltage. The presteady-state current records from rat SGLT1 in Fig. 6A were obtained when the membrane voltage was stepped from the holding (-100 mV) to test voltages 50, -10, -50, and -150 mV. The presteady-state currents were completely blocked by 10 µM phlorizin (data not shown) and were not observed in noninjected oocytes.


Figure 6: Characterization of the presteady-state currents in the absence of sugar. A, presteady-state current records. The presteady-state current records were obtained by subtracting the capacitive (I(1)e) and the steady-state currents (I) from the total current as described in B, and in Loo et al.(8) . V was -100 mV. The traces at V = 50, -10, -50, and -150 mV are presented beginning 1 ms after applying the pulse. The inset shows the pulse protocol. B, kinetics of the presteady-state current relaxation. The time constants of relaxation for the ON and OFF current transients () for each tested membrane potential (V) were obtained by fitting the measured current (I) to the equation: I = I(1)e + I(2)e + I, where I(1) is the oocyte capacitive current with time constant (1), I(2) is the rat SGLT1 transient current with time constant (2), before decaying to the steady-state currents (I). V was -100 mV. C, charge-voltage relationship of the current transients. The integrals of the ON and OFF transient currents (Q) due to rat SGLT1 were plotted as a function of the applied test voltage V. The smooth curve was obtained by fitting the average () of these charges to the Boltzmann equation: (Q - Q) = Q(max)/[1 + exp(V - V(0.5))zF/RT]. Q(max) = (Q - Q) is the maximal charge transfer, Q and Q are the charge movements at the depolarizing and hyperpolarizing limits, F is the Faraday's constant, R is the gas constant, T is the absolute temperature, V(0.5) is the potential for 50% Q(max), and z is the apparent valence of the movable charge. Shown are the data from a single oocyte (V = -50 mV) with parameters: z = 0.85; V(0.5) = -46 mV, and Q(max) = 11 nanocoulombs.



The dependence of the relaxation time constant of the ON transients () on test voltage V(t) is presented in Fig. 6B. decreased monotonically from 13.5 ± 2 ms at -50 mV to 2.6 ± 0.1 ms at 50 mV. In the OFF response, was independent of the test voltage V(t) and was 53 ± 2 ms over the voltage range -50 to 50 mV. Error bars are S.E. from three oocytes. of the oocyte capacitive current ((1)) was independent of the membrane potential (0.6 - 0.8 ms).

Fig. 6C shows the dependence of the total charge (Q, integral of the current transients) on membrane voltage. The curve was drawn according to the Boltzmann relation (see legend to Fig. 6C) to estimate the parameters Q(max) (maximal charge transferred), z (apparent valence of the movable charge), and V(0.5) (voltage for 50% Q(max)). The maximal charge transferred is: Q(max) = Q - Q, where Q and Q are the charges transferred at the tested voltage limits. Since Q(max) depends on the level of expression, to compare oocytes with differing levels of expression, the data were normalized between 0 and 1 using the relation: (Q - Q)/Q(max). Mean values from five different oocytes were V(0.5) = -43 ± 3 mV, z = 1.0 ± 0.15, and Q(max) = 9.0 ± 2.5 nanocoulombs.


DISCUSSION

The archetypical member of the Na-dependent family of transport proteins is SGLT. SGLTs have been cloned from rabbit, rat, human, and pig. This family also includes the transporters for myo-inositol and nucleosides(18) . In this study, we characterized the kinetics of the Na/glucose cotransporter cloned from rat kidney. Our goal is to understand the structure-function relations of Na-dependent glucose transport by comparing and contrasting the kinetics of highly homologous proteins of this gene family.

Steady-state Parameters

The estimated apparent affinities for Na and glucose (K and K) for the rat SGLT1 show moderate differences compared to those of rabbit and human transporters for these ligands. Fig. 2B shows that in the voltage range more positive than -50 mV there is a steeper voltage dependence for the binding of the Na-ions (40 mM at -30 mV) to the rat transporter. This accounts for higher voltage sensitivity of the apparent K for alphaMDG in the same voltage range, shown in Fig. 1A.

Lee et al.(4) found that the K was 397 µM at -60 mV and our value of 300 µM is in agreement with the value reported. In this study, the steady-state current induced by alphaMDG was three times higher than the study of Lee et al.(4) , and we were able to obtain the voltage dependence of the K for sugar and sodium. The stoichiometry from the Hill analysis was 2 Na:1 sugar molecule, and is similar to that of the rabbit and human(9, 10) .

Lee et al.(4) also observed a K two orders of magnitude less than the value of 10 µM for rabbit SGLT1(9) . The K based on inhibition of 50 µM [C]alphaMDG uptake was 0.17 µM. Our estimate of the real inhibitory constant K was 0.012-0.03 µM. It was recently observed(19) , that the Na-dependent glucose transport system in sheep tracheal epithelium also has a high affinity for phlorizin (K 0.020 µM). The species differences in the affinity to phlorizin observed here are almost certainly due to differences in the amino acid sequence. For the rat SGLT1, the estimated real K can also be regarded as binding/dissociation constant and used in future determination of the number of phlorizin molecules binding per cotransporter molecule. The rat, rabbit, and human clones all exhibit a phlorizin sensitive Na leak current, which is about 15-20% of the maximal alphaMDG-induced current.

Presteady-state Parameters

In the rat SGLT1, as in the rabbit (11, 20) and human (8) Na-dependent glucose transport systems, we observed transient charge movements in sugar free solutions which were completed within 50 ms. These currents were abolished in all three clones by either addition of sugar substrates or the competitive inhibitor phlorizin. The estimated Q-V curves and the resulting parameters (z, Q(max), and V(0.5)) give the functional relation between the moved charge and the membrane potential. z is the average number of net elementary charges (q) apparently moved through a distance () across the membrane (field) in each transporter molecule. This means, that either a single elementary charge moves completely through the membrane electric field, or two elementary charges each move 50% of this distance, or any other combination of negative or positive charges move such that q(i)(i) = 1. The voltage dependence of the charge movement is shown in Fig. 6C, where Q is plotted as a function of the test potential for the rat SGLT1, and is compared for all three cotransporters in Fig. 7A. There is a displacement of about 40 mV to more negative potentials of the Q-V curves for the rat/human transporters in comparison to the rabbit. Table 1shows a comparison of the parameters (z, Q(max), and V(0.5)) from fitting the Q-V relations to the Boltzmann equation. The apparent valence of the movable charge is the same for all three transporters (z 1). V(0.5), the voltage at 50% Q(max), was similar (approx-40 mV) for the rat and the human clones, whereas the rabbit clone was about 40 mV more positive. Fig. 7B presents the time constants () of the presteady-state current relaxation of the three clones in the membrane voltage range -50 to 50 mV. for the ON currents of human or rat SGLT1 transients decrease progressively as the test voltage was made more positive. In contrast, for the rabbit SGLT1 transients increased, reached a maximum at 10 mV, and decreases with more depolarizing potentials. Compared to the -V curve of the rabbit, the curves of the human/rat transporters are shifted approx50 mV to more negative potentials.


Figure 7: Comparison of the presteady-state current due to rat, rabbit and human SGLT1 transporters. Data for rabbit SGLT1 () are based on the estimates shown in Fig. 3, A and B, of Panayotova-Heiermann et al.(11) , data for human SGLT1 () are taken from Fig. 3B of Loo et al.(8) . Data for rat SGLT1 (circle) were obtained from a single oocyte as described in Fig. 6.





The maximal charge Q(max) depends on the level of expression of SGLT1 in the membrane since Q(max) = qzC(T), where C(T) is the total number of transporters. The maximal steady-state inward Na current induced by saturating sugar concentrations (I(max)) is proportional to Q(max)(8, 21) . I(max) = kqzC(T), where k is the apparent turnover number of the transporter. k for rat SGLT1 was 30 s and comparable to that of the human and rabbit (Table 1).

Kinetic Model

The mechanism of Na-dependent sugar transport via rat SGLT1 can be explained by a six-state ordered nonrapid equilibrium kinetic model with mirror symmetry similar to the proposed models for the rabbit (20) and human (8) transporters. For reviews see Wright et al.(21, 22) . The model assumes that (i) the transporter has a valence of -2; (ii) on both membrane surfaces, the transporter can be empty [C], loaded with Na [CNa(2)], or fully loaded with Na and sugar [SCNa(2)]; (iii) the transporter binds two Na-ions before binding sugar; (iv) C, CNa(2), and SCNa(2) can cross the membrane; membrane voltages affect Na binding to the transporter, and translocation of the empty transporter across the membrane. Simulations predict that for rabbit SGLT1 at -50 mV 85% of the transporter molecules in the membrane are bound to Na and facing out ([CNa(2)](o)), 5% are empty and facing out ([C](o)), and 10% are facing in ([C](i)). According to our kinetic model, the presteady-state currents observed after a depolarizing voltage step are due to the charge transfer involved in the dissociation of external Na and the reorientation of the unloaded SGLT1 protein in the membrane:

Computer simulations resulted in a set of rate constants which account quantitatively and qualitatively for the observed presteady- and steady-state kinetics. The results suggest that differences in the kinetics between the rabbit and rat/human cotransporters are due to differences in k and k (Table 2). Such changes in the rate constants must be due to differences in structure between the isoforms. Aligning the primary amino acid sequences show that there are different residues at 129 out of 665 positions, and, when conservative substitutions were taken into account (K = R; S = T; D = E; Y = F = W; and I = V = L = M) this reduces to differences at 76 positions. These 76 are evenly distributed between the N- and C-terminal halves of the protein (Fig. 8), and are mostly confined to hydrophobic loops between the putative transmembrane helices. The cytoplasmic hydrophilic N-terminal and the external loops between helices 5/6 and 11/12 contain 43 of the 76 nonconserved residues.




Figure 8: Sequence alignment of the human, rat, and rabbit SGLT1 cotransporters. The full sequence is given for the human SGLT1, for the rat and rabbit cotransporters only the nonconserved residues are included. Identical and residues similar to that in human (D = E, R = K, S = T, I = V = L, and Y = F = W) are replaced by dashes(-). The rabbit residues that are significantly different from those in rat and human, i.e. are polar in either rabbit or rat and human, are shown in bold italics. The location of the putative transmembrane domains is indicated by the lower case letters and underlined (). The N- and C-terminals in this secondary structure model (7) are placed on the cytoplasmic side of the plasma membrane.



A clue about the residues that may be important in determining kinetic differences comes from consideration of the residues that are identical in pairs of the three transporters. There are 25 residues shared between human and rat, 17 shared between human and rabbit, and 21 between rabbit and rat. Overall, there are 37 residues that are different between the rabbit and the rat and human, and 25 of these are polar (indicated in bold type in Fig. 8). At 10 positions the residues are charged in either the rabbit or in the human and rat, at 7 positions the residues are serines or threonines in either rabbit or human and rat, and at 5 other positions the residues are polar in either rabbit or human and rat. The polar residues are mostly in hydrophilic segments, and half are clustered in external loops between helices 5/6 and 11/12. There are no significant differences between the residues in putative transmembrane domains of the cotransporters (in transmembrane helix 4 the substitution 176DN is of no functional significance)(11) . This suggest that the polar residues in hydrophilic domains of the protein play an important role in determining differences in kinetics between species, k and k, by determining the three-dimensional protein structure through electrostatic interactions. This could be tested by examining functional properties of clones after either mutating the polar residues or swapping hydrophilic loops between species.

Our conclusion is that the kinetic differences between the human, rat, and rabbit transporters primarily are due to two partial reactions involving binding/dissociation of Na ions and translocation of the empty carrier. These differences are probably due to polar residues clustered between helices 5/6 and 11/12.


FOOTNOTES

*
This research was supported by National Institutes of Health Grant DK4460. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Physiology, UCLA School of Medicine, 10833 Le Conte Ave., Los Angeles, CA 90095-1751. Tel.: 310-825-6905; Fax: 310-206-5661; mariana@physiology.medsch.ucla.edu.

(^1)
The abbreviations used are: SGLT, sodium-dependent cotransporter for D-glucose; alphaMDG, alpha-methyl-D-glucopyranoside; Pz, phlorizin, phloretin-2`-beta-D-glucoside.


ACKNOWLEDGEMENTS

We thank Dr. M. Hediger for supplying us with the DNA-construct encoding for the rat SGLT1, Dr. B. Hirayama for suggestions on the manuscript, Dr. E. Turk for sequence alignments, and M. Contreras for assistance with the oocytes.


REFERENCES

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