Conformational Dynamics of hSGLT1 during Na⁺/Glucose Cotransport

The Na⁺/glucose cotransporter, SGLT1, is a member of a large family of proteins (SLC5) that uses the Na⁺ electrochemical gradient to transport substrates (sugars, amino acids, osmolytes, neurotransmitters, ions, and water) against their concentration gradients. We have proposed that the protein functions by an alternating access mechanism, and a simplified six-state ordered kinetic model has been proposed (Parent et al., 1992b; Loo et al., 1998). In previous studies, we characterized the voltage-dependent partial reactions of SGLT1 in the absence of glucose using charge and fluorescence measurements on an hSGLT1 mutant Q457C where sugar transport is abolished after labeling of Cys457 by the fluorophore tetramethylrhodamine-6-maleimide (TMR6M) (Loo et al., 1998, 2005; Meinild et al., 2002). Our conclusion was that the presteady-state currents (in the absence of glucose) are associated with Na⁺ binding, and isomerization of the empty carrier involves a series of four conformational changes (Loo et al., 2005).

The goal of the present study is to examine the conformations of the transporter under sugar transport conditions. Our kinetic model predicts that the conformational occupancy distribution of the transporter depends on the sugar concentration. We expressed hSGLT1 mutant G507C in Xenopus laevis oocytes and recorded changes in transporter currents (steady state and presteady state) and rhodamine fluorescence after step jumps in membrane voltage. The mutant transporter has similar steady-state and presteady-state kinetics as hSGLT1, before and after labeling of Cys507 by TMR6M. The charge and fluorescence measurements provide independent and complementary views of protein conformational changes. Our model predicts that the charge movements in the Na⁺/glucose cotransport cycle are associated with external Na⁺ binding and translocation of the empty transporter, whereas changes in fluorescence could originate from any (or all) of the conformational transitions of the transport cycle. By using a fully functional protein, we are now able to study the partial reactions involved in sugar binding and translocation. We expected that the fluorescence measurements would reveal conformations not detected by charge measurements. Our data do uncover a novel, slow conformational change with sugar transport that is not associated with charge movement, and simulations indicate that this is due to a slow electroneutral step in the transport cycle associated with the release of Na⁺ from SGLT1 in the internal membrane surface. We found that sugar shifted the conformations of SGLT1, resulting in a reduction in the occupancy probability in the outward-facing, and increasing the occupancy probability in the inward-facing, Na⁺-bound conformations. Phlorizin, the high affinity inhibitor, locked the transporter in the phlorizin-bound form and proportionally reduced the occupancy probabilities in all other conformations.

Mature Xenopus laevis oocytes were isolated, defolliculated, and injected with hSGLT1 or hSGLT1 mutant G507C cRNA (see Loo et al., 1993, 1998). The G507C mutant was constructed using standard PCR techniques and confirmed by DNA sequencing. Oocytes expressing hSGLT1 G507C were labeled with TMR6M (Invitrogen; Loo et al., 1998; Meinild et al., 2002). We determined the maximal fluorescence change (ΔF_max, see below) as a function of the incubating [TMR6M]_o (from 1 to 100 μM) and duration of labeling (from 1 to 30 min), and found that the extent of labeling of Cys 507C by the fluorophore was saturating in 100 μM [TMR6M]_o for 10 min in NaCl buffer at the normal resting membrane potential. We assume that all the functional transporters are labeled. After labeling, oocytes were washed free of dye and kept in NaCl buffer in the dark until use. MTSEA (2-aminoethyl methanethiosulfonate hydrobromide; Toronto Research Biochemicals) was prepared and used as previously described (Loo et al., 1998). Oocytes were bathed in a NaCl buffer containing (in mM) 100 NaCl, 2 KCl, 1 CaCl₂, 1 MgCl₂, 10 HEPES, pH 7.4. αMDG or phlorizin was added to the superfusing NaCl buffer at the reported concentrations. Na⁺ concentration was varied by equimolar replacement of Na⁺ with choline.

Sugar uptake into oocytes was measured using 50 μM ¹⁴C-α-methyl-d-glucopyranoside (¹⁴C-αMDG, Amersham Biosciences), a nonmetabolized sugar analogue that is transported by human SGLT1 (see Ikeda et al., 1989). The initial rates of sugar uptake into oocytes were measured at 20–23°C for 10–60 min. Experiments were repeated at least twice on oocytes isolated from different donor frogs. Uptake was expressed in pmol per oocyte per minute (mean ± SEM).

Electrophysiological and fluorescence experiments were performed simultaneously, using two-electrode voltage clamp fluorometry (Loo et al., 1998, 2005; Meinild et al., 2002). A standard pulse protocol was applied where membrane potential was held at −50 mV (V_h) and stepped to various test potentials (V_t from +90 to −150 mV in 20 mV decrements) for 100 (or 500) ms before returning to V_h. The current records were the averages of three sweeps, and the fluorescence records were averages of either 3 or 10 sweeps. To minimize the effects of photobleaching, the 500-ms fluorescence records were averages of three sweeps. Records were filtered at 500 or 50 Hz, depending on the sampling interval (0.1 or 0.5 ms per sample). Note that in the present studies, we do not record the fast components of ΔF observed in our previous study using cut-open oocyte voltage clamp fluorometry (Loo et al., 2005). Fluorescence intensity is expressed as arbitary units (au). In TMR6M-labeled oocytes expressing mutant G507C bathed in NaCl buffer, ΔF_max/F_total is ≈1%, where ΔF_max is the maximal fluorescence intensity change and F_total is the total fluorescence intensity. Fluorescence data have been corrected for photobleaching and rundown (Meinild et al., 2002). All experiments were performed at room temperature (20–23°C).

To obtain the current–voltage (I-V) relations of the sugar-induced current, the pulse protocol was applied with 100-ms pulses. The sugar-induced current was obtained by subtracting the current in NaCl buffer (measured at 100 ms) from the current with sugar (αMDG) added to the external solution. The substrate-induced currents (at each voltage) were fitted to the equation

where I_max is the maximal current, [S]_o is the external substrate (αMDG or Na⁺) concentration, K_0.5 is the half-maximal substrate concentration ([S]_o at 50% I_max), and n is the Hill coefficient. For the kinetics of sugar activation, n was fixed at 1 (Birnir et al., 1991; Parent et al., 1992a; Mackenzie et al., 1998).

Total membrane current in response to a voltage pulse consisted of the bilayer capacitive transient, the presteady-state currents of SGLT1, and the steady-state currents. In the absence of external sugar, the steady-state currents consisted of the background (endogenous) currents of the oocyte and the Na⁺ leak (uniporter mode) mediated by SGLT1 (Parent et al., 1992b; Loo et al., 1998). We found previously that the presteady-state currents of hSGLT1 (in the absence of external sugar) contained three components, with time constants (τ) ranging from fast (0.2–1.5 ms), medium (3–20 ms), and slow (30–160 ms), and the method of isolating the three components has been described in detail (Loo et al., 2005). The focus of this study is the medium component, as the fast component is beyond the resolution of the two-electrode voltage clamp (≈1 ms). Since the kinetics of the medium component (τ-V and Q-V relations) obtained from records corrected for the slow component (describe above) are similar to those obtained from uncorrected records (Loo et al., 2005), the medium component was estimated from uncorrected records using 100-ms voltage pulses (Loo et al., 1993; Hazama et al., 1997; Quick et al., 2001; Meinild et al., 2002). In these experiments, total current relaxation (I_tot) was fitted to the equation

where I_ss is the steady-state current, I_cmexp(−t/τ_cm) is the bilayer capacitance current with initial value I_cm and time constant τ_cm, and I_medexp(−t/τ_med) is the medium component of SGLT1 presteady-state current with initial value I_med and time constant τ_med. The medium component of presteady-state current (I_medexp(−t/τ_med) was isolated by subtraction of the steady-state (I_ss) and membrane bilayer capacitance (I_cmexp(−t/τ_cm)) from the total current. The medium charge movement associated with SGLT1 was obtained from the integral of the medium component of presteady-state current. This analysis was extended to the presteady-state current (or carrier transients) in the presence of sugar. At steady state, the current (I_ss) consisted of the oocyte background current, Na⁺-uniporter current (of SGLT1), and the Na⁺/sugar cotransport current.

In experiments where the slow and medium components were isolated (Fig. 2), 100- and 500-ms pulses were applied (Fig. 2, A and B). The slow time constant (τ_slow) was estimated from the 500-ms current records after the medium component has decayed (the period was five times the time constant of the medium component, see Fig. 2 of Loo et al., 2005 for detailed description). The early phase was obtained by extrapolation of the exponential fit to the peak of the capacitive transient, typically two sample points after onset of the voltage pulse. The slow charge was obtained from the integral of the slow component of presteady-state current. The medium charge was then estimated using 100-ms pulses after subtraction for the slow component and steady-state current.

The charge vs. voltage (Q-V_m) relations for medium and slow charge could, to a first approximation, be fitted to a single Boltzmann function (Loo et al., 1993; Hazama et al., 1997):

where Q_max = Q_dep − Q_hyp, Q_dep and Q_hyp are the Q (absolute value) at depolarizing and hyperpolarizing limits, V_m is membrane potential, F is the Faraday, R is the gas constant, T is the absolute temperature, V_0.5 is the membrane potential at 50% Q_max (or the midpoint voltage), and zδ is the maximum steepness factor for the dependence of Q on voltage, or the apparent valence of the voltage sensor. zδ is the product of the apparent valence of the movable charge (z), and the fraction of the membrane electric field traversed by the charge (δ). We denote zδ_Q for zδ obtained from charge movement.

The Boltzmann relation was also used to empirically fit the dependence of the change of fluorescence intensity (ΔF) on membrane voltage (Loo et al., 1998; Meinild et al., 2002). The parameters obtained were the maximal fluorescence intensity change (ΔF_max), the membrane voltage at 50% ΔF_max (⁠

Fits of data to equations were performed using either Sigmaplot 2002 (SPSS), or Clampfit 8.1 (Axon Instruments, Inc.). On data obtained on a single oocyte, the statistics are given by the estimates and the standard error of the fit. When data is from a population, the statistics are given by the means and standard errors of the means with the number of samples. While data are shown for representative experiments, all experiments were performed on at least three oocytes from different batches.

The differential equation for the eight-state model for Na⁺/glucose cotransport (Fig. 9) is shown as Eq. 4 (below). C_i is the occupancy probability in state i, and C₁ + C₂ + C₃ + C₄ + C₅ + C₆ + C_a + C_b = 1. Excluding the substrate-binding reactions, the rate constants k_ij for transitions from C_i → C_j are given by k_ij = k_ij^o exp(−ε_ijF/RT), where k_ij^o is a voltage-independent rate, ε_ij is the equivalent charge movement (up to the transition state from C_i → C_j), and F, R, and T have their usual physicochemical meanings (Parent et al., 1992b). Na⁺ and sugar binding to the protein on the external and internal membrane surfaces is represented by pseudo-rate constants k₁₂ = k₁₂^o [Na]_o² exp(−ε₁₂FV/RT), k₂₃ = k₂₃^o[αMDG]_o, k₆₅ = k₆₅^o[Na]_i², and k₅₄ = k₅₄^o[αMDG]_i. There are four components of presteady-state current associated with voltage-sensitive reactions (shaded region of Fig. 9): C₂⇄C₁, C₁⇄C_a, C_a⇄C_b, and C_b⇄C₆. The presteady-state current (I_ij) due to C_i⇄C_j was calculated by I_ij = e(ε_ij + ε_ji)(k_ijC_ij − k_jiC_j), where e is the elementary charge (Parent et al., 1992b; Loo et al., 2005). The total SGLT1 current (I) is I = N_T (I₁₂ + I_1a + I_ab + I_b6), where N_T is the total number of transporters in the oocyte plasma membrane. The simulations shown in Figs. 10–13,¹¹¹² and Table I were performed for wild-type hSGLT1 and TMR6M-labeled mutant G507C at 20°C using the parameters of Table II with [Na⁺]_o = 100 mM, [Na⁺]_i = 5 mM, [αMDG]_i = 0, and N_T = 10¹⁰ transporters. All the simulations were performed at 0–100 mM [Na⁺]_o and 0–100 mM [sugar]_o.

External phlorizin was assumed to bind to SGLT1 in the Na⁺-bound conformation (Parent et al., 1992b): [CNa₂]′W[CNa₂Pz]′, thus in the presence of phlorizin, there are nine states (Fig. 9). Simulations of the phlorizin-binding model (using the rate constants of Table II) were performed in a similar manner to the eight-state model, with modifications to include the phlorizin-bound state [CNa₂Pz]′(C₇): C₁ + C₂ + C₃ + C₄ + C₅ + C₆ + C_a + C_b + C₇ = 1; d/dt C₇ = −k₇₂C₇ + k₂₇C₂ ; and d/dt C₂ = −(k₂₁ + k₂₅ + k₂₃ + k₂₇)C₂ + k₁₂C₁ + k₅₂C₅ + k₃₂C₃ + k₇₂C₇.

Computer simulations were performed using Berkeley Madonna 8.0.1. The voltage pulse protocol was simulated by determining the initial occupancy probabilities at the holding potential (V_h = −50 mV), and as membrane potential was stepped to each test value (V_t ranging between +50 and −150 mV), the time course of the occupancy probabilities, cotransporter currents, and fluorescence (ΔF) were obtained by numerically integrating the differential equation (Eq. 4) using the Runge-Kutta Method. Steady-state kinetic parameters were simulated by generating the I-V relations as functions of [Na⁺]_o and [sugar]_o. Kinetic parameters I_max, K_0.5, and n (at each V_m) were obtained by fitting the I vs. [sugar]_o (or [Na⁺]_o) relations to Eq. 1.

For presteady-state simulations, the transient cotransporter currents for the ON and OFF responses at each test voltage (V_t) were integrated to obtain the charge (Q). The Q vs. V_t relations were fitted with the Boltzmann relation (Eq. 3) to obtain the maximal charge (Q_max), apparent valence of the voltage sensor (zδ_Q), and midpoint voltage (V_0.5). The eigenvalues of the matrix (Eq. 4) were obtained using MATLAB 6.0 (The MathWorks Inc.). The time constants were the reciprocals of the eigenvalues (Loo et al., 2005).

Fluorescence experiments were simulated by assuming that changes of fluorescence intensity (ΔF) with step jumps in membrane voltage are due to changes in occupancy probabilities: ΔF ≈ qy₁ΔC₁ + qy₂ΔC₂ + qy_aΔC_a + qy_bΔC_b + qy₃ΔC₃ + qy₄ΔC₄ + qy₅ΔC₅ + qy₆ΔC₆, where qy_j is the apparent quantum yield of the fluorophore (TMR6M) when SGLT1 is in conformation C_j. The simulations were performed with qy₁ = 3, qy₂ = 1, qy_a = 3, qy_b = 3, qy₄ = 3, qy₅ = 3, and qy₆ ≈ 5–6. In practice, ΔF was determined by the relative quantum yields qy₅/qy₂ ≈ 3, and qy₆/qy₂ ≈ 6 (see Discussion below).

The Dixon analysis (for the determination of the inhibitory constant K_i for phlorizin, Table I) was simulated by determining the intersection of the straight lines−1/I vs. [phlorizin]_o relations at [αMDG]_o = 1 and 2 mM, where I is the sugar-coupled current (at −150 mV) at each [phlorizin]_o (see Panayotova-Heiermann et al., 1995). [Phlorizin]_o used was 0, 50, 100, 200, 500, 1,000, 5,000, and 10,000 nM.

We first briefly describe the steady-state kinetic properties of hSGLT1 G507C, and the effects of MTSEA and TMR6M. The kinetics of the presteady-state currents in the absence of glucose and the fluorescence changes of the TMR6M-labeled mutant protein with step jumps in membrane voltage are then presented. These provide the basis for describing the conformations of SGLT1 under sugar-transporting conditions by charge and fluorescence measurements (Part II).

The rates of ¹⁴C-αMDG (50 μM) uptake into oocytes were comparable for oocytes expressing the mutant transporter and wild-type hSGLT1. In a representative experiment on the same batch of oocytes (each determination being the mean of 8–10 oocytes and expressed as the mean ± SEM), in the presence of external Na⁺ (100 mM), uptake by oocytes injected with hSGLT1 G507C-cRNA was 143 ± 8 pmol/oocyte/h versus 176 ± 10 pmol/oocyte/h for oocytes injected with wild-type hSGLT1-cRNA. This uptake rate was 100-fold greater than that of noninjected oocytes (1.4 ± 0.1 pmol/oocyte/h). Uptake of αMDG by hSGLT1 G507C was dependent on external Na⁺ and blocked by the SGLT1-specific inhibitor phlorizin. When external Na⁺ was replaced by choline, the rate of sugar uptake (1.6 ± 0.1 pmol/oocyte/h) was similar to that of noninjected control oocytes (0.6 ± 0.1 pmol/oocyte/h). Phlorizin (500 μM) in the external solution also reduced the rate of ¹⁴C-αMDG uptake by the mutant to that of controls.

The total current from an oocyte expressing hSGLT1 G507C in NaCl buffer is shown in Fig. 1 A. Membrane potential (V_m) was held at −50 mV (V_h) and stepped to a series of test values for 100 ms (from +50 to −150 mV) before returning to V_h. The current relaxation consisted of an initial membrane capacitive transient (time constant τ ≈ 0.8 ms) followed by the decay of the presteady-state current to steady state (with τ ≈ 3–20 ms) (Loo et al., 1993, 2005). The current records when 1 mM αMDG was added to the bathing medium are shown in Fig. 1 B. At each test voltage, αMDG increased the steady-state current, and there was a shift in the profile of the presteady-state current records. This is particularly noticeable in the OFF response when the test voltage was returned to V_h (the effect of sugar on presteady-state current will be presented below).

The current vs. voltage (I-V) relation of the αMDG-induced current tended toward saturation at large negative membrane voltages (−150 mV) and approached zero at +50 mV (Fig. 1 C). At each voltage, the I vs. [αMDG]_o relation was hyperbolic (e.g., Fig. 1 D shows the relation at −50 mV). The half-maximal concentration for αMDG (⁠

The dependence of the αMDG-induced current on [Na⁺]_o was sigmoid. The Hill coefficient was 1.5 and independent of membrane voltage (unpublished data). Fig. 1 F shows the dependence of the half-maximal concentration for Na⁺ (⁠

The rate of ¹⁴C-αMDG uptake for oocytes expressing hSGLT1 mutant G507C preincubated in MTSEA (1 mM for 15 min in NaCl buffer) was 81 ± 14% (n = 5) of the control nontreated oocytes, indicating that the mutant hSGLT1 G507C retained the ability to transport sugar after Cys507 was modified by MTSEA. We confirmed that the Cys507 was derivatized by MTSEA from the reduction in maximal fluorescence change (ΔF_max, see Fig. 3 below) after the oocytes were preincubated in the reagent. In four experiments, compared with control nonpreincubated oocytes, ΔF_max (measured with the oocytes bathed in NaCl buffer) was reduced 93% (from 9.1 ± 1.0 au to 0.06 ± 0.14 au). In control experiments on oocytes expressing wild-type hSGLT1, exposure to MTSEA had no effect on the kinetics of hSGLT1 (see also Loo et al., 1998), and after labeling by TMR6M, fluorescence changes induced by voltage jumps were not observed in noninjected oocytes and hSGLT1-expressing oocytes (unpublished data).

The affinity for sugar was unaffected by either MTSEA or TMR6M. In representative experiments on the same oocytes (at −50 mV),

We have previously found that in two-electrode voltage clamp experiments on hSGLT1, the presteady-state currents contained medium and slow components with time constants of 3–20 and 30–100 ms (Loo et al., 2005). Similar time constants were also observed in mutant hSGLT1 G507C (Fig. 2). Like wild-type hSGLT1, the transient currents of the mutant did not quite reach steady state at 100 ms (Fig. 2 A), and there was a slow decay to steady state revealed in the 500-ms pulses (Fig. 2 B).

The τ-V relation for the medium component is shown in Fig. 2 C. For the ON pulse, when membrane potential was stepped from V_h to V_t, τ_med was 20 ± 1 ms at −150 mV and decreased to 4.0 ± 0.2 ms at +50 mV (filled symbols). For the OFF pulse, when V_t was returned to V_h, τ_med was independent of the test voltage (open symbol), and was 12 ± 1 ms (n = 10). The medium charge transfer (Q_med), obtained by integrating the medium component of presteady-state current, was the same for the ON and OFF. The Q_med-V relation is shown in Fig. 2 D. The line was the fit of the data by a Boltzmann relation with a maximal charge (Q_max) of 12 nC, apparent valence (zδ_Q) of 1.0, and midpoint voltage (V_0.5) of −44 mV.

The τ-V relation for slow charge was similar to the medium (Fig. 2 C). τ_slow for ON was maximal at hyperpolarizing voltages (τ = 70 ms at −150 mV) and decreased as the test voltage was made positive (τ = 15 ms at +50 mV). τ_slow for OFF was also independent of voltage and was 39 ± 5 ms (n = 10). The Q_slow-V curve obeyed the Boltzmann relation with a Q_max of 7 nC, zδ_Q of 1.0, and a V_0.5 of −62 mV (Fig. 2 E). When the medium and slow charges were added to obtain the total charge (Q_total), the Q_total-V curve followed the Boltzmann relation with a Q_max of 19 nC, zδ_Q of 1.0, and V_0.5 of −50 mV (Fig. 2 F).

We focused our attention on the medium component and estimated medium charge from 100-ms pulses uncompensated for slow charge. Previously we found the τ_med-V and Q_med-V relations obtained from current records corrected for slow charge were similar to those obtained from uncorrected records (Loo et al., 2005). For the medium component, the maximal charge (Q_max) ranged from 5 to 30 nC, depending on the level of expression of mutant G507C in the oocyte plasma membrane. zδ_Q was 1.0 ± 0.1 (n = 6). V_0.5 ranged between −30 and −47 mV, with a mean of −38 ± 3 (n = 6).

TMR6M did not affect the kinetics of the presteady-state currents. In a representative experiment on the same oocyte where we monitored the medium charge movement before and after TMR6M, zδ_Q, V_0.5, and Q_max were 0.9 ± 0.1, −30 ± 1 mV, and 18 ± 1 nC before, and 0.9 ± 0.1, −28 ± 1 mV, and 18 ± 1 nC after TMR6M. The population means for zδ_Q and V_0.5 after labeling by TMR6M were 1.0 ± 0.1 (n = 6), and −35 ± 3 mV (n = 6). V_0.5 ranged between −28 and −45 mV. The τ-V relations (for medium charge) were the same before and after labeling by TMR6M (unpublished data).

The time course of the fluorescence signal (ΔF) from a TMR6M-labeled hSGLT1 G507C-expressing oocyte in response to a series of voltage pulses (from V_h −50 mV) is shown in Fig. 3. Fluorescence intensity increased with depolarizing voltages and decreased with hyperpolarizing voltages. Fluorescence intensity returned to baseline when the test pulse was stepped back to V_h. Like charge movement, the fluorescence records consisted of medium and slow components. The slow component was much more apparent in the depolarizing direction, especially the OFF response (Fig. 3, A vs. B).

The medium and slow components (ΔF_med and ΔF_slow) at each voltage were isolated by fitting the time course of the 500-ms fluorescence records (ΔF_total) with a sum of two exponential functions, and ΔF versus voltage relations for medium and slow components were obtained. The ΔF_med-V and ΔF_slow-V were sigmoidal and were fitted by the Boltzmann relation (Fig. 3, C and D). zδ_F and

The main difference between the medium and slow components was their amplitude. Maximal fluorescence change for the medium component (ΔF_max^med) was 64 ± 1% (n = 7) of the total maximal fluorescence change (ΔF_total^max).

The dependence of the relaxation time constants for ΔF on voltage differed between medium and slow components. For medium, τ was 15 ± 1 ms at −150 mV and decreased to 8.7 ± 0.1 ms at +90 mV for ON (filled symbols, Fig. 3 E). For OFF, τ was independent of test voltage (open symbols, Fig. 3 E) and was 14.4 ± 0.3 ms (n = 12). For slow ΔF (Fig. 3 F), τ (139 ± 8 ms, n = 10) was relatively independent of voltage, for ON and OFF τ = 150 ± 14 ms.

Sugar increased the steady-state current (due to Na⁺/sugar cotransport, see Fig. 1) and shifted the profile of the presteady-state currents. Fig. 4 A shows the total current records for the OFF-pulse when membrane potential was stepped from various test values (V_t, from +50 to −150 mV) back to the holding potential (−50 mV) from an oocyte in 0, 1, and 10 mM αMDG. The most pronounced effect of sugar was the reduction in presteady-state current with hyperpolarizing voltages. For example, at 10 mM [αMDG]_o, the OFF currents for hyperpolarizing voltages was completely inhibited (Fig. 4 A). As in the absence of sugar, the ON and OFF charges were equal and opposite, and the Q-V relations at each [αMDG]_o fitted the Boltzmann relation (unpublished data). With increasing [αMDG]_o (from 0 to 100 mM), there was a reduction and eventual elimination in the maximal charge (Q_max) with a K_0.5 of 1.1 mM (Fig. 6 and Fig. 7 A). The midpoint voltage (V_0.5) shifted to more positive values (maximal shift was +60 mV, from −45 mV in NaCl alone to +15 mV in 10 mM αMDG) with a K_0.5 of 1.3 mM (Fig. 7 C). zδ_Q (=1.0) was unaffected by αMDG.

The fluorescence records were also dependent on αMDG (Fig. 5). As [αMDG]_o increased, the amplitudes of the fluorescence changes (ΔF) decreased at every test voltage, but in contrast to the presteady-state current, ΔF was not abolished at saturating [αMDG]_o. This is illustrated in Fig. 6, which compares the current and fluorescence records in 100 mM [αMDG]_o. Compared with the maximal fluorescence change (ΔF_max) in NaCl buffer alone (Fig. 6 A), ΔF_max at 100 mM [αMDG]_o (Fig. 6 B) was only reduced 50%, whereas Q_max was reduced more than 95%. The K_0.5 for the reduction in ΔF_max with [αMDG]_o was 1.1 ± 0.2 mM, and was similar to the K_0.5 for the reduction of Q_max (Fig. 7 A). V_0.5 for ΔF shifted to more positive values with [αMDG]_o. The K_0.5 (1.3 mM) was also similar to the K_0.5 for the shift of the V_0.5 for charge (Fig. 7 C). zδ_F (=0.8), as zδ_Q (=1.0), was unaffected by αMDG.

The relaxation time constants (τ) of the medium component of the presteady-state current showed a small decrease with increasing [sugar]_o, but only at hyperpolarizing voltages. For example, we determined τ in the presence and absence of 0.1 mM aMDG. In the case of G507C, there was no significant change in the ON and OFF time constants at either at +30 or −130 mV (τ_ON was 5.1 ± 0.4 at +30 mV, and 13.4 ± 0.6 ms at −130 mV, n = 4; and τ_OFF was 10.5 ± 0.4 ms, n = 6). For wild-type hSGLT1, there was also no significant change in τ_ON at +30 mV, but there was a reduction from 19 to 14 ms at −130 mV. At higher sugar concentrations we were unable to accurately determine the time constants of the medium component in the hyperpolarizing direction or the slow component owing to the reduction of the presteady-state current (Fig. 7 A) and the positive shift in V_0.5 (Fig. 7 C).

The time constant (τ) of the medium component of ΔF was also unaffected by αMDG. On average, for ON, at +30 mV, τ was 10.7 ± 0.2 ms (n = 5). At −150 mV, ΔF was too low for reliable estimates of τ (compare Fig. 5). On average, the time constant for OFF was 17.1 ± 0.3 ms (n = 5). When large depolarizing voltages were applied at saturating sugar concentrations (>10 mM, compare Fig. 5 and Fig. 6 B), the time course of ΔF was slow, with τ ranging between 600 and 900 ms. In three experiments at 100 mM αMDG where 500-ms pulses were applied, the mean was 800 ± 100 ms.

Phlorizin reduced, and at high concentrations eliminated, the presteady-state current, but did not alter the profiles of the OFF current records (Fig. 4 B). The maximal charge (Q_max) was reduced with [phlorizin]_o, with an inhibitory constant (K_i) of 0.3 μM (Fig. 7 B). There was no change in the V_0.5 (Fig. 7 D) and zδ_Q (not depicted)

Fig. 8 shows the effect of phlorizin on the fluorescence records. The inhibitor reduced ΔF at every test voltage. At the highest concentration applied (100 μM), ΔF was completely inhibited. The K_i for the reduction of ΔF_max with [phlorizin]_o was 0.4 μM, similar to the K_i for the inhibition of charge (Fig. 7 B). There was also no effect of phlorizin on the V_0.5 (Fig. 7 D) and zδ for fluorescence (not depicted).

The time constants of the presteady-state current and ΔF were unaffected by phlorizin. For example, in the experiment of Fig. 4 B, as [phlorizin]_o varied from 0 to 1 μM, on average, for the ON presteady-state currents at +50 and −150 mV, τ was 4.8 ± 0.3 ms (n = 5) and 17.3 ± 0.4 ms (n = 5), respectively. For OFF, on average, τ was 10.6 ± 0.3 ms (n = 5). Likewise, in the experiment of Fig. 8, on average, for the ON fluorescence records at +30 mV, τ was 11.1 ± 0.2 ms (n = 5). For OFF, τ was 14.6 ± 0.6 ms (n = 5).

The goal of this study is to examine sugar interactions with SGLT1 and to place this in context of a kinetic model for Na⁺/sugar cotransport. We used a rhodamine-labeled mutant G507C of SGLT1 that transports sugar with similar kinetics as wild type. The kinetics of wild-type hSGLT1 and mutant G507C determined from the electrical and optical measurements (steady-state sugar-induced current, presteady-state current, and ΔF measurements) are summarized in Table I. Apart from the slight increase in

Our strategy was to perturb the steady-state distribution of conformations of the protein in the absence and presence of sugar or the competitive inhibitor phlorizin by step jumps in membrane voltage, and measure the presteady-state currents and changes in rhodamine fluorescence. The charge and fluorescence experiments provide insights into conformational changes induced by ligands (Na⁺ and sugar) and membrane voltage. In the absence of sugar, we have previously shown that charge measurements, namely, the Q-V relations, are correlated with the steady-state distribution of hSGLT1 between conformations C₂ and C₆ (C₂ is the outward-facing Na⁺-bound state, and C₆, the inward-facing empty transporter) (see Fig. 9 and Loo et al., 1998, 2005; Meinild et al., 2002). In addition, we have shown the fluorescence changes (ΔF) reflect differences in local environment of the fluorophore as the protein undergoes changes of conformations (Loo et al., 2005).

The key observations here are that αMDG and phlorizin reduce the presteady-state current and maximal fluorescence, and αMDG shifts the midpoint voltages (V_0.5) for charge and fluorescence. More importantly, at saturating [αMDG]_o, presteady-state current was completely inhibited, whereas fluorescence change (ΔF_max) was not (Fig. 6). The decrease in presteady-state current (and Q_max) is matched by the generation of the αMDG-induced steady-state current, i.e., Na⁺/sugar cotransport (Loo et al., 1993). The reductions in Q_max and ΔF_max and the shifts of the V_0.5's with [αMDG]_o occurred with similar kinetics (⁠

Previously, we have developed and refined a model to account for the observed steady-state and presteady-state kinetics of SGLT1 (Parent et al., 1992b; Loo et al., 2002) Basically, this is a six-state, ordered, nonrapid equilibrium, alternating-access model with mirror symmetry and a Na⁺/glucose transport stoichiometry of 2. We postulated that the ligand-free carrier is negatively charged (valence −2). There are six kinetic states, empty (C₁ and C₆), Na⁺ bound (C₂ and C₅), and Na⁺- and sugar-bound (C₃ and C₄) states in the external and internal membrane surfaces (Fig. 9). The Na⁺ ions bind to the protein before glucose, and the substrates are transported simultaneously. To simplify the model we assumed the Na⁺ ions bind to two identical Na⁺ binding sites with the same rate constant. The only voltage-dependent steps are translocation of the ligand-free protein between the two sides of the membrane and the binding of external Na⁺. The effects of voltage on these reaction steps were described by the Eyring rate theory. The presteady-state currents are associated with the voltage-dependent steps: binding of external Na⁺ (C₁⇄C₂, 30% of total charge; Loo et al., 1993; Hazama et al., 1997) and translocation of the empty carrier (C₁⇄C₆, 70% of total charge).

All steady-state and presteady-state parameters (I/V curves, K_0.5 and I_max values for Na⁺ and sugar as a function of membrane voltage and given cis and trans concentrations) were computer generated simultaneously with numerical values assigned for the 12 of the 14 rate constants, and the results were compared and contrasted with the experimental datasets. Optimization of the simulations was accomplished by progressively adjusting each rate constant to obtain a global fit to the experimental results. A single set of rate constants, reasonable but not necessarily unique, was obtained that accounted for the overall behavior of the cotransporter, including the I/V curves in the presence and absence of sugar, the steady-state kinetics of inward Na⁺/sugar cotransport as a function of the external ligand concentrations and voltage (Parent et al., 1992b).

Subsequently, we tested and refined the model by detailed investigations of: (a) the presteady-state behavior of SGLT1 as a function of external Na⁺ concentration, membrane voltage, and temperature (Loo et al., 1993, 2005, Hazama et al., 1997); (b) the stoichiometry of Na⁺ and glucose cotransport (Mackenzie et al., 1998); (c) Na⁺ transport in the absence of substrate (Loo et al., 1999); (d) the kinetics of reverse Na⁺/glucose cotransport (Quick et al., 2003; Eskandari et al., 2005); (e) the conformational states of SGLT1 as a function of ligand concentrations and membrane voltage using optical techniques on a TMR6M-labeled hSGLT1 mutant (Q457C) where Na⁺/sugar cotransport is abolished after labeling (Loo et al., 1998, 2005; Meinild et al., 2002) and (f) electron microscopic techniques to determine the density of SGLT1 in the membrane (Zampighi et al., 1995). These comprehensive studies support the validly of the model but require the presence of two additional states (C_a and C_b) between C₁ and C₆ to account for the presteady-state behavior (shaded region of Fig. 9; Loo et al., 2005). The major discrepancies between the model and the observed behavior are the fast presteady-state kinetics in the submillisecond range, and these are thought to be due to assumptions about Na⁺ binding to two identical sites with similar rate constants (see Loo et al., 2005 and below). In this study we have employed the eight-state model (Fig. 9) along with electrical and optical methods to examine the conformational states during the Na⁺/glucose cotransport cycle. Since the binding of the high-affinity inhibitor phlorizin is Na⁺ dependent, this is incorporated into the model as C₂⇄C₇ ([CNa₂]′ + Pz⇄[CNa₂Pz]′) with ON and OFF rates k₂₇ and k₇₂ (Fig. 9). Phlorizin binding to the cytosolic surface of SGLT1 ([CNa₂]′′+ Pz⇄[CNa₂Pz]′′) is neglected owing to the low affinity (K_i > 1 mM; Quick et al., 2003; Eskandari et al., 2005).

As a starting point, we used the rate constants for the voltage-dependent steps from Loo et al. (2005). The rate constants for the sugar binding and translocation steps (k₂₃, k₃₂, k₃₄, and k₄₃) are from Hirayama et al. (1996). The rate constants for Na⁺ and sugar binding on the internal membrane are from Eskandari et al. (2005). We extend the model to mutant G507C by reducing the rates for sugar binding (k₂₃, k₅₄) for mutant G507C to account for the lower

The eight-state model accounted quantitatively for the kinetics of steady-state Na⁺/glucose cotransport as a function of extracellular ligand concentrations and voltage (Table I). The sugar-induced currents increased and saturated with hyperpolarizing voltages (compare Fig. 1 C). The simulated K_0.5 for αMDG and Na⁺ (⁠

Ligands and voltage alter the occupancy probability (P_o) of each state (Fig. 10 A). In external NaCl buffer, the transporter is predominantly in C₂ at negative membrane voltages (98% occupancy in C₂ at −150 mV), and in C₆ at positive voltages (e.g., 73% in C₆ at +50 mV). External sugar alters the distribution by reducing the occupancy in C₂ and increasing C₅ occupancy at large negative membrane voltages. At large positive voltages (e.g., +50 mV), C₆ is predominant, regardless of [sugar]_o (Fig. 10, A, B and D). For example, at 10 mM [αMDG]_o, C₅ is the highest occupancy state (57%) at V_m −150 mV, and C₆ (69%) at V_m +50 mV.

This analysis predicts large changes in the distribution of SGLT1 with jumps in membrane potential, and for the voltage-dependent transitions, a presteady-state current.

Fig. 11 A shows the simulated presteady-state currents for the OFF-pulses in response to the standard 100-ms voltage pulse protocol (V_h = −50 mV; compare Fig. 1 A). The currents are progressively reduced with increasing [sugar]_o, and at 10 mM, they are inhibited 82% (Fig. 11 D). The simulated Q-V relations for medium change (at each [sugar]_o) could be fitted by Boltzmann relations. Without sugar, V_0.5 = −32 mV and zδ_Q = 0.9. Increasing [sugar]_o reduced the maximal charge (Q_max) with a

The τ-V relations are simulated by the model. In the absence of sugar, there are four time constants, two fast (τ_fast < 1 ms), one medium (τ_med 3–25 ms), and a slow (τ_slow 30–100 ms; Loo et al., 2005). τ_med and τ_slow are voltage independent for hyperpolarizing voltages and decreased with depolarizing voltages (see Fig. 16 C of Loo et al., 2005). Simulations indicate that the time constant τ_med for ON (as V_m is stepped from −50 to +50 mV) is relatively unaffected at [sugar]_o below the K_0.5; e.g., at +50 mV, τ_ON decreased by <1 ms when [sugar]_o increased from 0 to 1 mM. Experimentally, we found in the range 0–1 mM, the time constant (at V_t = 30 mV) was independent of [αMDG]_o (see Results). The simulations indicated a decrease in τ_med (by ≈5 ms) in the hyperpolarizing direction. However, since the presteady-state current in response to hyperpolarizing voltage pulses was abolished by external sugar (see Fig. 1, A and B), accurately resolving this difference is beyond the resolution of the method.

There is good agreement between our model simulations and the experimental data as functions of voltage and the concentrations of sugar and phlorizin (Fig. 12). In the presence of 100 mM external Na⁺, the ON responses for 100-mV jumps in membrane potential from −50 mV to +50 and −150 mV matched the experimental observation in the presence and absence of external sugar (0, 1, and 10 mM) in terms of (a) the magnitude of the changes in fluorescence; (b) the asymmetry of the response to depolarizing and hyperpolarizing voltage steps; and (c) the reduction in ΔF with increasing [sugar]_o (Fig. 12). These fits were within the normal variation recorded from experiment to experiment. A discrepancy between the simulations and the data is in the relative magnitude of the fast and medium components for the OFF responses when the test voltage returned from +50 mV to the holding potential. The underestimated size of the fast component is likely to be due to our simplifying assumptions about Na⁺ binding (see below).

We also simulated ΔF as a function of membrane potential in the presence and absence of sugar and phlorizin (Fig. 13) to obtain the presteady-state parameters ΔF_max, V_0.5, and zδ_F as functions of sugar and phlorizin concentrations. The ΔF-V relations saturated at −150 and +90 mV and fitted the Boltzmann relation (with a zδ_F of 0.7 and V_0.5 of −11 mV). With saturating sugar (10 mM αMDG), ΔF_max was reduced by 53%, with a K_0.5 of 0.5 mM (Table I). V_0.5 also shifted with increasing [sugar]_o (maximum shift was +51 mV) with a K_0.5 of 1.1 mM (Table I). Thus the observed small reduction of ΔF_max (≈50%) compared with Q_max (>95%, Fig. 6 B) is reproduced by the simulations.

In the absence of sugar, with a large depolarizing voltage pulse (e.g., from −50 to +50 mV), the fluorescence changes are due to the transition C₂→C₆ (Fig. 10 A), as the voltage jump dictates a new equilibrium state distribution highly favoring C₆ (Fig. 14 C; see also Fig. 10 A). Thus the apparent quantum yield of C₆ must be higher than that of C₂, as there is an increase in ΔF (Figs. 3 and 13). This series of conformational changes involve all the voltage-dependent transitions (Fig. 9, shaded region). On the other hand, at saturating [αMDG]_o (e.g., 10 mM; Fig. 10 D), C₅ is the most highly populated state at V_h (−50 mV), and a depolarizing voltage favors C₆ over C₅ (see Fig. 10 D and Fig. 14 D). So if the quantum yield of C₆ > C₅, on depolarization, we would observe an increase in ΔF (Fig. 14 B). The change in ΔF is slower than in Na⁺ alone (Fig. 14 A), as is observed experimentally (Figs. 5 and 12). The charge movements arising from the concurrent relaxation of the voltage-dependent transitions (shaded region, Fig. 9) are negligible, owing to the negligible occupancy of C₂ at V_h (Fig. 10).

Phlorizin locked the transporters in fixed state (C₇ in Fig. 9) and prevented the protein from running through the transport cycle. This is illustrated by the occupancy probabilities as [phlorizin]_o is varied. The increase in the occupancy of the phlorizin-bound state ([CNa₂Pz]′) is balanced by reduction of the occupancy probabilities (P_o) in the other conformations (Fig. 10, A, C, and E). The profile of the P_o-V_m curves is not affected by phlorizin. The simulated inhibitory constant (⁠

The effects of phlorizin on presteady-state currents and fluorescence (ΔF) is simulated in Fig. 11 (C and E) and Fig. 13 (C and E) for 0.4 and 1 μM phlorizin. The amplitudes of the presteady-state current and ΔF are reduced at every test voltage. Analysis of the Q-V and ΔF-V relations as [phlorizin]_o was varied from 0 to 100 μM showed that maximal charge (Q_max) and maximal fluorescence (ΔF_max) were reduced by phlorizin, with K_i's of 277 nM for charge and 399 nM for fluorescence. There were no changes in the V_0.5 (and zδ) values for both charge and fluorescence.

Our estimate of the rate constants for phlorizin binding and dissociation for human SGLT1 (k₂₇ = 50,000 M⁻¹s⁻¹, k₇₂ = 0.01 s⁻¹) was obtained with the requirements that (a) the ratio k₇₂/k₂₇ is the dissociation constant K_D (K_D = k₇₂/k₂₇ = 0.2 μM from Dixon analysis; Panayotova-Heiermann et al., 1995; Hirayama et al., 2001), and (b) the phlorizin-bound state ([CNa₂Pz]′) does not decay significantly during 2-s depolarizing voltage pulses. These estimates are within the range of values obtained previously by Toggenburger et al. (1982) on rabbit small-intestinal brush border membrane vesicles: k₂₇ ≈18,000–150,000 M⁻¹s⁻¹, k₇₂ = 0.4 s⁻¹, and K_D = 2.7 μM.

(a) We have obtained a single set of kinetic parameters that accounts for the global experimental observations on steady-state and presteady-state kinetics, for both charge and fluorescence measurements in the presence and absence of sugar or phlorizin.

(b) In view of the close agreement between the data and simulations on charge movements in the presence of sugar, this suggests that there are no significant voltage-dependent steps in the transport cycle other than those associated with external Na⁺ binding/dissociation and conformational change of the empty transporter (shaded region of Fig. 9). This is in agreement with our previous study on reverse Na⁺/sugar cotransport using the giant patch (Eskandari et al., 2005).

(c) At saturating [αMDG]_o, when membrane voltage is stepped from −50 to +50 mV, the charge movement is too small to be detected because of the low occupancy in states C₂, C₁, C_a, C_b, and C₆ (Fig. 10 D and Fig. 14 D), which is observed experimentally (Fig. 6 B). However, there is a fluorescence change (bold line of Fig. 14 B) due to the transition: C₅→C₆ (bold lines in Fig. 14 D). This is a rate-limiting step for Na⁺/ sugar cotransport, and we have thus experimentally isolated a conformational change that has not been previously described. The differential sensitivities of the electrical and optical measurements allow us to measure the fluorescence change associated with the electroneutral steps.

(d) Simulations show that the model accounts for the reductions in the Q_max and ΔF_max and the shifts in V_0.5 for charge and fluorescence produced by external sugar (or phlorizin). While the apparent valence (zδ) is similar for both charge movement and fluorescence changes (zδ ∼ 0.7–0.9), this may depend on the nature and position of the fluorophore and its local interactions with SGLT1. The apparent valence (zδ_Q) for charge movement is the sum (=z₁δ₁ + z₂δ₂ + .. + z_nδ_n) of the products of moveable charges (z_i) and the displacement (δ_i) of each charge in the membrane electric field, whereas the apparent valence (zδ_F) for fluorescence depends on the apparent quantum yields of the fluorophore in different protein conformations. While the apparent valence of SGLT1 and mutant charge movement have been found to be 1, the observed values of zδ_F varied: zδ_F = 0.4 for TMR6M-Q457C (Loo et al., 1998, 2005; Meinild et al., 2002), 0.7 for TMR6M-D454C (Diez-Sampedro et al., 2004), and 0.9 for TMR6M-G507C (this study).

(e) The previous six-state model could account for the steady-state kinetics of SGLT1 and the steady-state kinetics of TMR6M-G507C obtained in the present study (Parent et al., 1992b; Eskandari et al., 2005; present study). However, the model failed to describe the presteady-state currents in the absence and presence of sugar and phlorizin, especially their voltage dependence. In particular, the model predicted τ-V relations that are bell shaped (see Fig. 7 B of Hazama et al., 1997), contrary to experimental observations (Fig. 2 C; see also Fig. 4 A of Loo et al., 2005). The eight-state model accounts for the voltage dependence of τ by asymmetric equivalent charge movements (ε_1a ≈ 0, ε_a1 = 0.5, ε_ab = 0.15, ε_ba ≈ 0, ε_b6 = 0.05, ε_6b = 0.7). An asymmetric voltage dependence between forward and backward reactions could arise from asymmetric Eyring energy barriers for the transition state (Zagotta et al., 1994). The molecular mechanism for the asymmetry and the location of the voltage sensor(s) in cotransporters such as SGLT1 is unknown. The asymmetry has been observed in the gating currents of shaker Potassium channels (Zagotta et al., 1994; Rodriguez et al., 1998; Schoppa and Sigworth, 1998), but the mechanism is also unknown. One possibility could be a change in the membrane electric field profile between forward and backward reactions (Blunck et al., 2005).

(f) We have previously noted that the model for presteady-state current (Fig. 9, shaded region) failed to account for the kinetics of the fast rising phase (τ = 0.17 ms, Loo et al., 2005), and is due to our assumption of two Na⁺ ions binding to identical sites with the same rate constant (Falk et al., 1998; Loo et al., 2005). In this study, we found that the model does not correctly predict the fast monotonic decay of presteady-state current and fluorescence for hyperpolarizing pulses from very positive voltages. The simulations predict a biphasic time course in the OFF current when membrane potential is stepped from a large positive test value to V_h (from +50 to −50 mV). This can be seen in Fig. 11 A, and is a consequence of the high rate (k_6b = 300 s⁻¹) and high equivalent charge movement (ε_6b = 0.7) from C₆→C_b (see Loo et al., 2005). The discrepancies between model simulations and data are in the submillisecond domain. Resolving the fast kinetic events in Na⁺ binding remains a major challenge to understanding Na⁺/glucose cotransport.

(g) The fluorescence changes are primarily associated with the differences in relative quantum yields of the fluorophore in conformations C₂, C₆, and C₅ (qy₅/qy₂ = 3, qy₆/qy₂ = 6) because the occupancy probabilities of the other states are low.

Since our initial observation of presteady-state currents in the Na⁺/glucose cotransporter (Birnir et al., 1991), they have been observed in many solute carrier (SLC, HUGO Nomenclature) gene families, including SLC1 (high affinity glutamate transporter; Grewer and Rauen, 2005), SLC6 (Na⁺/Cl⁻/GABA; Mager et al., 1993), SLC13 (Na⁺-sulfate/carboxylate; Chen et al.,1998), SLC15 (H⁺/oligopeptide; Mackenzie et al., 1996), SLC20 and SLC34 (types II and III Na⁺/phosphate; Forster et al., 2002), and SLC28 (Na⁺/nucleoside; Larrayoz et al., 2004, Smith et al., 2004). Common observations are the reduction of presteady-state currents by substrates and the linear relationship between maximal charge (Q_max) and maximal substrate-coupled current (I_max), with the ratio (I_max/Q_max) providing an estimate of the turnover rate of cotransporter (for reviews see Forster et al., 2002; Loo et al., 2002; Grewer and Rauen, 2005). We have used charge measurements to probe the conformations of SGLT1 under sugar transport. The positive shift of V_0.5 with [sugar]_o indicates that the rate-limiting step is C₅→C₆. In contrast, we recently found that for hPEPT1 (human H⁺/dipeptide transporter), glycylsarcosine reduced Q_max and shifted V_0.5 to more negative values (Sala-Rabanal et al., 2006). This negative shift indicates that the rate limiting step is the return of the empty transporter from inward-facing to outward-facing conformation (C₆→C₁), unlike the C₅→C₆ for hSGLT1. Thus charge measurements in the presence of substrates provide unique and novel insights into the rate-limiting step for cotransporters.

Since our initial application of voltage clamp fluorometry to study SGLT1, voltage clamp fluorescence experiments have been performed on the Na⁺/Cl⁻/γ-aminobutyric acid (GAT1; Li et al., 2000; Meinild, A.-K., N. MacAulay, U. Gether, and D.D.F. Loo. 2006. FASEB J. 20:A839), serotonin (SERT; Li and Lester, 2002), glutamate (EAAT3; Larsson et al., 2004), and Na⁺/P_i (Virkki et al., 2006) cotransporters. In these studies, with the exception of Li and Lester (2002), the ΔF-V relations are affected by substrates, indicating that substrate interactions alter the distribution of conformations.

We have used voltage-clamp fluorometry to examine the partial reactions involving sugar binding and transport by the human Na⁺/glucose cotransporter. This was accomplished using a voltage-jump perturbation protocol in the presence and absence of external sodium, sugar, and phlorizin. Based on simulations of the experimental data using our eight-state, ordered, nonrapid equilibrium, alternating access model (Fig. 9) we conclude that partial reactions involving sugar binding (k₂₃, k₃₂, k₄₅, and k₅₄) and Na⁺/sugar cotransport (k₃₄ and k₄₃) are neither voltage dependent nor rate limiting. Furthermore, our analysis of presteady-state currents and fluorescence in the presence and absence of sugar or phlorizin demonstrate that: (a) external sugar increases the occupancy probability of inward-facing conformations (C₅ and C₆) at the expense of outward-facing conformations (C₂ and C₃); and (b) the high affinity blocker phlorizin locks the cotransporter in an inactive conformation (C₇). Finally, under the maximum driving force for inward Na⁺/glucose cotransport (saturating voltage and external Na⁺ and sugar concentrations) the rate-limiting step for transport is the electroneutral dissociation of Na⁺ from the cotransporter at the internal face of the membrane (C₅ to C₆). In general, our model for cotransport satisfactorily accounts for all the observed steady-state and presteady-state kinetics. The model does not completely account for submillisecond presteady-state kinetics due in part to our assumption that external binding of the two Na⁺ ions can be simplified by using single lumped rate constants (k₁₂ and k₂₁). A current challenge is to parse out the rapid kinetics of Na⁺ binding to the two binding sites.

We thank Mary Lai-Bing, Amanda Johnson, Kari Edwards, Linh Nguyen, and Teresa Ku for the preparation, injection, care of oocytes, and assistance with the uptake experiments. We especially thank Dr. Eric Turk for his help with mutagenesis.

This research was supported by National Institutes of Health grant DK19567.

Olaf S. Andersen served as editor.