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¹ Department of Physiology, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095
² Department of Anesthesiology, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095

Correspondence to Donald Loo: dloo{at}mednet.ucla.edu

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Conformational changes of the human Na⁺/glucose cotransporter (hSGLT1) were studied using voltage-jump methods. The cotransporter was expressed in Xenopus laevis oocytes, and SGLT1 charge movements were measured in the micro- to millisecond time scale using the cut-open oocyte preparation and in the millisecond to second time scale using the two-electrode voltage clamp method. Simultaneous charge and fluorescence changes were studied using tetramethylrhodamine-6-maleimide-labeled hSGLT1 Q457C. In 100 mM external [Na⁺], depolarizing voltage steps evoked a charge movement that rose initially to a peak (with time constant = 0.17 ms) before decaying to steady state with two time constants ( = 2–30 and 25–150 ms). The time to peak (0.9 ms) decreased with [Na⁺], and was not observed in 0 mM [Na⁺]. In absence of Na⁺, charge movement decayed monotonically to steady state with three time constants (0.2, 2, and 150 ms). Charge movement was accompanied by fluorescence changes with similar time courses, indicating that global conformational changes monitored by charge movement are reflected by local environmental changes at or near Q457C. Our results indicate that the major voltage-dependent step of the Na⁺/glucose transport cycle is the return of the empty carrier from inward to outward facing conformations. Finally, we observed subtle differences between time constants for charge movement and for optical changes, suggesting that optical recordings can be used to monitor local conformational changes that underlie the global conformational changes of cotransporters.

Key Words: Na⁺/glucose cotransport • presteady-state kinetics • charge movement • fluorescence • conformational changes

¹ Since the V_0.5 for charge is very negative in the absence of Na (–200 mV), some SGLT charge is being subtracted using the P/4 protocol. However, it does not invalidate the conclusions of the present study because the time course of the fast charge movement was not altered since the time constants were independent of voltage (Fig. 6 C).

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The Na⁺/glucose cotransporter (SGLT1) is a member of a large family of proteins that uses the electrochemical potential gradient of Na⁺ to accumulate substrates into cells. Cotransport occurs by an alternating access mechanism via a series of conformational changes induced by substrate binding and membrane voltage (Parent et al., 1992; Hazama et al., 1997; Loo et al., 1998; Meinild et al., 2002). A simplified six-state kinetic model has been proposed to account for the kinetics of SGLT1 (Parent et al., 1992; Hazama et al., 1997; Loo et al., 1998; Mackenzie et al., 1998; Meinild et al., 2002). The transporter has six kinetic states consisting of the ligand-free (C₁, C₆), Na⁺-bound (C₂, C₅), and Na⁺- and sugar-bound (C₃, C₄) states at the external and internal membrane surfaces (see Fig. 15 A). Two external Na⁺ ions bind to the transporter before glucose, and the substrates are transported simultaneously, and glucose is released before Na⁺ on the inside. The model accounted not only for the steady-state kinetics of SGLT1, but also predicted presteady-state transients associated with the transporter in the absence of glucose (Parent et al., 1992; Loo et al., 1993). In the model, the protein is negatively charged (valence of –2), and membrane voltage is assumed to influence: (a) the reorientation of the ligand-free or empty carrier between inward and outward facing conformations, and (b) the binding of external Na⁺ to a site in the membrane electric field. Thus, there is a predicted charge movement associated with the voltage-dependent partial reactions C₁C₆ (empty carrier alternating between outward and inward facing conformations) and C₂C₁ (external Na⁺ binding/dissociation).

In 1993, we reported transporter capacitive currents, carrier currents, with stepped jumps in membrane voltage (in the presence of Na⁺ but absence of sugar), and hypothesized that most of these voltage-dependent transient currents (presteady-state currents or charge movement) are due to the voltage-sensitive conformational changes of the membrane protein (Loo et al., 1993). These presteady-state currents have been used to gain insight into the partial reactions of cotransporters (for reviews see Loo et al., 2002; Forster et al., 2002), and to estimate the total number of transporters in the cell membrane (Zampighi et al., 1995). The origin of the cotransporter transients, however, has been questioned, and it has been proposed that the currents are due solely to ion binding to sites within the membrane electric field (e.g., Su et al., 1996).

By combining presteady-state measurements with optical techniques using extrinsic fluorescent probes covalently bound to engineered cysteine residues in the transporter (Loo et al., 1998; Meinild et al., 2002), we have established that the presteady-state currents of SGLT1 are due to Na⁺ binding/dissociation and transitions of the empty transporter between outward- and inward-facing conformations (Loo et al., 1998; Meinild et al., 2002). We have also shown that the fluorescent changes in the tetramethylrhodamine-6-maleimide (TMR6M)–labeled hSGLT1 mutant Q457C reports local conformational changes (at Cysteine 457) associated with ligand binding (Na⁺ and sugar) and voltage jumps.

In this study using both charge and fluorescence measurements, we have set out to extend the voltage- and Na⁺-induced perturbations in the conformation of SGLT1 over a wider time scale, ranging from microseconds to seconds. Our previous studies have examined the conformational changes of SGLT1 with time constants in the range of 3–35 ms. The motivations for performing charge and fluorescence measurements at an expanded time scale were our simulations on the six-state kinetic model for SGLT1, which predicted a fast rising phase of charge movement with depolarizing potentials (Parent et al., 1992; Hazama et al., 1997), and our observation that steady-state conditions are not reached at the end of our standard 100-ms voltage pulses.

The concurrent employment of an independent optical method to monitor voltage-induced conformational changes in the transporter overcomes some of the inherent limitations of charge measurements alone. Our results demonstrate that there are at least three components of voltage-induced perturbations in hSGLT1 with time constants ranging from 0.2 to 200 ms. The slow ( = 30–200 ms) and medium (3–20 ms) components contribute equally to total charge transfer, while the fast (0.2–1.5 ms) component contributes the most to total fluorescence changes. Under Na⁺-free conditions, all three components are still evident, but the time constants decrease in magnitude. We conclude that most of the SGLT1 charge movement is due to the reorientation of intrinsic charge in the membrane protein and that there are at least two intermediate conformations (C_1a and C_1b) between the two final states C₁ and C₆ in the external and internal membrane surfaces (C₁C_1aC_1bC₆). External Na⁺ modulates charge transfer between C₁ and C₆ by increasing the occupancy of the Na⁺-bound state (C₂ = C₁Na₂) and reducing the occupancies of C₁, C_1a, C_1b, and C₆. This suggests that C₁ is meta-stable; the highest probability states are C₂ in the presence of Na⁺, and C₆ in the absence of Na⁺. The implication is that after the Na⁺/glucose transport step, the high external Na⁺ concentration shifts the transporter from the cytoplasm (C₆) into the state with the highest affinity for glucose (C₂) to initiate another cycle of Na⁺/glucose transport across the membrane. Our results also suggest that the voltage sensitivity of Na⁺/glucose cotransport resides mainly in return of the empty carrier from the inward- to outward-facing conformation (C₆ to C₁).

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Strategy
Our general strategy was to express SGLTs in Xenopus laevis oocytes and to record changes in transporter currents and extrinsic fluorescence over a wide time scale (microsecond to seconds) after rapid jumps in the membrane potential. In the absence of sugar, the transient transporter currents are capacitive, due to a reorientation of the protein charge in the membrane electric field, and/or the binding and dissociation of external Na⁺ in a well within the membrane field. To examine the role of Na⁺, external [Na⁺] was varied between 0 and 100 mM by isomolar replacement of NaCl with cholineCl. Na⁺ binding and dissociation at the cytoplasmic face of the protein may be neglected as the cytoplasmic concentration is either low (10 mM in intact oocytes) or absent in the cut-open oocyte preparation (see below) and the internal Na⁺ binding affinity is low (Eskandari et al., 1999; Sauer et al., 2000; Quick et al., 2003).

The two-electrode voltage clamp, with a settling time 0.6–1.0 ms (Loo et al., 1993), was used to record transients in the millisecond to second range. To examine kinetic events in the microsecond to millisecond range, the cut-open oocyte preparation, with a settling time of 80 µs (Taglialatela et al., 1992; Stefani and Bezanilla, 1998) was used. The wild-type human SGLT1 (hSGLT1) was used to record transporter charge movements, and the hSGLT1 mutant transporter, Q457C, was used to correlate fluorescence and charge movement (Loo et al., 1998; Meinild et al., 2002). Previously, it has been shown that the mutant protein is able to transport sugar, but sugar transport is abolished on alkylation of Cys457 with methanethiosulfonate reagents, or after labeling by TMR6M (Loo et al., 1998; Meinild et al., 2002). Since the TMR6M-labeled mutant transporter can bind Na⁺ and sugar, it can be used for studying the conformational changes associated with ligand binding and voltage jumps. The human isoform was used because the distribution of the protein between outside-facing and inside-facing conformations was at a midpoint at the normal resting potential (holding potential) of the oocyte, –50 mV. This means that over the practical range of voltage jumps that can be used with oocytes, –150 to +50 mV, the full charge movement may be recorded by hyperpolarizing the membrane to –150 mV and depolarizing to +50 mV. It should also be borne in mind that the midpoint V_0.5 for SGLT1 shifts by 100 mV per 10-fold reduction in external [Na⁺] (Loo et al., 1993; Hazama et al., 1997; Quick et al., 2001), and this means that it is not feasible to obtain the full charge vs. voltage curve at [Na⁺] < 10 mM.

Preparation and Maintenance of Oocytes
Mature Xenopus laevis oocytes were isolated, defolliculated, injected with human SGLT1 or human SGLT1 Q457C cRNA (Loo et al., 1993, 1998). hSGLT1 Q457C was labeled with 200 µM TMR6M (Loo et al., 1998).

Combined Electrophysiological and Fluorescence Experiments
Electrophysiological and fluorescence experiments were performed simultaneously, using either two-electrode (Loo et al., 1998; Meinild et al., 2001) or cut-open oocyte voltage clamp fluorometry (Cha and Bezanilla, 1997, 1998). The current records were the averages of 3–4 sweeps, and the fluorescence records were averages of either 4 or 20 sweeps. Interpulse interval was 1 s. Records were filtered at 2 kHz, 500 Hz, or 50 Hz, depending on the sampling interval (5 µs to 750 µs per sample). In two-electrode voltage clamp experiments, the bath contained 100 mM NaCl buffer (in mM, 100 NaCl, 2 KCl, 1 CaCl₂, 1 MgCl₂, 10 HEPES, pH 7.4). In cut-open oocyte experiments, external and guard solutions contained (in mM) 100 Na-methanesulfonate, 1 CaCl₂, 10 HEPES (pH 7.3), and internal solution contained (in mM) 100 K-methanesulfonate, 1 EGTA, 10 HEPES (pH 7.3). The Na⁺ concentration was varied by equimolar replacement of Na⁺ with choline. Fluorescence data have been corrected for rundown (Meinild et al., 2002). All experiments were performed at room temperature (20–23°C).

Data Analysis
Isolation of Presteady-state Currents.
In response to a voltage pulse, the total membrane current consisted of the membrane bilayer capacitive transient, the presteady-state currents of SGLT1, and the steady-state current. Using 100-ms voltage pulses, we have reported that the relaxation of the presteady-state current exhibited a single time constant between 2 and 30 ms (Loo et al., 1993; Hazama et al., 1997; Quick et al., 2001; Meinild et al., 2002). In pilot studies, we found additional components with time constants between 0.2–2 and 35–160 ms. For clarity of presentation, we operationally defined the fast, medium, and slow components (for charge and fluorescence) as those with time constants between 0.2–2, 2–20, and 30–160 ms, respectively. Since at each voltage step their time constants differed by an order of magnitude, the components were estimated separately using test voltage pulses of different durations.

Slow Component.
The time constant of the slow component was estimated from the current relaxations after the medium component has decayed. 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.

Medium Component.
The medium component was estimated using 100-ms voltage pulses after subtraction for the slow component (above) and the steady-state current. The compensated current was fitted to the function

where I_cmexp(–t/_cm) is, to a first approximation, the capacitive current. The charge was obtained from the integral of the medium component.

Phlorizin Subtraction.
An alternative procedure for isolating the SGLT1 transient was to use phlorizin subtraction (Loo et al., 1993; Hazama et al., 1997; Meinild et al., 2001). The transients were obtained by pointwise-subtraction of the current relaxations in 100 µM external phlorizin (in the presence of Na⁺) from those obtained in the absence of phlorizin (in Na⁺). In Na⁺-free situations (e.g., Fig. 5, C and D), they were obtained by subtraction of the current relaxations in 100 µM phlorizin and 100 mM Na⁺ from the current relaxations in 0 mM Na⁺. The currents isolated by phlorizin subtraction were the total presteady-state currents. Subtraction could not be used with the TMR6M Q457C because of the low phlorizin affinity (K_i 100 µM for TMR6M Q457C vs. 200 nM for hSGLT1).

Cut-open Oocyte Experiments.
The background leak currents and the oocyte bilayer capacitive transients were first compensated with the voltage clamp amplifier (CA-1; Dagan Corporation). The P/4 protocol was used to isolate the presteady-state currents (Bezanilla and Armstrong, 1977; Bezanilla and Stefani, 1998) using a subtracting holding potential (V_shp) of –150 mV where there was negligible SGLT1 charge movement.¹

Fitting of Q–V and F–V Relations.
The charge vs. voltage (Q–V_m) relations 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), z is apparent valence of the movable charge, and is the fraction of the membrane electric field traversed by the charge. z is the steepness factor for the dependence of Q on voltage. The Boltzmann relation was also used to fit the dependence of the change of fluorescence intensity (F) on membrane voltage (Loo et al., 1998; Meinild et al., 2002). The parameters obtained were the maximal fluorescence intensity change (F_max), the membrane voltage at 50% F_max (V_0.5), and the product (z) of the apparent valence of the fluorescence voltage sensor (z) and the dielectric distance ().

Fits of data to equations were performed using either Sigmaplot 2002 (SPSS) or Clampfit 8.1 (Axon Instruments). Unless otherwise noted, statistics were obtained from the error of the fit. While data are shown for representative experiments, all experiments were performed on at least three oocytes from different batches.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Part I. Charge Movement
The first part describes the time course of voltage-induced capacitance currents (the presteady-state current) through SGLT1. The study extends our previous experiments to encompass sub-millisecond and second time domains. Charge measurements are presented for wild-type human SGLT1 and the TMR6M-labeled hSGLT1 mutant Q457C. The latter was used to monitor the conformational changes of the transporter by fluorescence measurements.

Slow and Medium Charge Movements in hSGLT1.
The raw current records from an oocyte expressing hSGLT1 performed with the two-electrode voltage clamp are shown in Fig. 1 A. The membrane potential was held at –50 mV (V_h) and stepped to a test value (V_t, from +50 to –150 mV, and representative traces are shown) for 100 ms before returning to V_h. The current contained (a) an initial spike, with a time constant of 0.7–1.0 ms; (b) hSGLT1 presteady-state currents, which decayed to an apparent steady state with time constants of 3–25 ms; and (c) the steady-state current, comprising of the background current of the oocyte and a hSGLT1-mediated Na⁺-uncoupled (or leak) current due to the uniporter function of the transporter in the absence of sugar (Umbach et al., 1990; Loo et al., 1999). Only the initial spike and the background currents were observed in noninjected oocytes. Inspection of the recordings on oocytes with high levels of expression reveals that the steady state has not been reached at 100 ms, especially in the hyperpolarizing direction (Fig. 2 A). We therefore applied voltage pulses of up to seconds in duration.

View larger version (16K): [in this window] [in a new window]   FIGURE 1. Time course of the raw or total currents (capacitive and hSGLT1 presteady-state currents) of an oocyte expressing hSGLT1. The experiment was performed using a two-electrode voltage clamp, and all data were obtained from a single oocyte. Membrane potential was held at –50 mV (Vh) and then stepped to a series of test values Vt (from +50 to –150 mV in 20-mV decrements; representative traces are shown for +50, –10, –50, –90, and –150 mV) before returning to Vh. The pulse duration was 100 ms in A and 500 ms in B, and the currents, which are the average of three sweeps, have been filtered at 500 Hz (A) and 50 Hz (B). An upwards deflection of the current trace represents an outward current.

View larger version (30K): [in this window] [in a new window]   FIGURE 2. Medium and slow components of the presteady-state currents of hSGLT1 (ON response). Shown are the current records for the ON pulse from Vh (–50 mV) to Vt +50, –10, –50, –90, and –150 mV for the 100-ms (A) and the 500-ms (B) duration pulses. The data were from the same oocyte as Fig. 1. To isolate the slow charge, the steady-state current was removed, and the transient current (for the 500 ms pulses) was fitted to a single exponential function. The fit was restricted to the region between 5med (med 3–20 ms) and 500 ms. The starting point was 25 ms at +50 mV, 60 ms at –10 mV, and 100 ms at Vt more negative than –90 mV. The fit was extrapolated to the peak of the total current trace (dashed lines in A and B), typically two data samples after onset of the voltage pulse (to 0.2 ms in A). The isolated slow charge is shown in D. To obtain the medium component, the slow component (dashed line) was subtracted from the 100-ms current records (A). The difference was fitted to I(t) = Icm exp(–t/cm) + Imed exp(–t/med). Panel C shows the medium charge obtained after subtraction of the membrane capacitance (Icm exp(–t/cm)). For clarity, the current trace at –50 mV (Vh) has been omitted in D.

The procedure for isolating the components of the presteady-state current is illustrated in Fig. 2 for the ON response. The fit of the slow component began after a period of five times the relaxation time constant of the medium component (_med). At 5_med (typically 100 ms), the medium component has decayed 99% (from the initial value), and the remaining slow relaxations were well described by a single exponential function (Fig. 2 B). Fig. 2 D shows the slow component extrapolated to the beginning of the pulse.

The medium component was obtained from the 100-ms current records by first subtracting the slow component (dashed line in Fig. 2 A) from the total current, and then subtracting the membrane capacitive currents (Fig. 2 C).

Fig. 3 shows the OFF currents when the test potential was returned to V_h. The slow relaxations (between 100 and 500 ms) could be fitted by a single exponential function (Fig. 3 B). Fig. 3 (C and D) shows the isolated slow and medium components. All charge movements associated with hSGLT1 in the presence of Na⁺ (the medium and slow components and the rising phase described in Fig. 6 below) were blocked by 100 µM phlorizin, the specific high-affinity competitive inhibitor of SGLT1 (unpublished data).

View larger version (32K): [in this window] [in a new window]   FIGURE 3. Medium and slow components of the presteady-state currents of hSGLT1 (OFF response). Shown are the current records for the OFF pulse when membrane potential was stepped from the test potential (+50, –10, –50, –90, and –150 mV) back to the holding (–50 mV) for the 100 ms (A) and the 500 ms (B) duration pulses. The data were from the same oocyte as Fig. 1, and the protocol for isolation of the medium (C) and slow components of charge movement (D) is as described in Fig. 2. As med for OFF was independent of test voltage (med = 15 ms, see Fig. 4 A), the initial point for the fit (Fig. 3 B) was 96 ms for all the OFF pulses.

View larger version (27K): [in this window] [in a new window]   FIGURE 4. Kinetics of charge movement of hSGLT1: voltage dependence of the medium and slow components. Data was from the experiment of Fig. 1. (A) –V relations. The filled symbols represent the ON where membrane potential was stepped from Vh (–50 mV) to different test potentials (Vt). The open symbols represent the time constant of the relaxation of the OFF where membrane potential was returned from Vt to Vh (–50 mV). Error bars are standard errors (SE) of the fit when the SE exceeds the size of the symbol. The OFF responses were independent of the previous test potential (Vt), and open symbols represent the mean of 10 values with Vt varying between +50 and –150 mV. (B) Q–V relations for the medium charge. At each Vm, the medium charge (Q) was obtained as the time integral of the medium presteady-state current for the ON (circles) and OFF (squares) responses. The curve is the fit of the mean of the medium ON and OFF charge to the Boltzmann relation with Qmax = 8.7 ± 0.2 nC, z = 1.0 ± 0.1, and V0.5 = –33 ± 1 nC. (C) Q–V rela tions for slow charge. Slow charge (Q) was obtained from the time integral of the slow presteady-state current for the ON and OFF responses. The curve is the fit of the mean of the slow ON and OFF charge to the Boltzmann relation with Qmax = 7.2 ± 0.1 nC, z = 1.0 ± 0.1, and V0.5 = –67 ± 1 nC. (D) Q–V relations for total charge. Total charge is the sum of the medium and slow components (described in B and C). Filled and open symbols represent the total ON and OFF, respectively. The smooth curve is the fit of the total OFF charge to the Boltzmann relation with Qmax = 17.2 ± 0.3 nC, z = 0.9 ± 0.1, and V0.5 = –44 ± 1 mV.

The medium and slow charges (Q_med and Q_slow) were added to obtain the total charge (Fig. 4 D). In the hyperpolarizing direction, the total ON charge was equal in magnitude but opposite in sign to the total OFF charge. At the two most positive test voltages applied, +50 and +30 mV, the ON charge was slightly less than the OFF charge. The difference suggests that the kinetics of the ON response might be too fast to be accurately measured using the two-electrode voltage clamp (see Fig. 7). The curve in Fig. 4 D was the fit of the OFF charge to the Boltzmann relation with z = 1.0 and V_0.5 = –44 mV.

Slow and Medium Charge Movements in Q457C.
Medium and slow components of charge movement were observed in the 100- and 500-ms current records of the TMR6M-labeled Q457C (unpublished data), and their characteristics were similar to those of wild-type hSGLT1. The –V relation for the medium component (_med) was the same as hSGLT1; for test voltages more negative than –50 mV, _med was 20 ms and was independent of voltage, and in the depolarizing direction, _med decreased to 3 ms at +50 mV (compare Fig. 4 A). Like wild-type hSGLT1, for hyperpolarizing voltages, the slow component of TMR6M-labeled Q457C was independent of voltage for the ON and OFF pulses. The time constant (_slow) ranged between 60 and 200 ms, depending on the level of expression of hSGLT1-Q457C; the higher the expression, the larger was _slow. This variation in _slow was possibly due to an underestimation of _slow at low levels of expression seen with this mutant. As in hSGLT1, the magnitudes of the medium and slow charges for the OFF response (in TMR6M Q457C) were comparable and contributed equally to the total charge. The Q–V relation for total charge could be fitted by the Boltzmann relation with z = 1.0 and V_0.5 in the range –40 to –65 mV.

Rising Phase of Charge Movement in hSGLT1.
At a high sampling rate (8 µs/sample), we have observed a rising phase with the two-electrode voltage clamp (Fig. 5 A). In this record, the presteady-state current was obtaining by phlorizin subtraction. With onset of the depolarizing voltage pulse, the current rose initially to a peak (at 1.5 ms) before decaying toward the steady state. The rising phase was not observed for hyperpolarizing voltages (Fig. 5 A).

View larger version (31K): [in this window] [in a new window]   FIGURE 5. Rising phase of charge movement. The experiment was performed on hSGLT1 using a two-electrode voltage clamp. Current records were obtained by phlorizin subtraction. Vh was –90 mV, data were digitized at 8 µs per sample, and pulse was 6 ms. (A) Presteady-state currents in 100 mM [Na+]o. (B) Dependence of the rising phase on [Na+]o. The current records were obtained at Vt = +50 mV in 100, 25, and 12 mM [Na+]o. (C) –V relations for the medium component in 100 and 0 mM [Na+]o. Data were obtained from one oocyte. The open symbols were obtained from the OFF response and represent the mean of 10 values (with test potential varying between +50 and –150 mV). (D) Q–V relations for the medium component in 100 and 0 mM [Na+]o. Q was obtained from the mean of the ON and OFF charges using 100-ms pulses in 100 mM Na+ and 30-ms pulses for 0 mM Na+. The curve (for 100 mM Na+) was drawn using the Boltzmann relation with Qmax = –20.3 ± 0.4 nC, z = 1.1 ± 0.1, and V0.5 = –47 ± 1 mV.

When external Na⁺ concentration ([Na⁺]_o) was reduced from 100 to 12 mM, the time to peak (t_peak) was reduced (Fig. 5 B). For the +50 mV pulse illustrated, t_peak was 1.5 ms at 100 mM [Na⁺]_o, 0.9 ms at 25 mM [Na⁺]_o, and 0.7 ms at 12 mM [Na⁺]_o. A plot of t_peak vs. [Na⁺]_o (at 100, 50, 25, 12, and 6 mM) yielded a sigmoidal relation with a Hill coefficient between 1 and 2 (unpublished data).

From peak current, the time constant of the medium component of decay of transient current decreased with external Na⁺ concentration (Fig. 5 B), and this is illustrated for 100 and 0 mM [Na⁺]_o in Fig. 5 C. At –150 mV, decreased from 18 to 3.5 ms, and from 4.5 to 1.9 ms at +50 mV (see inset). In the working voltage range (+50 to –150 mV), the "maximal" charge recorded (the difference in charge between the limits +50 and –150 mV) was reduced 50% on removing Na⁺ (Fig. 5 D). We were unable to estimate the Q_max, z, and V_0.5 under Na⁺-free conditions because the charge did not saturate at the largest hyperpolarizing voltage applied (–150 mV). This observation is in agreement with the previous finding that the midpoint voltage (V_0.5) of the Q–V curve (for the medium component) became more negative by 100 mV per 10-fold decrease in [Na⁺]_o (Hazama et al., 1997; Quick et al., 2001).

The cut-open oocyte voltage clamp, with a settling time of <80 µs, was used to examine the rising phase of charge movement in more detail. Representative current records for the hSGLT1-Q457C labeled with tetramethylrhodamine-6 maleimide (TMR6M) are shown in Fig. 6. V_h was –80 mV, and the traces (shown for V_t +50 and –150 mV) were obtained with a sampling interval of 5 µs. In Na⁺, the current rose from an initial low value (close to 0 nA) to a peak (at 0.9 ms for V_t +50 mV) before decaying toward steady state. The time duration at peak current was rather broad, 0.5–1 ms. As in two-electrode voltage clamp experiments (Fig. 5 A), the rise to peak current became less pronounced as V_t was made less positive (not depicted). In the hyperpolarizing direction, the current showed a simple relaxation to steady state. In the OFF response, current decayed from an early time point (Fig. 6 A). In the absence of Na⁺, the rising phase was not observed for depolarizing or hyperpolarizing pulses.

View larger version (21K): [in this window] [in a new window]   FIGURE 6. The rising phase of charge movement in hSGLT1-Q457C. The experiment was performed on TMR6M-labeled Q457C using the cut-open oocyte voltage clamp. The currents have been compensated for membrane capacitance and background current using the P/4 protocol with a Vshp of –150 mV. External and guard solutions contained 100 mM Na-methanesulfonate and internal solution contained 100 K-methanesulfonate. Data was digitized at 5 µs per sample. Vh was –80 mV. The Vt values were +50 and –150 mV. (A) Presteady-state currents in 100 mM [Na+]o. Current trace at +50 mV was averaged from 10 sweeps. The other records were single sweeps. (B) Presteady-state currents 0 [Na+]o. The records were single sweeps. Current and time scales are the same for A and B. (C) –V relations for fast charge. Data is from the experiment of A and B (Vh = –80 mV). The filled symbols are from the ON, and open symbols from the OFF response. The circles (•) are obtained from the decay of the presteady-state current with hyperpolarizing pulses in 100 mM [Na+]o. The triangles () were from the rising phase of the presteady-state current with depolarizing pulses and were obtained using a two exponential fit with the constraint that the time constant of decay was the same as those obtained from the same oocyte with 100-ms pulses. The squares () are the time constants of current decay in the absence of Na+.

Part II. Fluorescence Changes
Slow Fluorescence Changes
The time course of rhodamine fluorescence for a 100-ms voltage pulse using the two-electrode voltage clamp is shown in Fig. 7 A for TMR6M-labeled Q457C in 100 mM [Na⁺]_o. There was a change of fluorescence intensity (F, a decrease for depolarizing and an increase for hyperpolarizing voltages) that returned to the holding level at the end of the pulse. The larger the voltage step, the larger was the F, but the time constant was 10 ms and independent of voltage (see Loo et al., 1998; Meinild et al., 2002). With longer (500 ms) pulses, we observed an additional component (Fig. 7 B). In Fig. 7 B, the 100-ms records (from Fig. 7 A) were split (at 100 ms) and overlaid on the 500-ms records to agree at the onset of the ON and OFF pulses. The dashed traces indicate the F at 100 ms, and a comparison with the fluorescence level at 500 ms shows that the amplitude of the slow component increased with the size of the test voltage step in both depolarizing and hyperpolarizing directions. The amplitude of the slow component during the OFF response increased with the duration of the ON pulse, indicating that the slow component developed sequentially after relaxation of the 10-ms component.

View larger version (34K): [in this window] [in a new window]   FIGURE 7. Slow (Fslow) and medium (Fmed) components of F. The experiment was performed using a two-electrode voltage clamp on a TMR6M-labeled Q457C. Bath solution contained 100 mM Na+. (A) Time course of F for a 100-ms pulse. Vh was –50 mV and the Vt values are indicated next to the traces. (B) The corresponding F records for a 500-ms pulse, and superimposition of the short and long (100- and 500-ms) pulses. The traces from the 100-ms pulses (from A) have been split to overlap with the 500-ms pulses at the onset of the ON and OFF. The dotted lines indicate the F at 100 ms. (C) Fmed and Fslow. The time course of total F (shown for Vt of +90 and –190 mV) was fitted (smooth curves) to two exponential components: F = Fmed (1 – exp(–t/med)) + Fslow (1 – exp(–t/slow)), where Fmed, Fslow, med, and slow are the amplitudes and time constants of the medium and slow components. Parameters obtained from the fit were as follows: at +90 mV, Fmed = –1.17 au, med = 9.9 ms, Fslow = –0.38 au, slow = 102 ms; at –190 mV, Fmed = –1.33 au, med = 7.9 ms, Fslow = –0.36 au, slow = 91 ms. Dashed curves represent the Fmed and Fslow components (from the fit) with the time constants next to the traces. The 's were independent of Vt (between +50 and –150 mV). med was 7.9 ms and 8.5 ms (n = 10) for the ON and OFF responses, respectively. slow ranged between 60 and 150 ms with a mean of 92 ms for the ON, and 138 ms for the OFF. (D) Fmed–V (filled circles) and Fslow (open circles) relations. F (at each Vt) was obtained from fitting the relaxation of F to two exponential components (as in C). The Fmed–V and total F–V relations (sum of Fmed and Fslow) were fitted with the Boltzmann relation with Fmax = 3.00 ± 0.15 au, z = 0.4 ± 0.1, V0.5 = –61 ± 4 mV; and Fmax = 3.86 ± 0.24 au, z = 0.4 ± 0.1, V0.5 = –61 ± 5 mV.

Fig. 7 D shows a plot of the amplitudes of F_slow and F_med vs. voltage. The F_med–V relation was sigmoidal with a z of 0.4 and V_0.5 of –61 mV. The slow component increased with the size of the voltage jump, but we were unable to obtain a Boltzmann fit to the F_slow–V relation because of the low amplitudes and scatter. Nevertheless, inspection of the OFF records (Fig. 7 B) suggests that V_0.5 is similar for the slow and medium components, i.e., the symmetry between the OFF responses to depolarizing and hyperpolarizing test pulses is similar for the medium and slow component. The maximal observed fluorescence change, the difference between the F's at the largest depolarizing and hyperpolarizing voltages (+90 and –190 mV), was much larger for F_med than F_slow (ratio was 5). The sum of the two components, F_total, gave a z of 0.4 and V_0.5 of –61 mV (Fig. 7 D).

The 100- and 500-ms fluorescence records at various [Na⁺]_o are shown in Fig. 8 A. They are overlaid (as in Fig. 7 B). The slow component is evident at each Na⁺ concentration. The dependence of F_med on Na⁺ is shown in Fig. 8 B. The F_med–V relations obeyed the Boltzmann relation. z was 0.4 and independent of [Na⁺]_o. As [Na⁺]_o was reduced, there was no apparent change in maximal fluorescence (F_med^max), but there was a shift of V_0.5 to more negative values: from –30 mV at 100 mM Na⁺ to –99 mV at 25 mM Na⁺. In Na⁺-free solutions, the F_med–V curve did not saturate even at the most negative voltage applied (–150 mV). When V_0.5 was plotted against [Na⁺]_o, the slope yielded a 100-mV shift in V_0.5 per 10-fold decrease in Na⁺ concentration (unpublished data), indicating that in Na⁺-free solutions, the V_0.5 is more negative than –200 mV. The dashed line represents the Boltzmann relation in the absence of Na⁺, with V_0.5 of –200 mV, F_max of 1, and z of 0.4. The close agreement with the Na⁺-free data indicates that in Na⁺, the medium fluorescence can be largely attributed to the empty transporter.

View larger version (34K): [in this window] [in a new window]   FIGURE 8. Dependence of the slow and medium components of F on [Na+]. (A) Time course of F when [Na+]o was 100, 25, and 0 mM. [Na+]o was varied by choline replacement. The records from 100- and 500-ms pulses are overlaid, with the 100-ms records split (at 100 ms) to align with the 500-ms records at the onset of the ON and OFF pulses (see Fig. 7 B). Data were collected from a single oocyte, and all three panels share the abscissa and ordinate scales. The 500-ms records were fitted to F = Fmed (1 – exp(–t/med)) + Fslow (1 – exp(–t/slow)). The 's obtained are independent of Vt. For ON, respectively at 100, 50, 25, and 0 mM [Na+]o, med was 11.6 ± 0.2 ms (n = 9), 11.4 ± 0.3 ms (n = 10), 11.3 ± 0.4 ms (n = 9), and 10.9 ± 0.3 ms (n = 10); and slow was 149 ± 21 ms (n = 7), 167 ± 28 ms (n = 6), 159 ± 44 ms (n = 6), and 146 ± 22 ms (n = 8). For OFF, med was 8.0 ± 0.2 ms (n = 10), 9.6 ± 0.4 ms (n = 10), 10.0 ± 0.4 ms (n = 9), and 10.2 ± 0.4 ms (n = 10); and slow was 154 ± 41 ms (n = 7), 184 ± 38 ms (n = 8), 119 ± 45 ms (n = 7), and 146 ± 48 ms (n = 7). (B) F–V relations for the medium component (Fmed). Fmed and Fslow were obtained by curve fitting (described in A). At each [Na+]o, the Fmed–V curves were fitted (smooth curves) to the Boltzmann relation. At 100 mM [Na+]o, z = 0.4 ± 0.03, and V0.5 = –30 ± 3 mV. At 50 mM [Na+]o, z = 0.4 ± 0.07, and V0.5 = –59 ± 6 mV. At 25 mM [Na+]o, z = 0.4 ± 0.15, and V0.5 = –99 ± 31 mV. For comparison, the curves have been normalized to the maximal extrapolated (slow-compensated) fluorescence change (Fmax) observed in 100 mM [Na+]o and have also been shifted to align at the extrapolated depolarizing limit (see Loo et al., 1993; Meinild et al., 2001). The dotted line at 0 Na+ was the Boltzmann relation with the same Fmax and z (0.4) as 100 mM [Na+], and V0.5 of –200 mV.

Fast Fluorescence Changes
A fast component of fluorescence was observed using the cut-open oocyte voltage clamp. This is illustrated in Fig. 9 A, which shows the time course of F in 100 mM [Na⁺]_o. The experiment was performed on the same oocyte as Fig. 6 with membrane potential held at –80 mV. With onset of the voltage pulse, there was a fast initial change of fluorescence. The kinetics became faster when Na⁺ was removed from the external solution (Fig. 9 C). In both Na⁺ and Na⁺-free solutions, increasing the pulse duration to 40 ms revealed an additional component with a time constant 10 ms (Fig. 9, B and D). The amplitude of F was dependent on [Na⁺]_o; maximal change of fluorescence (between +50 and –150 mV) decreased 64% (from 6.3 to 4.0 arbitrary units) when Na⁺ was removed (Fig. 9 D).

View larger version (38K): [in this window] [in a new window]   FIGURE 9. Fast fluorescence changes. Data was obtained using the cut-open oocyte on a TMR6M-labeled Q457C. External and guard solutions contained 100 mM Na-methanesulfonate and internal solution contained 100 mM K-methanesulfonate. Subtracting holding potential (Vshp) was –150 mV. The records were the averages of 20 sweeps. Data was digitized at 5 µs per sample (A) and 50 µs per sample (B). C and D show the time course of F in Na+-free solution (choline replacement). Digitizing rate was 5 µs per sample (C) and 50 µs per sample (D).

View larger version (25K): [in this window] [in a new window]   FIGURE 10. Voltage dependence of the fast and medium components of F. Time course of F (for a 5-ms pulse) at Vt of +50 and –150 mV (Vh = –80 mV) in 100 mM [Na+]o (+Na) or 0 Na+ (–Na). Data is from the experiment of Fig. 9. The fluorescence records were fitted to two exponential components (denoted by fast and medium). The number next to each trace is the time constant of the fast component (fast). A and B show the time courses of F (for ON [A] and for OFF [B]). (C) –V relations for fast (ON). Open symbols (0 Na+) were obtained from 5-ms pulses, and filled symbols (+Na) were from 40-ms pulses. (D) –V relations for fast (OFF). Data were from 5-ms pulses. (E and F) –V relations for med (for ON [E] and for OFF [F]). Data were from 40-ms pulses.

View larger version (18K): [in this window] [in a new window]   FIGURE 11. Na+ dependence of the fast and medium components of F. The fast and medium fluorescence amplitudes were obtained by fitting the time course of F to two exponential components (compare, Fig. 7). (A) F–V relation for the fast component. The curve is the fit of the data in 100 mM Na+ to the Boltzmann relation with Fmax = 7.0 au, z = 0.6, and V0.5 = –48 mV. The F–V curve in 0 Na+ has been shifted to align with that of 100 mM Na+ at +50 mV. (B) F–V relation for the medium component. The curve is the fit of the data in 100 mM Na+ with Fmax = 3.8 ± 1.9 au, z = 0.5 ± 0.3, and V0.5 = –100 ± 47 mV. The F–V curve in 0 Na+ has been shifted to align with that of the 100 mM Na+ at +50 mV.

Part III. Correlation between Charge Movement and Fluorescence Changes
Medium and Slow Charge and Fluorescence Changes
The time course of slow charge and fluorescence in the presence of Na⁺ (100 mM) are compared in the experiment of Fig. 12 A. Shown are the records in response to 750-ms hyperpolarizing voltage pulses from –50 mV (V_h) to –110, –130, and –150 mV. For F, the time constants (of the ON response) were 9 and 169 ms. For comparison of the slow components, the concurrent charge and fluorescence records were normalized to 75 and 750 ms. Within this interval, there was a close agreement between the two time courses. We were unable to compare slow charge and fluorescence at large depolarizing voltages (more positive than +10 mV) because of the high endogenous chloride currents of the oocytes. In the absence of Na⁺, there was also a fair agreement between slow charge and slow fluorescence (Fig. 12 B).

View larger version (27K): [in this window] [in a new window]   FIGURE 12. Correlation between slow charge and fluorescence. Experiment was performed using a two-electrode voltage clamp on TMR6M Q457C. (A) Correlation between slow charge and fluorescence (red traces) in 100 mM [Na+]o. The charge record has been normalized to agree with F at 40 and 750 ms. Shown are records at hyperpolarizing voltages of –110, –130, and –150 mV from Vh = –50 mV. F contained two voltage-independent time constants of 9 ± 1 ms (n = 10) and 169 ± 19 ms (n = 7) for the ON, and 9 ± 1 ms (n = 10) and 154 ± 9 ms (n = 9) for the OFF response. slow for charge (ON) was 104 ± 10 ms (n = 3). (B) Correlation between slow charge and slow fluorescence in Na+-free solution. Pulse duration was 300 ms, and Vh was –50 mV. The current records have been normalized to agree with the fluorescence at 50 and 300 ms (as in A). F (for the ON pulse) contained two voltage-independent time constants of 11 ± 1 ms (n = 8) and 144 ± 15 ms (n = 8). slow for charge (ON) was 63 ± 8 ms (n = 3).

View larger version (19K): [in this window] [in a new window]   FIGURE 13. Correlation between medium charge and fluorescence. Experiment was performed using a two-electrode voltage clamp on a TMR6M-labeled Q457C in 100 mM NaCl buffer. Vh was –50 mV. The time course of Q and F are compared at +30 and –150 mV. Q was obtained from the total current by subtraction of the steady-state current and the oocyte membrane capacitive transient. The traces have been normalized to agree at the end of the voltage pulse (75 ms). The numbers next to the traces are the time constants of Q and F.

View larger version (20K): [in this window] [in a new window]   FIGURE 14. Correlation between fast charge and fluorescence. The experiment was performed using the cut-open oocyte on TMR6M-labeled Q457C (from the oocyte of Fig. 6). Membrane potential was held at –80 mV and stepped to +50 mV. (A) Comparison of the rising phase of the presteady-state current (I) and fluorescence (F) in 100 mM [Na+]o. I is from Fig. 6 A and F is from Fig. 9 A. (B) Comparison of charge and F in absence of Na+. I is from Fig. 6 B and F is from Fig. 9 C.