Correspondence to Donald Loo: dloo{at}mednet.ucla.edu
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ABSTRACT |
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Key Words: Na+/glucose cotransport presteady-state kinetics charge movement fluorescence conformational changes
1 Since the V0.5 for charge is very negative in the absence of Na (
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INTRODUCTION |
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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 335 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 ( = 30200 ms) and medium (320 ms) components contribute equally to total charge transfer, while the fast (0.21.5 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 (C1a and C1b) between the two final states C1 and C6 in the external and internal membrane surfaces (C1
C1a
C1b
C6). 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, C1a, C1b, 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).
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MATERIALS AND METHODS |
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The two-electrode voltage clamp, with a settling time 0.61.0 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 V0.5 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 34 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 (2023°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 0.22 and 35160 ms. For clarity of presentation, we operationally defined the fast, medium, and slow components (for charge and fluorescence) as those with time constants between 0.22, 220, and 30160 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
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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 (Ki
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 (Vshp) of 150 mV where there was negligible SGLT1 charge movement.1
Fitting of QV and FV Relations.
The charge vs. voltage (QVm) relations could, to a first approximation, be fitted to a single Boltzmann function (Loo et al., 1993; Hazama et al., 1997
):
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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.
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RESULTS |
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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 (Vh) and stepped to a test value (Vt, from +50 to 150 mV, and representative traces are shown) for 100 ms before returning to Vh. The current contained (a) an initial spike, with a time constant of 0.71.0 ms; (b) hSGLT1 presteady-state currents, which decayed to an apparent steady state with time constants of 325 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.
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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 Vh. 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).
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The medium and slow charges (Qmed and Qslow) 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 V0.5 = 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 QV relation for total charge could be fitted by the Boltzmann relation with z
= 1.0 and V0.5 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).
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When external Na+ concentration ([Na+]o) was reduced from 100 to 12 mM, the time to peak (tpeak) was reduced (Fig. 5 B). For the +50 mV pulse illustrated, tpeak 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 tpeak 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 Qmax, z
, and V0.5 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 (V0.5) of the QV 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. Vh was 80 mV, and the traces (shown for Vt +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 Vt +50 mV) before decaying toward steady state. The time duration at peak current was rather broad, 0.51 ms. As in two-electrode voltage clamp experiments (Fig. 5 A), the rise to peak current became less pronounced as Vt 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.
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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.
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Fig. 7 D shows a plot of the amplitudes of Fslow and
Fmed vs. voltage. The
FmedV relation was sigmoidal with a z
of 0.4 and V0.5 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
FslowV relation because of the low amplitudes and scatter. Nevertheless, inspection of the OFF records (Fig. 7 B) suggests that V0.5 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
Fmed than
Fslow (ratio was
5). The sum of the two components,
Ftotal, gave a z
of 0.4 and V0.5 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 Fmed on Na+ is shown in Fig. 8 B. The
FmedV 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 (
Fmedmax), but there was a shift of V0.5 to more negative values: from 30 mV at 100 mM Na+ to 99 mV at 25 mM Na+. In Na+-free solutions, the
FmedV curve did not saturate even at the most negative voltage applied (150 mV). When V0.5 was plotted against [Na+]o, the slope yielded a 100-mV shift in V0.5 per 10-fold decrease in Na+ concentration (unpublished data), indicating that in Na+-free solutions, the V0.5 is more negative than 200 mV. The dashed line represents the Boltzmann relation in the absence of Na+, with V0.5 of 200 mV,
Fmax 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.
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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).
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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 (Vh) 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).
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DISCUSSION |
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In the case of SGLT1, our previous studies have established that the distribution of the human isoform (hSGLT1) between two stable conformations, Na+-bound outward facing (C2) and the Na+-free inward facing (C6, Fig. 15 A), is voltage dependent. hSGLT1 charge movement as a function of membrane voltage is described by a Boltzmann relation with a V0.5 of 50 mV (Loo et al., 1993
; Quick et al., 2001
). The voltage dependence of the C2-C6 distribution was independently confirmed with optical studies of hSGLT1 (Loo et al., 1998
; Meinild et al., 2002
). Here we have expanded the study of the transitions between C2 and C6 using a voltage perturbation protocol where we monitored SGLT1 charge and fluorescence from microseconds to seconds. The resolution of our previous voltage clamp experiments on SGLT1 expressed in oocytes has been limited due to the speed of the clamp (
1 ms) and the limited duration of the voltage pulse (typically 100 ms). We now have recorded transients that have revealed hitherto unexpected features of the transitions between C2 and C6, and these provide new insights into the partial reactions of the transporter. Confidence in the new charge recordings is enhanced by the concurrent optical recordings since fluorescence measurements are subject to different constraints than charge measurements; charge is extracted from SGLT1 capacitive currents within a background of plasma membrane capacitive currents, SGLT1 ionic currents (uniporter currents), and membrane leakage currents (Parent et al., 1992
; Loo et al., 1999
), whereas SGLT1 optical records are measured against a constant background fluorescence (Loo et al., 1998
; Meinild et al., 2002
).
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Charge Movement
Slow Charge.
Whereas previous studies (Loo et al., 1993; Hazama et al., 1997
; Quick et al., 2001
) have documented SGLT1 charge movement between C2 and C6 with time constants in the range 220 ms (medium component), we now have clearly demonstrated an additional slow component with time constants in the range of 100 ms (Fig. 4 A; Table I). The slow charge movements were found in the presence and absence of Na+ (Fig. 12, A and B), indicating that they are associated with the unloaded carrier. A major consequence of this new slow component is that we have previously overestimated the turnover rate of SGLT1. The turnover rate is estimated from the ratio (Imax/Qmax) of the maximal rate of sugar transport (Imax) and maximal charge (Qmax). Previous estimates for human SGLT1, 57 s1, were based on the medium component (Loo et al., 1993
). Since the total charge (Qmax) is doubled with inclusion of the slow component, the turnover rate of human SGLT1 is reduced by 50%, to 28 s1.
Our finding of a slow charge (in the presence and absence of Na+) in human SGLT1 is consistent with reports of time constants up to 50 ms in rabbit SGLT1 (Krofchick and Silverman, 2003). Since the rabbit currents were obtained from 100-ms pulses, they are undoubtedly underestimated (Krofchick and Silverman, 2003
). In long pulse protocols on rabbit SGLT1, we have found that the slow component required
80 ms at +20 mV and
500 ms at 100 mV to reach steady state (unpublished data).
The time constants of the slow component (Table I) has important operational consequences for the protocol used in charge movement studies; i.e., the test voltage pulses need to be separated by interpulse intervals greater than five times the slowest time constant (or 500 ms). This may, in part, explain the variations in the kinetics of charge movement observed using different protocols (present study; Chen et al., 1996
; Krofchick and Silverman, 2003
).
Medium Charge.
The medium charge, with between 3 and 35 ms (Table I), has been previously described for human, rabbit, and rat SGLT1 (Loo et al., 1993
, 1998
; Panayotova-Heiermann et al., 1995
; Chen et al., 1996
; Hazama et al., 1997
; Quick et al., 2001
; Meinild et al., 2002
; Krofchick and Silverman 2003
; Krofchick et al., 2004
). In the present study, the current records for the medium component (for hSGLT1 and TMR6M-labeled hSGLT1 Q457C) were corrected for the slow component (Fig. 2, A and C), and the
V and QV relations obtained are similar to those described previously on hSGLT1 and the TMR6M-labeled hSGLT1 Q457C from uncorrected records (Loo et al., 1993
; Chen et al., 1996
; Hirayama et al., 2001
; Quick et al., 2001
; Meinild et al., 2002
). In the case of the rabbit isoform, the time constant for medium charge can be simulated by the kinetic model for SGLT1 (Hazama et al., 1997
).
The slow and medium charge movements were equal in magnitude (Fig. 4, B and C). Their time constants showed similar voltage dependences; they were voltage independent for hyperpolarizing voltages and decreased with depolarizing voltages (Fig. 4 A). For both ON and OFF, z was identical between medium and slow charge, but V0.5 was 33 mV for medium and 67 mV for slow. The total charge vs. voltage (QV) relation was described by a Boltzmann function with two states and a single steepness factor (z
) of 1.0. Since total charge consists of three components, the QV relation could, in principle, be fitted by a Boltzmann function with four states. Chen et al. (1996)
obtained a z
of 0.70.8 for human SGLT1 (in 90 mM [Na+]o), and as slow charge was not reported, this represented the sum of the fast and medium components. Krofchick et al. (2004)
modeled the QV relation of rabbit SGLT1 (for total charge with fast, medium, and slow components) by a Boltzmann function with three states (and two steepness factors) and obtained z
values of 0.45 and 0.97 and V0.5 values of 7 and 49 mV. A proper analysis of Boltzmann distributions with multiple states requires experimental protocols that take into account the range of the time constants.
Fast Charge.
Our previous consideration of fast hSGLT1 charge movements, i.e., components with time constants <2 ms, have been fraught with uncertainty due to large membrane capacitive transients with a time constant of 0.5 ms. This has been clarified by use of the phlorizin subtraction protocol (Fig. 5, A and B) and the cut-open oocyte voltage clamp with a settling time of 80 µs (Fig. 6). We observed two components of fast charge: (1) a fast decay for hyperpolarizing pulses in the presence of Na+, and both hyperpolarizing and depolarizing voltage pulses in the absence of Na+; and (2) a rising phase with large depolarizing pulses in the presence of Na+. As will be discussed (below), these fast components indicate that there are two fast conformational changes of hSGLT1 between C2 and C6 (Fig. 15), and the dominant component depends on the experimental condition. The time constant of the fast decay is 0.21.5 ms for ON, and 1.6 ms for OFF (Table I). A fast decay (with
0.40.8 ms in the presence and absence of Na+) has been reported by Chen et al. (1996)
using the cut-open oocyte, and suggested by Krofchick and Silverman (2003)
(
0.51.0 ms) using phlorizin subtraction and two-electrode voltage clamp.
The fast rising phase is a novel finding of this study. The time constant was 200 µs, Na+ dependent and voltage independent (Fig. 5 B; Fig. 6 B). Hints of a rising phase have been observed experimentally in human SGLT1 using the two-electrode voltage clamp (Loo et al., 1993) and in model simulations for rabbit SGLT1 (Hazama et al., 1997
). The rising phase has not been reported by other investigators, largely because kinetics were studied using conditioning prepulses of only 40 ms (Chen et al., 1996
) and with hyperpolarizing pulses and/or with OFF responses (Chen et al., 1996
; Krofchick and Silverman, 2003
). The rising phase has confounded our attempts to estimate the contribution of fast charge to total charge. A lesson gained from the present study is that given the wide range of time scales, for kinetic studies, voltage pulses of different durations need to be applied and extra caution taken to ensure that steady-state conditions are reached for accurate estimation of presteady-state kinetics.
Dependence of Charge on Na+.
The kinetics of charge movement was dependent on external Na+. With decreasing [Na+], there was a shift of QV curve. For example, for the medium component, V0.5 shifted negative (100 mV/10-fold reduction in [Na+]o) with no change in z
(Loo et al., 1993
, Fig. 3; Hazama et al., 1997
, Fig. 3; Quick et al., 2001
, Fig. 3 B). There was little change in maximal charge (Qmax) until [Na+] was below 25 mM (Loo et al., 1993
, Fig. 3; Hazama et al., 1997
, Fig. 3; Quick et al., 2001
, Fig. 3 B). In the absence of Na+, there was an apparent loss of charge (Fig. 5, A, B, and D) due to the shift of the QV curve out of working range (between 150 and +50 mV). The medium charge observed was 50% of that observed in 100 mM Na+ (Fig. 5 D).
The time constants for all three components of charge decreased with [Na+] (Table I). For example, as [Na+] is reduced from 100 to 0 mM, for fast decay, decreased from 1.6 to 0.2 ms (Fig. 6 C); for medium charge,
decreased from 14 to 3 ms (Fig. 5 C); and for slow charge,
decreased from 104 to 63 ms. For the rising phase with depolarizing pulses, the time to peak decreased with reducing [Na+], in part due to a decrease in the time constant of the medium component (Fig. 5 B).
Q457C-TMR6M Fluorescence
Similar to charge, there were three components of fluorescence (Table I): slow ( 100 ms), medium (10 ms), and fast (1 ms). The maximal fluorescence (
Fmax) of the slow component was 20% of the medium. The fast fluorescence was extracted from the medium using the cut-open oocyte preparation (Fig. 10), and the dependence of medium fluorescence on [Na+] observed in two-electrode voltage experiments (Fig. 8 B) was actually due to the Na+ sensitivity of the fast component (Fig. 11 A). Since
Fmax for fast fluorescence increased threefold between 0 and 100 mM Na+ (Fig. 11 A), we estimate that in 100 mM Na+, 60% of the total
F was fast, 20% medium, and 20% slow. In contrast,
50% of the total charge was medium and
50% slow (fast charge was small). Thus there is a small charge associated with a large change in fast fluorescence.
In Na+, V0.5 was 48 mV for the fast (Fig. 11 A), and 100 ± 50 mV (Fig. 7 B and Fig. 11 B) for the medium and slow components of fluorescence. For medium and fast fluorescence, z was
0.4 (see Fig. 4, Meinild et al., 2002
; Fig. 7 D; Fig. 11), and the time constants for all three components of fluorescence were voltage independent, with
ON and
OFF generally equal (Fig. 10; Table I). We were unable to precisely determine the Boltzmann parameters for the slow component, largely due to the low expression of this mutant.
F as a Function of Na+.
The time constants for fluorescence showed some dependence on Na+. For the slow component, was 169 ms vs. 144 ms in Na+ free (Table I). For medium component,
was 19 ms in presence and absence of Na+. For the fast component,
was 1.5 ms vs. 0.2 ms in Na+ free. Charge and fluorescence changes as a function of Na+ suggest that most if not all charge and fluorescence changes are due to conformational changes of the unloaded carrier, i.e., C1C6 transitions. A more thorough investigation of the fast transients was made possible by the fluorescence experiments. In the absence of Na+, there was a fast component of the fluorescence,
300500 µs, that is independent of voltage (Fig. 10 C), but the ON response is slowed by the presence of external Na+ to 2 ms (Fig. 10 C). This observation is consistent with the Na+ dependence of the rising phase of charge movement (Fig. 6 C). A fast component of the fluorescence change,
400 µs is nevertheless observed for the OFF response in Na+, but only for the depolarizing voltage pulses (Fig. 10 D).
Charge vs. Fluorescence Measurements
Charge and fluorescence measurements provide complementary views of the global/local conformational changes. The following observations indicate that to each charge state, there is a corresponding fluorescence state, and a closely associated conformational change to each component of charge movement: (a) the V0.5 for total charge (50 mV) was similar to that for total fluorescence (present study; Meinild et al., 2002
); (b) the shift of the V0.5 with varying [Na+], 100 mV/10-fold reduction in [Na+], was the same for charge as for fluorescence (present study; Meinild et al., 2002
); (c) the time constants of each component of charge movement were in the same range as those for fluorescence; and (d) the time constants of fast charge and fluorescence showed a decrease with reducing [Na+].
The major difference between charge and fluorescence measurements was their dependence on voltage. (a) For the voltage dependence of the Boltzmann relation, z was 0.4 for fluorescence vs. 1.0 for charge. (b) We have found that z
, which provides a measure of the voltage dependence of the global protein conformations, was the same (
1) for SGLT1 mutants (unpublished data). On the other hand, z
for fluorescence, which depended on the local environment of the fluorophore, depended on the location of TMR6M; e.g., z
for medium fluorescence of TMR6M-labeled D454C was 0.6 (Díez-Sampedro et al., 2004
). (c) The time constants for charge were voltage dependent (for medium and slow components), while those for fluorescence were voltage independent. We do not yet understand the mechanism whereby the time constants of the fluorescence changes were independent of voltage but the steady-state
FV relations were voltage dependent.
While there was good agreement between the time course of charge and fluorescence for the fast and slow components (in the hyperpolarizing direction), there was a difference in the medium component (Fig. 13); in the hyperpolarizing direction, fluorescence led charge movement, indicting that the change in local environment of the fluorophore at residue 457 preceded the conformational changes that generate the charge movement. The situation is reversed in the depolarizing direction with charge movement leading fluorescence. These differences between charge and fluorescence depended on the location of the fluorophore on the protein. Preliminary experiments on mutant protein hSGLT1 I443C (labeled with TMR6M) indicated a close agreement between charge and fluorescence for depolarizing and hyperpolarizing pulses (unpublished data). Further studies on the relation between charge movement and fluorescence at different locations in the protein may enable us to obtain a spatial and temporal map of the conformational changes of the cotransporter.
Concurrent charge and fluorescence experiments have been performed on the glucose (Loo et al., 1998; Meinild et al., 2002
),
-aminobutyric acid (GAT1; Li et al., 2000
), serotonin (SERT; Li and Lester, 2002
), and glutamate transporters (EAAT3; Larsson et al., 2004
), and the general agreement between charge and fluorescence and their dependence on substrates confirmed, in real time, that conformational changes are involved in transporter function. These studies have also revealed differences between charge and fluorescence, e.g., there were fluorescence changes in GAT1 (Li et al., 2000
), SERT (Li and Lester, 2002
), and EAAT3 (Larsson et al., 2004
) without a corresponding charge movement. In SGLT1, the changes in the local environment at residue 457C (as monitored by fluorescence) closely mirrored the global conformational changes of the protein.
The slow time constant for SGLT1 is in the range for the Na+/Cl/GABA family, such as GAT1 (Mager et al., 1993, 1996
; Loo et al., 2000
; Whitlow et al., 2003
). The turnover rate is comparable to the turnover rate for SGLT1, indicating that reorientation of the empty transporter is the rate-limiting step for both families of cotransporters.
Kinetic Model for Na+/Glucose Cotransport
In the absence of external glucose and low internal [Na+], the six-state model for Na+/sugar cotransport is reduced to the three-state model for charge movement, and the three-state model accounted qualitatively for the medium QV (Boltzmann) and V relations, and the shift of these relations with reducing [Na+]o (Fig. 5, C and D; Loo et al., 1993
; Hazama et al., 1997
). The model also predicted a rising phase of charge movement with depolarization at high [Na+] (Hazama et al., 1997
). A rising phase is developed when the charge transfer associated with the empty carrier (C1
C6) is faster and greater than the charge associated with Na+ binding/dissociation (C2
C1). Na+ binding/dissociation is apparently the slower step because of the fast "back" reaction of Na+ binding (C2
C1). In absence of Na+, the "back" reaction is absent, and the rising phase is not observed. This also explains the decrease of the time to peak of the medium component as external [Na+] is reduced (Fig. 5 B). The rising phase is not observed at hyperpolarizing voltages because the fastest charge is moved during the first step (C6
C1).
A rising phase has been observed in the gating currents of Shaker K+ channels (Bezanilla et al., 1991; Stefani et al., 1994
). Like the charge movement of SGLT1, the development depended on membrane voltage; the rising phase was not observed for small depolarizations (
20 mV) from the holding potential (80 mV), but became pronounced with large depolarization voltages more positive than +50 mV (Fig. 1 of Bezanilla et al., 1991
). The development of the rising phase (of charge movement) in SGLT1 and (gating current) in Shaker K+ channels is due to the increase in the rate of charge transfer with positive membrane voltages and the majority of the charge being carried by the later step (Bezanilla et al., 1991
; Parent et al., 1992
; Loo et al., 1993
; Stefani et al., 1994
; Hazama et al., 1997
).
A simplifying assumption of the three-state model (for C2C6 transitions) involves the Na+ binding step; the two Na+ binding reactions (C1+Na+C1Na; C1Na+Na+
C2) are lumped into one, C1+2Na+
C2 (Hazama et al., 1997
; Parent et al., 1992
; Loo et al., 1993
). This one-step approximation at high [Na+] is supported by the observation of high cooperativity between the two Na+ binding sites (Parent et al., 1992
; Falk et al., 1998
; Mackenzie et al., 1998
; Meinild et al., 2002
). However, the simplified model runs into difficulties when external [Na+] is lowered. The model predicted a smaller shift of the V0.5 with reducing [Na+] than experimentally observed (Hazama et al., 1997
). In computer modeling, Falk et al. (1998)
found that replacement of the two Na+ binding steps by a single step results in a Na+-dependent pseudo rate constant from C1 to C6, and this leads to incorrect predictions as external [Na+] is reduced. Thus for low Na+ concentrations, the two Na+ binding steps need to be included.
In view of the additional fast and slow components of charge observed in the absence of Na+ (for a total of three time constants), the single step transition between C1 and C6 needs to be extended. Two additional intermediate states (in addition to end states C1 and C6) are required for the empty transporter: C1C1a
C1b
C6. Together with the binding of two Na+ ions to C1 to form C2 a total of at least seven states are required to describe the presteady-state kinetics of SGLT1 (Fig. 15 B). Thus, in saturating [Na+] at hyperpolarizing voltages (eg., 150 mV) where there is a high occupancy in C2, when membrane voltage is stepped to large positive values, the protein goes through more than three intermediate states before reaching C6 (C2a, C2b, C1, C1a, C1b). The estimation of 16 rate constants (each with unknown voltage dependences [z
's] for a total of 32 parameters) of a seven-state kinetic scheme is a formidable task.
Our computer simulations suggest that with the current knowledge no one set of kinetic parameters can account for the charge movement between C2 and C6. In order to illustrate the simulations we present one example in Fig. 15 C. Here we make the assumptions that (a) the two sodium ions bind to the transporter in one step; (b) the rates of the conformational changes are fast (C2C1), medium (C1
C1a), slow (C1a
C1b) and fast (C1b
C6); (c) all the transitions from C2 to C6 are voltage dependent; and (d) for transitions from C6 to C2, only the first (C6
C1b) and last (C1
C2) steps are voltage dependent. Fig. 16 A shows the predicted charge movements in 100 mM Na+ for 10-ms voltage pulses ranging from 80 (Vh) to +90 and 150 mV, and Fig. 16 C shows the time constants as a function of the test potential. Four components of charge are predicted: two fast and difficult to discriminate between, and medium and slow components with voltage dependencies similar to those observed experimentally (compare Fig. 4 A). The fast components (0.22 ms) are relatively insensitive to voltage. The medium component has time constants decreasing from 25 to 3 ms from 150 to +50 mV. The slow component has time constants ranging from 90 to 25 ms from 150 to +50 mV. A rising phase is also predicted for depolarizing voltage steps with a peak current at 0.5 ms. In the absence of Na+, the rising phase is not present. Only three components of charge are predicted, and there is a decrease in the time constants for the medium and slow components (Fig. 16, C and D). It can also be deduced from Fig. 16 (A and B) that on removing Na+, the V0.5 for charge movement does not shift to the measured value (200 mV). The failure of the model to predict the change in V0.5, similar to the failure of the three-state model (Hazama et al., 1997
), is likely due to our assumption that the binding of two Na+ ions can be represented as a single rate constant (see also Falk et al., 1998
).
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Our assumptions about the relative rates of the conformational transitions of the empty carrier (from C1C1a
C1b
C6) are an extension of our original two-state model (Hazama et al., 1997
) and the three-state models proposed by Chen et al. (1996)
and Krofchick and Silverman (2003)
to account for the fast and medium charge movements. Both of the latter groups postulated an intermediate state C1a between C1 and C6 to account for the fast charge in the absence of Na+, and a medium transition between C1 and C1a. Our results require an additional intermediate C1b and a slow transition between C1a and C1b.
The simulation in Fig. 16 also makes the following predictions. (a) The observed asymmetry of voltage dependence of the forward and backward reactions of the transitions between C1 and C6. The medium and slow conformational changes are voltage dependent in the depolarizing direction (C1C1a
C1b) but voltage independent in the hyperpolarizing direction (C1
C1b
C1b). This is consistent with our assumption that for transitions from C1 to C6, the major voltage-dependent step (major charge movement) is C1
C1a, and for transitions from C6 to C1, the major voltage-dependent step is C6
C1b.
(b) In the presence of Na+, the system behaves energetically as though it has two equilibrium states, C2 and C6. This suggests that C1, C1a, and C1b are unstable intermediates or states with extremely low occupancy. The instability of C1 is also supported by our previous findings that in the absence of Na+, the estimated V0.5 values for the medium QV and FV relations are
200 mV (Meinild et al., 2002
). Stabilization of the unloaded carrier in the outward-facing conformation by the activating cation (Na+ or H+) appears to be a common mechanism for ion-coupled transporters, e.g., the Na+/Cl/
-aminobutyric acid (GAT1; Hilgemann and Lu, 1999
), H+/dipeptide (PEPT1; Mackenzie et al., 1996
), glutamate (EAAT; Wadiche et al., 1995
), Na+/Pi cotransporter (NaPi; Forster et al., 2002
), and H+/lactose transporters (Abramson et al., 2003
).
(c) The rising phase of charge movement is due to the transition C2C1
C1a. The implication is that the charge associated with the C1 to C1a transition is greater than that with the dissociation of Na+, C2
C1. The observation of a rising phase (with large depolarizing steps) requires that the major charge be associated with the empty transporter.
(d) A charge associated with Na+ binding/dissociation. This is not observed experimentally, although it is indicated in the simulation (compare Fig. 15, A and C). This suggests that the predicted charge is either extremely fast, within 80 µs (settling time of the cut-open oocyte voltage clamp), or that the amount of charge associated with this step is very small (beyond the resolution of the current measurements).
Conclusions
Our application of perturbation methods to SGLT1 by recording charge and fluorescence signals upon step jumps in membrane voltage have permitted the study of partial reactions of the transport cycle. In the absence of sugar and Na+, we have obtained evidence for four states (C1, C1a, C1b, and C6). Transitions between C1 and C6 carried most of the charge, and are the major voltage-dependent steps in the overall Na+/glucose cotransport cycle. C1, C1a and C1b are unstable conformations. Transitions between C1 and C6 contribute the most to charge and F measurements, i.e., the major voltage-dependent step in the overall transport cycle is the "return" of the unloaded carrier between the interior and exterior surfaces of the membrane. The kinetic states (obtained from charge movement studies) are also supported by the fluorescence measurements.
In general, TMR6M bound to Q457C exhibited fluorescence changes with a time course similar to charge movement. This suggests that global conformational changes monitored by charge movement are reflected by conformational changes at or near Q457C. However, there are subtle differences: the local conformations occurred BEFORE charged transitions from C6 to C2 and AFTER charged transitions from C2 to C6. Finally, the combination of charge and fluorescence recordings at different locations in the protein may be used to construct a spatial map of the conformational changes of the transporter.
Major unresolved questions include the charge associated with Na+ association and dissociation with the transporter (very fast and/or very small), the order of the binding and dissociation of the two Na+ ions, and the nature of the cooperativity between the two Na+ binding sites. Resolving these issues has important implications for both the kinetics of SGLT1 charge movement and the kinetics of Na+/glucose cotransport.
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ACKNOWLEDGMENTS |
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This research was supported by National Institutes of Health grant DK19567.
Olaf S. Andersen served as editor.
Submitted: 15 July 2004
Accepted: 2 November 2004
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REFERENCES |
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