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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS
1Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, Cincinnati, Ohio; 2Membrane Biology Program and Renal Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts; and 3Institute of Biochemistry and Molecular Medicine, University of Berne, Bern, Switzerland
Submitted 21 September 2007 ; accepted in final form 18 December 2007
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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70 µM), temperature dependent (Q10 5), and energized by the Na+ electrochemical potential gradient. We obtained a Na+-L-ascorbic acid coupling ratio of 2:1 from simultaneous measurement of currents and fluxes. L-Ascorbic acid and Na+ saturation kinetics as a function of cosubstrate concentrations revealed a simultaneous transport mechanism in which binding is ordered Na+, L-ascorbic acid, Na+. In the absence of L-ascorbic acid, SVCT1 mediated pre-steady-state currents that decayed with time constants 3–15 ms. Transients were described by single Boltzmann distributions. At 100 mM Na+, maximal charge translocation (Qmax) was 25 nC, around a midpoint (V0.5) at –9 mV, and with apparent valence –1. Qmax was conserved upon progressive removal of Na+, whereas V0.5 shifted to more hyperpolarized potentials. Model simulation predicted that the pre-steady-state current predominantly results from an ion-well effect on binding of the first Na+ partway within the membrane electric field. We present a transport model for SVCT1 that will provide a framework for investigating the impact of specific mutations and polymorphisms in SLC23A1 and help us better understand the contribution of SVCT1 to vitamin C metabolism in health and disease. cotransporters; sodium dependent; intestinal absorption; model simulation; renal reabsorption; Xenopus oocyte
We and others previously demonstrated that human SVCT1 is a Na+-dependent transporter that favors L-ascorbic acid over D-isoascorbic acid and that the oxidized form of vitamin C, dehydroascorbic acid, is excluded (5, 34). Here we explored its molecular mechanisms by testing the hypothesis that the Na+ dependence and rheogenicity of SVCT1 arise from coupled transport of two Na+ and one L-ascorbic acid per transport cycle and that L-ascorbic acid transport is driven by the electrochemical gradient for Na+. Our approach was to measure radiotracer (L-[14C]ascorbic acid, 22Na) fluxes and currents in Xenopus oocytes expressing SVCT1 and to model the kinetic mechanisms of SVCT1 with the aid of computer simulation.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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50 ng of human SVCT1 cRNA and incubated these for 2–4 days before performing voltage-clamp or radiotracer experiments at 22–24°C (except where noted). Media used for functional assays in oocytes. Functional assays were performed using a standard Na+ uptake medium containing (in mM): 100 NaCl, 2 KCl, 1 CaCl2, 1 MgCl2, and 10 HEPES (pH 7.5 with Tris base). For Na+-free or low-Na+ media, NaCl was replaced by equimolar choline chloride (ChoCl).
Voltage-clamp experiments.
We used the two-microelectrode voltage clamp (Dagan CA-1B amplifier) to measure currents associated with SVCT1 in oocytes. Microelectrodes (resistance 0.5–5 M
) were filled with 3 M KCl. Voltage-clamp experiments comprised two protocols: 1) continuous current recordings were made at a holding potential (Vh) of –50 mV, low-pass filtered at 20 Hz, and digitized at 1 Hz; 2) oocytes were clamped at Vh = –50 mV, and step changes in membrane potential (Vm) were applied (from +50 to –150 mV in 20-mV increments), each for a duration of 100 ms, before and after the addition of substrate. Current was low-pass filtered at 500 Hz and digitized at 5 kHz. Steady-state data were obtained by averaging the points over the final 16.7 ms at each Vm step. Test solutions were always washed out with substrate-free medium (100 mM ChoCl, pH 7.5) for at least 2 min. Steady-state data from protocols 1 or 2 were fit by a modified Hill relationship (Eq. 1) for which I is the evoked current, Imax the derived current maximum, S the concentration of substrate S (Na+ or L-ascorbic acid), K
the substrate concentration at which current was half-maximal, and nH the Hill coefficient for S.
 | (1) |
 | (2) |
 | (3) |
 | (4) |
Radiotracer uptake experiments. We determined the Na+-L-ascorbic acid coupling stoichiometry by measuring radiotracer fluxes under voltage clamp (–50 mV), a method we have described elsewhere for other Na+-coupled transporters (15, 18, 19, 36). We used L-[1-14C]ascorbic acid at final specific activity 0.3–3 GBq/mmol and 22Na at a final specific activity 0.34 MBq/mg (both from Perkin-Elmer Life Sciences). At the end of the 5-min uptake period, oocytes were rinsed with ice-cold ChoCl medium and solubilized with 5% SDS before 14C or 22Na content was assayed by liquid scintillation counting.
Statistical and regression analysis.
Statistical analyses were performed using SigmaStat version 3.5 (Systat Software) with a critical significance level of 
= 0.05. We used two-way ANOVA for between-group comparisons. Linear and nonlinear regression analyses using the least-squares method were performed using SigmaStat, and errors presented are the SEs of the estimates. Pre-steady-state current data were fit by Eq. 3 using Clampfit version 9.2 in the pCLAMP software suite (Axon Instruments).
Modeling of SVCT1-mediated pre-steady-state currents.
We propose an eight-state model (see Fig. 5) to account for our experimental data for SVCT1. In the absence of L-ascorbic acid, SVCT1 exhibits pre-steady-state currents that are sensitive to changes in extracellular Na+ concentration. We tested whether transitions between three carrier states, namely the empty carrier at the external face C1 = [C]', or at the internal face C8 = [C]'', and the outward-facing Na+-bound configuration C2 = [CNa]', can account for the pre-steady-state currents observed for SVCT1 by computer simulation of the partial reaction. We did not consider the inward-facing Na+-bound configuration C7 = [CNa]'' since intracellular Na+ concentration ([Na+]) is generally low. Our partial-reaction model is essentially identical to that used to describe the Na+-glucose cotransporters SGLT1 (9, 14, 28) and SGLT3 (formerly pSGLT2) (17) and similar to that used to describe a myo-[H+]inositol cotransporter (12). The rate of change in concentration of each carrier state is given by the difference of forward and reverse reactions
 | (5) |
 | (6) |
 | (7) |
Cy is described by the rate kxy, which comprises its voltage-independent rate constant k
; C1 + C2 + C8 = 1. Thus the effect of membrane potential on Na+ binding/dissociation and translocation of the empty carrier is described by the following Eyring-theory reaction rates. We assumed one Na+-binding event (n = 1) and carrier valence (zC) of –1, based on experimental observations (see Figs. 1G and 4C).
 | (8) |
 | (9) |
 | (10) |
 | (11) |
and 
are phenomenological coefficients describing the fraction of the membrane electric field sensed by Na+ binding/dissociation at the external face (') and at the internal face ('') and by reorientation of the empty carrier (). Microscopic reversibility requires that ' + '' + = 1 (13). We did not consider [CNa]'' (state 7), so '' = 0. [Na]o is the extracellular Na+ concentration; and µ = FV/RT where F, R, and T have their usual thermodynamic meanings. We simulated the pre-steady-state current using Berkeley Madonna modeling software, integrating the carrier-state concentration equations (Eqs. 5–7) such that the pre-steady-state current (It) is given by Eq. 12.
 | (12) |
k
k
and k
by simulating It best approximating the compensated pre-steady-state currents observed for SVCT1. Initial carrier-state concentrations were derived using root equations that were simultaneously solved for carrier-state proportions in the case of zero charge movement (i.e., at –50 mV).
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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in this example was 76 ± 7 µM. The mean K
determined from seven independent experiments was 72 µM (SD 20 µM; 95% confidence limits 54 and 91 µM). The Hill coefficient for L-ascorbic acid (n
was 1 and did not vary with membrane potential (Vm). The derived maximum current (I
varied significantly with Vm (Fig. 1C). The I
/Vm relationship was roughly linear between +50 and –10 mV. I peaked at between –30 and –50 mV and was then reduced at hyperpolarized Vm. K did not vary with Vm at negative potentials (Fig. 1D). The slight increase in K at positive Vm probably arises from the fact that the [Na+] of 100 mM is subsaturating at positive Vm (see below). Therefore, it is likely that SVCT1 binding of L-ascorbic acid is voltage independent.
The currents evoked by 100 µM L-ascorbic acid varied as a function of Na+ concentration (Fig. 1E). The current-voltage relationship at 100 mM Na+ resembled that described for I/Vm, roughly linear between +50 and –30 mV and peaking at –50 mV. A decrease in the evoked currents was observed at hyperpolarized potentials (–70 to –110 mV). At any given Vm, the L-ascorbic acid-evoked current was always greater at higher extracellular Na+ concentration ([Na+]o) in the range 20 through 100 mM Na+ (for clarity, data at 20 mM Na+ were not shown). The current-voltage relationship (and the peak current) shifted in the hyperpolarizing direction with decreasing [Na+]o. Therefore, the L-ascorbic acid-evoked currents are driven by the Na+ electrochemical potential gradient.
The reduction of L-ascorbic acid-evoked currents (and I at hyperpolarized potentials (more negative than around –70 mV) observed for human SVCT1 is more pronounced than what we previously observed for rat SVCT1 (33). We have observed a similar phenomenon for the H+-coupled peptide transporters PEPT1 (16) and PEPT2 (4). In the case of PEPT1, the inhibition at hyperpolarized potentials was associated with a 10-fold increase in K0.5 for the dipeptide. We considered that binding of L-ascorbic acid (given its 1– charge at neutral pH, see Fig. 2D) to SVCT1 may be hindered at extreme hyperpolarized potentials, but the voltage independence of K(Fig. 1D) does not support such a conclusion. Alternatively, activation of an additional ion conductance at hyperpolarized potentials may result in reduced net current. If this is the case, however, it is unlikely to involve Cl–, since the L-ascorbic acid-evoked currents were unaffected by replacement of Cl– with gluconate (data not shown).
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13% the magnitude of the currents evoked by 500 µM L-ascorbic acid in 100 mM Na+ medium. Such Na+ currents are consistent with SVCT1 mediating a modest Na+ uniport ("internal leak") pathway in the absence of L-ascorbic acid, a phenomenon that is common among mammalian Na+-coupled nutrient transporters, including the Na+-glucose cotransporter SGLT1 (18). Currents evoked by 100 µM L-ascorbic acid in an oocyte expressing SVCT1 demonstrated a sigmoidal dependence on Na+ concentration, with half-maximal Na+ concentration (K
of 50 mM and Hill coefficient (n
for Na+ of 2 (Fig. 1G), suggesting that two Na+ activate SVCT1-mediated L-ascorbic acid transport per cycle. In contrast, the Na+ uniport pathway (n
1) appears to only involve the binding of one Na+ and appears to require much higher Na+ concentrations for saturation. K
for Na+-L-ascorbic acid cotransport varied with membrane potential (data not shown): in the same experiment shown in Fig. 1E, we estimated K to range from 46 mM at –110 mV to well over 100 mM at –10 mV and at positive Vm.
In summary, the currents evoked by Na+ and L-ascorbic acid in oocytes expressing SVCT1 support the conclusions that: 1) SVCT1 is a high-affinity L-ascorbic acid transporter (K 70 µM), 2) SVCT1-mediated L-ascorbic acid transport is driven by the Na+ electrochemical potential gradient, 3) SVCT1 also mediates Na+ uniport in the absence of L-ascorbic acid, and 4) two Na+ are required to bind to SVCT1 to activate L-ascorbic acid transport, whereas only one Na+ per transport cycle participates in Na+ uniport.
Temperature dependence of the Na+-L-ascorbic acid cotransport activity of SVCT1.
The SVCT1-mediated L-ascorbic acid-evoked currents were highly temperature dependent and increased from –20 nA at 18°C to –98 nA at 30°C. The predicted Arrhenius activation energy (Ea) was 23 kcal/mol (Fig. 1H). The corresponding temperature coefficient Q10 (the factor by which activity was increased for every 10° increase in temperature) over this temperature range was 4.6 ± 0.2. These data support the "carrier" model of transport in which Na+-L-ascorbic acid transport comprises a series of ligand-induced conformational changes. In contrast, channels are expected to comprise fewer conformational changes and therefore be less temperature dependent.
Na+-L-ascorbic acid coupling stoichiometry of SVCT1.
We determined Na+-L-ascorbic acid coupling stoichiometry of SVCT1 by measuring 22Na+ and L-[14C]ascorbic acid fluxes in individual oocytes under voltage clamp and relating the fluxes to net charge translocation (Fig. 2). Two Na+ were transported per net positive charge, and the net positive charge influx was equivalent to the influx of L-ascorbic acid. Because L-ascorbic acid bears a charge of 1– at pH 7.5, these data demonstrate that the coupling ratio is two Na+ per one L-ascorbic acid under the conditions described. The Hill coefficient 
for Na+ of 2 (Fig. 1G) therefore indicates that there is strong cooperativity between the two Na+-binding sites involved in cotransport.
Ordered binding and simultaneous transport of Na+ and L-ascorbic acid by SVCT1. We examined the order of substrate binding to SVCT1 and the cotransport mechanism by determining the saturation kinetics parameters [K0.5 and maximum current (Imax)] for both Na+ and L-ascorbic acid as a function of the cosubstrate concentration (Fig. 3). Our data indicate that Na+ and L-ascorbic acid are translocated simultaneously, since K0.5 for each substrate was at its lowest in the presence of higher concentrations of the cosubstrate (11, 17, 19, 31). In contrast, for a consecutive carrier model, the K0.5 values are expected to rise as cosubstrate concentration is increased.
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was dependent on the L-ascorbic acid concentration, i.e., L-ascorbic acid concentration imposed a limitation on the maximal rate of transport even when Na+ was saturating. This observation rules out the possibility that SVCT1 first binds L-ascorbic acid. Therefore, one or both Na+-binding events must precede L-ascorbic acid binding, a conclusion that is supported by our observations that SVCT1 can mediate Na+ uniport (see Fig. 1) and Na+-sensitive pre-steady-state currents (see Fig. 4), each indicating that SVCT1 can bind Na+ in the absence of L-ascorbic acid. Moreover, we found that I was dependent on the Na+ concentration (Fig. 3D), indicating that the second Na+ must bind after the ascorbate-binding step. In contrast, considering a carrier model in dynamic equilibrium, we should expect that saturating L-ascorbic acid would always drive the transporter at its maximal rate were L-ascorbic acid bound last (11, 17, 19, 31). Our data are not consistent with a random-binding model in which case Imax for either substrate would be independent of the cosubstrate concentration.
We conclude from these data that the order of substrate binding by SVCT1 is Na+, L-ascorbic acid, Na+, followed by their simultaneous translocation (see Fig. 5). Our conclusion is further supported by the observation that the Na+ Hill coefficient (n was 2 in the case of Na+-L-ascorbic acid cotransport but only 1 in the case of Na+ uniport (Fig. 1F).
Pre-steady-state currents associated with SVCT1.
We observed pre-steady-state currents following step changes in Vm in oocytes expressing SVCT1 (but not in control oocytes) in the absence of L-ascorbic acid (Fig. 4). Pre-steady-state currents associated with SVCT1 decayed exponentially with time constants (
) of 3–15 ms (Fig. 4B) and were essentially abolished in the presence of saturating L-ascorbic acid (data not shown). Fitting of the /Vm relationship using a Gaussian function revealed a maximum time constant (max) of 15 ms at membrane potential (Vmax) approximately equal to –18 mV. Pre-steady-state currents were integrated with time to obtain charge (Q); the Q/Vm relationship at 100 mM Na+ could be described by a single Boltzmann function with Qmax of 25 nC and midpoint (V0.5) approximately equal to –9 mV (Fig. 4C). The apparent valence (z) of the movable charge was approximately equal to –1. Using Eq. 4, we estimated NT, the number of functional SVCT1 units in the plasma membrane, to be 1.7 x 1011/oocyte.
We also estimated turnover rates of the transport cycle as a function of Vm using the relation –I/Qmax taking I to be the current evoked by 1 mM (saturating) L-ascorbic acid. The maximal turnover rate for this oocyte was 9.7 ± 0.2 s–1 at –50 mV, dropping to 6 s–1 at –110 and +10 mV. The predominant effect of lowering the [Na+] was to shift the Q/Vm relationships to more hyperpolarized potentials without a significant effect on maximal charge (Fig. 4, C and E) since Qmax did not vary significantly with [Na+] (P = 0.41), whereas V0.5 shifted by 77 mV per 10-fold change in Na+ concentration (P < 0.001).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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70 µM), temperature dependent (Q10 5), and energized by the Na+ electrochemical potential gradient. We obtained a Na+-L-ascorbic acid(1–) coupling ratio of 2:1 from simultaneous measurement of currents and fluxes in individual oocytes at pH 7.5. The flux ratio is unlikely to differ within the environment of the acidic microclimate of the intestinal brush border (pH 6; see Ref. 24) or the renal proximal tubule (luminal pH 7), since the ascorbate is still charged 1– under these conditions (Fig. 2D); however, a modest drop in L-ascorbic acid transport activity is observed for SVCT1 at lower pH (33, 34). The Hill coefficient (nH) for Na+ activation of L-ascorbic acid-evoked currents was 2, indicating a high degree of cooperativity between the two Na+-binding events. L-Ascorbic acid and Na+ saturation kinetics as a function of the cosubstrate concentration revealed a simultaneous transport mechanism in which binding is ordered Na+, L-ascorbic acid, Na+ (Fig. 5). This same binding sequence was recently proposed for a second SVCT isoform, human SVCT2 (7). However, in the latter study, the evidence cited in support of this conclusion is evidence, rather, for a simultaneous transport mechanism, and Vmax data provided elsewhere in that paper (Fig. 3, C and D, of Ref. 7) may instead support the binding sequence Na+, Na+, L-ascorbic acid for SVCT2. Pre-steady-state currents and model simulation. In the absence of L-ascorbic acid, SVCT1 mediated rapidly decaying pre-steady-state currents that could be described by single Boltzmann distributions, with apparent valence of the movable charge approximately equal to –1. Maximal charge (Qmax) was conserved upon progressive removal of Na+, whereas the voltage midpoint (V0.5) shifted to more hyperpolarized potentials. Pre-steady-state currents have been observed for several other Na+-coupled (1, 9, 14, 17, 19, 27) or H+-coupled (8, 12, 16, 21, 22, 25) solute transporters. The results of model simulation have led investigators to attribute pre-steady-state currents to two steps in the transport cycle, namely reorientation of the empty charged carrier and binding/dissociation of the driving ion partway within the plane of the membrane electric field (and in some cases also to the binding/dissociation of the ion at the internal face).
We simulated transitions between three states in our model for SVCT1 (Fig. 5): the empty carrier configurations at the internal face (C8 = [C]'') and the external face (C1 = [C]') and the Na+-bound outward configuration (C2 = [CNa]'). Pre-steady-state currents associated with SVCT1 were adequately described by this three-state partial-reaction model using the rate constants and phenomenological coefficients (' and ) given in the legend to Fig. 5. Our model closely predicted the /Vm and Q/Vm relationships (Fig. 4, B–D) at 100 mM Na+. The model closely accounted for the shift in V0.5 to more negative potentials, without significant effect on Qmax, observed when [Na+]o was reduced (Fig. 4, C-E). That ' was much greater than (' = 0.83) indicates that most of the charge transfer arises from an ion-well effect of Na+ binding partway within the membrane electric field. In contrast, a simulation (data not shown) in which we set ' < (' = 0.32) closely predicted the /Vm and Q/Vm relationships at 100 mM Na+ but underestimated the Na+-dependent shift in V0.5 (only –27 mV/decade) and predicted instead a one-sixth reduction in Qmax between 100 and 5 mM Na+. Qmax effects of the driving-ion concentration were observed for the Na+-glucose cotransporter SGLT1 (14) and the H+-peptide cotransporter PEPT1 (16) and were accounted for by models in which ' < (' 0.3).
Our model predicted that most of the SVCT1 carriers are in the Na+-bound outward-facing configuration [CNa]' at Vm of –50 mV and [Na+]o = 100 mM (Fig. 4F). The voltage step to +50 mV results in a rapid transition [CNa]' [C]' [C]'' involving only a modest increase in the steady-state concentration of [C]'. Turnover rates of the transport cycle at 100 mM Na+ ranged from 6 to 10 s–1, peaking at –50 mV. From the comparison of turnover rates with the reciprocals of (Fig. 4B), we conclude that neither the reorientation of the empty carrier nor Na+ binding is likely to be a rate-limiting step in the transport cycle at any Vm. Model simulation confirms this conclusion: the rates k81 (which varies from 45 s–1 at +50 mV to 89 s–1 at –150 mV) and k12 (which at 100 mM Na+ varies from 43 s–1 at +50 mV to 1,150 s–1 at –150 mV) in our model always exceed the maximum observed turnover rate of 10 s–1, so carrier reorientation [C]'' [C]' and Na+ binding [C]' [CNa]' are not rate limiting.
Conclusions
In conclusion, we have demonstrated that SVCT1 is a voltage-dependent and rheogenic two Na+-one L-ascorbic acid(1–) cotransporter (symporter) with high affinity for L-ascorbic acid (K0.5 70 µM). SVCT1 exhibited marked temperature dependence (Q10 5). Our data fit an ordered simultaneous transport model with binding sequence Na+, L-ascorbic acid, Na+. Model simulation predicts that the rheogenicity of SVCT1 arises predominantly (83%) from an ion-well effect of Na+ binding partway within the membrane electric field and, to a much lesser extent (17%), also from the translocation of the empty charged (1–) carrier (i.e., ' > ). Neither of these steps limits the rate of Na+-L-ascorbic acid cotransport. Translocation of the fully loaded carrier (symport) and the internal Na+ leak (Na+ uniport) pathway are proposed to be electroneutral steps. Our kinetic model provides a framework for investigating the impact of specific mutations and polymorphisms in the SLC23A1 gene coding for SVCT1. It is anticipated that this work will help to elucidate the contribution of SVCT1 to vitamin C metabolism in health and disease.
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ACKNOWLEDGMENTS |
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This work was presented in part at Experimental Biology, April 2–6, 2005, San Diego, CA (20).
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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, the electrical potential µ, and (in the case of k12) [Na+], as well as the phenomenological coefficients 
= 983 M–1·s–1, k
= 54.0 s–1, k
= 112 s–1, and k
) for L-ascorbic acid of 1.3 ± 0.1, and half-maximal L-ascorbic acid concentration (K
= –91 ± 12 nA (r2 = 0.991; P < 0.001). For Na+ uniport (triangles), n
