Symmetry of H+ Binding to the Intra- and Extracellular Side of the H+-coupled Oligopeptide Cotransporter PepT1*

(Received for publication, June 26, 1996, and in revised form, January 15, 1997)

Stephan Nussberger §, Angela Steel , Davide Trotti , Michael F. Romero , Walter F. Boron and Matthias A. Hediger par

From the  Renal Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, and Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115 and the  Yale University, Department of Cellular and Molecular Physiology, School of Medicine, New Haven, Connecticut 06510

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

Ion-coupled solute transporters exhibit pre-steady-tate currents that resemble those of voltage-dependent ion channels. These currents were assumed to be mostly due to binding and dissociation of the coupling ion near the extracellular transporter surface. Little attention was given to analogous events that may occur at the intracellular surface. To address this issue, we performed voltage clamp studies of Xenopus oocytes expressing the intestinal H+-coupled peptide cotransporter PepT1 and recorded the dependence of transient charge movements in the absence of peptide substrate on changing intra- (pHi) and extracellular pH (pHo). Rapid steps in membrane potential induced transient charge movements that showed a marked dependence on pHi and pHo. At a pHo of 7.0 and a holding potential (Vh) of -50 mV, the charge movements were mostly inwardly directed, whereas reduction of pHo to below 7.0 resulted in outwardly directed charge movements. When pHi was reduced, inwardly directed charge movements were observed. The data on the voltage dependence of the transient charge movements were fitted by the Boltzmann equation, yielding an apparent valence of 0.65 ± 0.03 (n = 7). The midpoint voltage (V0.5) of the charge distribution shifted linearly as a function of pHi and pHo. Our results indicate that, as a first approximation, the magnitude and polarity of the transient charge movements depend upon the prevailing H+ electrochemical gradient. We propose that PepT1 has a single proton binding site that is symmetrically accessible from both sides of the membrane and that decreasing the H+ chemical potential (Delta µH) or increasing the membrane potential (Vm) shifts this binding site from an outwardly to an inwardly facing occluded state. This concept constitutes an important extension of previous kinetic models of ion-coupled solute transporters by including a more detailed description of intracellular events.


INTRODUCTION

Several ion-coupled solute transporters have been shown to exhibit pre-steady-state currents in response to step changes in membrane potential. These currents were found to be analogous to gating currents of voltage-dependent ion channels (1-5; 12). Based on Xenopus oocyte expression studies of Na+/glucose cotransporters, Wright and coworkers (3, 22) proposed that they are due to binding and dissociation of Na+ near the extracellular transporter surface in conjunction with a slow voltage-sensitive conformational change that leads to translocation of the charged empty carrier within the plane of the membrane before binding or after dissociation of Na+ (see Fig. 6A). The investigators reported that analogous events occur in the human H+-coupled oligopeptide cotransporter PepT1 and that transient charge movements can be attributed to the reorientation of the empty carrier within the membrane in response to H+ binding/dissociation (10). In contrast, Lester and colleagues (6) described the kinetics and transient charge movements of the GABA1 transporter GAT1, the 5-HT transporter, and the Na+-glucose transporter SGLT1 by a model which treated the transporters as a single file of binding sites with ends open to the external and internal solutions. Unlike previous reports (see Refs. 1-5, 10, and 17), this model does not consider conformational changes of the empty transport protein in response to voltage jumps. Thus, although the findings on the dynamic aspects of transporters have generated considerable interest and have stimulated numerous studies, the interpretations of the pre-steady-state currents vary, and the molecular events underlying these events are poorly understood. In addition, the effect of changes of the intracellular concentrations of the coupling ions (i.e. Na+ or H+) on the pre-steady-state kinetics have not yet been reported for an ion-coupled cotransporter.


Fig. 6. Hypothetical models of charge movement. A, model proposed by Wright and coworkers (10) describing the pre-steady-state currents of human PepT1 observed in response to step changes in membrane potential. The currents are predicted to be due to binding and dissociation of H+ near the extracellular transporter surface followed by a relatively slow voltage-sensitive conformational change of the charged empty carrier (see arrow). B, model of transient charge movements of rabbit PepT1, describing H+-binding/dissociation at the extra- and intracellular transporter surface. The empty PepT1 carrier (left) exists in two conformations that show the H+ binding site either in the inwardly or outwardly facing position (step 6). H+ binding to the outside (steps 1 and 2) or inside (steps 5 and 4) of the carrier induces a conformational change, resulting in the formation of outwardly or inwardly facing occluded states (right). According to the model, the occluded states cannot cycle between the inwardly and outwardly facing conformations in the absence of peptide substrate. The tau 2 values for dissociation/binding of H+ at the extracellular surface of PepT1 are predicted to be determined by the rate constants of steps 2, 1, and 6. Similarly, the tau 2 values for dissociation/binding of H+ at the intracellular surface are predicted to be determined by the rate constants of steps 4, 5, and 6. Since H+ dissociation (steps 1 and 5) is most likely faster than steps 2, 4 and 6, it probably contributes less to the tau 2 values. If pHo is lowered (Delta µH increased) and/or the membrane hyperpolarized (Vm decreased), the equilibrium will be shifted more toward the outwardly occluded state. Likewise, If pHi is lowered (Delta µH decreased) and/or the membrane depolarized (Vm increased), the equilibrium will be shifted more toward the inwardly occluded. A and B, the direction of the net movement of charge during the voltage-sensitive conformational change (step 6) of the charged empty carrier (left) is indicated by arrows. The ominus  indicates the center of the sum of movable charges, which is predicted to be negative.
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The intestinal brush border oligopeptide cotransporter PepT1 is the prototype of a new family of mammalian H+-coupled transporters. This transporter was previously cloned in our laboratory using an expression cloning approach. PepT1 mediates electrogenic transport of di-, tri-, and tetrapeptides into epithelial cells and has a coupling ratio of 1:1 for neutral peptides (7, 23; and for review, see Refs. 13 and 16).

In the present study, we have analyzed the pre-steady-state currents displayed by rabbit PepT1 and have determined their dependence on intra- and extracellular pH. Our data provide new insights into the H+-coupling mechanism of PepT1 and show a striking symmetry of the pre-steady-state characteristics obtained in response to either intra- or extracellular acidification.


MATERIALS AND METHODS

Xenopus Oocyte Expression

The rabbit intestinal oligopeptide transporter PepT1, was expressed in Xenopus laevis oocytes by microinjection of transporter cRNA as described previously (7). For intracellular acidification using the human high affinity glutamate transporter EAAC1 (9), PepT1 cRNA was coinjected into oocytes with EAAC1 cRNA at a ratio of 1:1 (w/w). Control oocytes were injected with 50 nl of deionized water. Experiments were performed 3 to 7 days after injection.

Voltage Jump Experiments

Transporter currents were measured by using the two-electrode voltage clamp technique (GenClamp 500, Axon Instruments, Foster City, CA) as described previously (7, 9). The resistance of the microelectrodes was 0.3-0.6 MOmega . The standard bath solution contained 100 mM NaCl, 2 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 3 mM Hepes, and 3 mM Mes. The pH was adjusted to the value of interest with Tris buffer. To lower the intracellular pH (pHi) of the oocytes, PepT1 cRNA-injected oocytes were incubated either in standard bath solution (see above) in the presence of either 2 mM Gly-Leu or 2 mM Trp-Gly or in acetate buffer (30 mM sodium acetate, 58 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, 5 mM Hepes, 5 mM] Mes, pHo 6.0). Alternatively, oocytes expressing PepT1 and EAAC1 were incubated in standard bath solution in the presence of 2 mM L-glutamate. Intracellular pHi changes were monitored using pH-sensitive microelectrodes filled with a hydrogen-selective ionophore (Fluka Chemie AG, ionophore I, coctail B) as described previously (7, 9). To measure charge movements, oocytes were clamped at a holding potential (Vh) of -50 mV, and step changes in membrane potential to transient voltages (Vt) were applied for 250 ms followed by a 1-s interpulse interval using the pCLAMP software package (Version 6.0, Axon Instruments, CA). Analog signals were averaged from three sweeps, low pass filtered at 5 kHz, digitized, and saved for further processing.

To analyze the pre-steady-state currents, the current traces were fitted to I(t) = I1 exp(-t/tau 1) + I2 exp(-t/tau 2) + Iss; where I1 is a capacitive current with time constant tau 1 associated with the oocyte plasma membrane (tau 1 is also observed in non-injected control oocytes), I2 is a transient current associated with PepT1 expression with time constant tau 2, and Iss is the steady-state current. The parameters tau 1, tau 2, I1, I2, and Iss were allowed to vary in all fits. The fractional error using a Simplex algorithm was set to 0.0001 (pClamp software package, Version 6.0, Axon Instruments). The transient charge movements Q were obtained from the time integrals of Itransient(t) = I2 exp(-t/tau 2) during the ON and OFF responses for all depolarizing and hyperpolarizing potentials and fitted by the Boltzmann equation, Q = Qmax/(1 + exp((V - V0.5)zF/RT) + Qhyp; where Qmax represents the total charge movement, z represents the effective valence, V0.5 represents the midpoint of the charge distribution, and Qhyp represents the charge movement for extreme hyperpolarizing potentials (for reviews, see Refs. 1-6 and 17). R, T, and F are the usual thermodynamic constants. In our experiments RT/F was 25.3 mV.


RESULTS

Dependence of the Pre-steady-state Kinetics on Extracellular pH (pHo)

Pre-steady-state currents of rabbit PepT1 expressed in Xenopus oocytes were recorded as a function of pHo (Fig. 1A). These experiments were performed in the absence of peptides to suppress the "normal" transport cycle of PepT1 (step 3 in Fig. 6B) and to isolate the partial reactions that account for the pre-steady-state currents. Oocytes were voltage clamped at a holding potential (Vh) of -50 mV, and pre-steady-state currents were induced by a series of test voltage pulses (Vt) (ON response). At pHo 7.0, stepping the oocyte membrane potential from Vh to hyperpolarizing potentials (Vt = -150, -110, and -70 mV) resulted in large inwardly directed transient currents, whereas for depolarizing pulses (Vt -30, +10, and +50 mV) only small outwardly directed currents were observed. Virtually the opposite observations were made when pHo was less than 6.0 where we measured relatively large outwardly directed pre-steady-state currents in response to depolarizing pulses. Stepping the membrane potential back to Vh (OFF response) produced opposite transient currents that were identical in magnitude to those obtained for the ON response (see below).


Fig. 1. Dependence of PepT1-mediated pre-steady-state currents on extracellular pHo. A, using the two-microelectrode voltage clamp technique, membrane currents of PepT1 cRNA-injected oocytes were recorded at pHo 6.0, 6.5, and 7.0 in the absence of substrate to isolate the partial reactions of the transport cycle which account for the pre-steady-state currents. The oocytes were held at a membrane potential of -50 mV, and a series of voltage pulses (Vt = -150, -110, -70, -30, +10, and +50 mV) was applied at each vertical line shown in the current records (see top right). The current recordings indicate that the pre-steady-state currents were voltage- and pHo-sensitive and switched direction with changing pHo. No significant voltage- or pHo-dependences were observed for PepT1-injected oocytes in the presence of substrate (2 mM Gly-Leu, pHo 7.0) (B) or for water-injected control oocytes (C). The steady-state current/voltage relationship at pHo 7.0 (D) was obtained as the differences between the currents measured in the presence and absence of Gly-Leu (2 mM). E, the transients recorded at pHo = 6.0, and Vt = +50 (1), +10 (2), and -30 mV (3) are plotted on a semilogarithmic scale for PepT1 cRNA injected oocytes. The data indicate that it is appropriate to describe the current traces by two exponential functions superimposed by a steady-state component (Iss).
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Addition of substrate completely abolished the transient currents of PepT1 for pHo between pH 5.5 and 7.5 and produced large inwardly directed steady-state currents that increased with hyperpolarization of the membrane. Fig. 1B shows a typical current recording in the presence of saturating Gly-Leu concentration (2 mM) in response to the voltage steps at pHo 7.0. Water-injected control oocytes (Fig. 1C) exhibited capacitive currents that were symmetrical with respect to hyper- and depolarization but did not reveal significant transient currents when compared with those depicted in Fig. 1A. Consistent with our previous current recordings for Gly-Sar (7), the steady-state I/V relationship for Gly-Leu taken at pHo 7.0 was membrane potential-dependent (Fig. 1D).

Relaxation time constants of pre-steady-state currents were obtained by fitting the pre-steady-state currents to a steady-state component and two exponential functions. Fig. 1E shows the transient currents displayed in Fig. 1A in response to depolarizing potentials at pHo 6.0 on a semilogarithmic scale and indicates that the data can be adequately described by two exponentials. The exponential functions represented the contributions of a transient capacitive component associated with the oocyte plasma membrane and a transient component associated with expression of PepT1. The time constant tau 1, which reflects the oocyte membrane capacitance, was insensitive to voltage and external pHo (Fig. 2A) but varied with the size of the oocytes. For example, relatively small PepT1 cRNA or water-injected control oocytes gave tau 1 values of 0.72 ± 0.06 ms (mean ± S.D.; n = 90, number of oocytes). By contrast, the largest oocytes gave tau 1 values of 1.35 ± 0.09 ms (n = 72) (data not shown).


Fig. 2. Comparison of the PepT1 transient current characteristics at different extracellular H+-concentrations. A, The capacitive time constants tau 1 associated with the oocyte plasma membrane and the transient time constants tau 2 associated with PepT1 expression were derived from current traces as depicted in Fig. 1. The dependences of tau 1 and tau 2 on extracellular pHo are shown during the onset of the pulse potential. The data represent the average of five independent experiments (oocytes). B, the charge movements during ON and OFF response of the voltage steps were obtained from the time integrals of the transient currents. The Qon and Qoff values are plotted for various membrane potentials between -150 and +50 mV and for pHo values between pHo 5.5 and 7.5. C, voltage and pHo dependence of charge movements. The data points represent the average of five PepT1 cRNA-injected oocytes. The data were fitted to the Boltzmann equation. The horizontal arrow intersects each curve at half completion of charge movement corresponding to V0.5 given by the Boltzmann equation. D, fitted values for V0.5 are plotted versus pHo. The least-squares fit corresponds to a change of -61 mV (r2 = 0.99) per pHo unit. E, turnover estimate of PepT1 calculated from the correlation of transient charge movements with currents resulting from a saturating dose of Gly-Leu at Vh = -70 mV. Linear regression yielded a slope of 6.6 s-1, which is an index of charge transfer at this potential.
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For the ON response, the relationship between tau 2 and Vm (Vt) was bell-shaped, and the maximum of tau 2 (tau 2max) increased with decreasing pHo (Fig. 2A), consistent with data of Mackenzie et al. (10) on human PepT1. At Vm = +50 mV, tau 2 increased from 5 to 15 ms (n = 5) when pHo was reduced from pH 7.5 to pH 6.0. The Vm at which tau 2 was maximal moved in the depolarizing direction with decreasing pHo, consistent with the view that reducing pHo promotes H+ binding to the extracellular transporter surface, resulting in a shift toward the "outwardly occluded state" (Fig. 6B). Thus, after reduction of pHo, a more depolarizing potential is required to maintain the transporter at tau 2max condition. At hyperpolarizing potentials (e.g. -120 mV), tau 2max was much smaller, and the tau 2 values were less dependent on pHo (Fig. 2A). However, as will be shown later, tau 2max at negative potentials goes back up again after intracellular acidification (Fig. 3B).


Fig. 3. Dependence of pre-steady-state currents and charge movements on intracellular pHi. A, voltage clamp records for a PepT1 cRNA injected oocyte before and after intracellular acidification. Measurements were made as described in Fig. 1 at pHo 7.0. Intracellular acidification was induced by PepT1-mediated uptake of Gly-Leu under voltage clamp conditions (Vh = -50 mV). Oocytes were incubated in Gly-Leu uptake buffer (2 mM Gly-Leu, pHo 5.5, 1 h). B, analysis of the current recordings revealed the voltage and pHi dependence of the relaxation constants tau 1 and tau 2 (lines 1 and 2). The observed changes in tau 2 were reversible when oocytes were held at Vh = -50 mV pHo 7.0 to allow recovery of pHi (line 3). The outline of the experiment is depicted on the top. C, PepT1 relaxation kinetics altered through intracellular acidification by incubation of oocytes in acetate buffer. Diffusion of acetate across the oocyte plasma membrane induced similar changes in the relaxation kinetics of PepT1 as shown in panel B. Parallel measurements of intracellular pHi using pH-sensitive microelectrodes are shown in panels D and E.
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Analysis of the transient currents during the OFF response exhibited relaxation constants tau 2 of 10-15 ms that were relatively insensitive to Vt and pHo (data not shown). These data are consistent with the findings on human PepT1 (10), Na+/glucose cotransporters (3, 22), and the GABA transporter, GAT1 (4). The Vt insensitivity during the OFF response is analogous to the Vh insensitivity of the transient currents during the ON response.

Charge Transfer Associated with PepT1 Expression

To further study the pHo-dependence of the pre-steady-state behavior of PepT1, we calculated the charge transfer Q for the ON and OFF responses from the time integrals of the transient currents. The charge movements were virtually identical for ON and OFF responses at all values of Vt and pHo (Fig. 2B), indicating strict charge conservation, a property that is in agreement with studies of Na+-coupled transporters (1, 3-5, 17). When plotted against Vt, Q saturated with both hyper- and depolarization of the membrane (data not shown).

Fig. 2C shows the voltage- and pHo-dependence of the transient charge movements. The data were fitted by a single Boltzmann distribution and represent the average of five oocytes. To correct for different levels of PepT1 expression in the oocyte plasma membrane, the data were normalized against the maximal charge movement Qmax. The slope z, which provides a measure of the equivalent charge moving within the membrane electric field in response to voltage jumps, shifted from 0.62 to 0.53 when changing pHo from 5.5 to 7.5. The midpoint potential (V0.5) of the Boltzmann equation shifted from approximately +60 to -63 mV when changing pHo from 5.5 to 7.5 and was linearly dependent on pHo (Fig. 2D). V0.5 changed by -61 mV (r2 = 0.99, n = 5) per pHo unit. The slope of this relationship, however, varied between different batches of oocytes. In another set of PepT1-injected oocytes V0.5 changed by -38 mV per pHo unit (r2 = 0.92, n = 7; Fig. 5). This variation may be related to different intracellular ion concentrations in different batches of oocytes. Comparison between the V0.5 values of rabbit and human PepT1 (10) revealed marked differences at similar pHo values. This may be due to differences in pHi (see below) or species variations as exhibited by rabbit and human SGLT1 (19) or wild-type and mutant SGLT1 (20). Taken together, our data suggest that V0.5 roughly follows the Nernst potential for protons, which is -58 mV per pH unit. Thus, as a first approximation, we hypothesize that V0.5 depends on the electrochemical potential for protons.


Fig. 5. Dependence of midpoint potential on extra- and intracellular pH. Fitted values for the midpoint potential V0.5 are plotted versus extracellualar pHo (solid lines). The least squares fits correspond to oocytes before and after intracellular acidification. The latter was achieved by incubation of oocytes in the presence of Gly-Leu (open squares) and acetate (closed circles). Coexpression of PepT1 and EAAC1 in oocytes show that V0.5 shifted toward more negative values in response to EAAC1-mediated uptake of L-glutamate (closed triangles).
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Fig. 4. Effect of glutamate-induced intracellular acidification on the pre-steady-state kinetics. A, coexpression of PepT1 and the Na+,K+ and H+-coupled high affinity glutamate transporter EAAC1. Intracellular pHi was lowered by incubation of PepT1/EAAC1 cRNA-injected oocytes with L-glutamate. Membrane potential was held at -50 mV, and PepT1 pre-steady-state currents were recorded in response to rapid steps in membrane potential. B, the voltage dependencies of the relaxation time constants tau 1 and tau 2 resemble those shown in Fig. 3. Lines and 2 show the tau 2 voltage relation before and after glutamate-induced intracellular acidification. Line 3 represents the recovery of pHi.
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As expected, Qmax increased with the expression levels of PepT1 (Fig. 2E) and with the oocyte size. The latter became clear when we plotted Qmax versus tau 1. The tau 1 value, which is proportional to the membrane capacitance and thus to the oocyte surface area, was linearly dependent on Qmax (data not shown). Three to five days after injection of PepT1 cRNA, analysis of transient currents of 10 oocytes revealed a median value for the total transient charge movement of 48 ± 7.0 nCi. This value is 4 to 10-fold higher than that determined for human and rat SGLT1, human EAAT2, and human PepT1 (3, 5, 10) but is comparable with previous estimates for rabbit SGLT1 (19, 20) and the rat GABA transporter GAT1 (4). As Fig. 2E shows, one oocyte displayed an exceptionally high Qmax value of -140 nC. The corresponding tau 1 value of that oocyte was also high (1.37 ms), indicating that the high charge movement was due to a larger number of transporters expressed over a large oocyte surface area. Given that Qmax is proportional to the carrier density N in the oocyte membrane according to Qmax N z e, where z is the effective valence of the moving charge (see below) and e is the elementary charge, we estimate an average expression level of 4.6 × 1011 PepT1 transporters. This level is on the same order of magnitude as that of other solute transporters (rabbit SGLT1, rat GAT1, and human PepT1; ~1011 transporters) (10, 19, 20). It is worth noting that this high estimate was the result of both relatively high charge movements and small z values (see below).

The turnover rate of PepT1 is the slope of the Qmax versus Imax plot (Imax was obtained at -70 mV at saturating peptide concentration (2 mM Gly-Leu, Fig. 2E)) and was 6.6 s-1 (r2 = 0.83) based on data from 12 oocytes. The turnover rate is thus similar to that of the GABA transporter GAT1 (4), the glutamate transporter EAAT2 (5), and other Na+-coupled solute transporters (3, 10). The turnover rate of human PepT1 (10) was about 10-15-fold greater than the 6.6 s-1 value we obtained for the rabbit cotransporter, probably because the human data were obtained at a Vh of -150 mV (10).

Dependence of the Pre-steady-state Kinetics on Intracellular pH (pHi)

PepT1-injected oocytes were voltage clamped at -50 mV, and pre-steady-state currents were recorded before and after intracellular acidification. Intracellular acidification was achieved using three different protocols: 1) a 60-min pre-incubation of PepT1-injected oocytes with 2 mM Gly-Leu (pHo 5.5), 2) a 10-min incubation of the oocytes in acetate buffer, and 3) a 10-min incubation with glutamate using oocytes expressing both PepT1 and EAAC1. Pre-steady-state currents were recorded at an extracellular pHo of 7.0. At this pHo, the inwardly and outwardly directed pre-steady-state currents were symmetrical upon depolarization or hyperpolarization of the membrane so that changes in their direction could be easily detected by loss of symmetry of the current traces.

Typical recordings before and after intracellular acidification are shown in Fig. 3A. Here, intracellular acidification was achieved by PepT1-mediated H+-coupled uptake of Gly-Leu under voltage clamp conditions. The decrease of pHi was monitored simultaneously on the same batch of PepT1-injected oocytes using pH-sensitive microelectrodes (Fig. 3D). Because intracellular acidification in the latter experiments was measured under non-clamping conditions, and because application of Gly-Leu elicited a membrane depolarization (data not shown) and the Gly-Leu elicited current is voltage-dependent (Fig. 1D), the reduction of pHi under clamping condition at the resting membrane potential (-50 mV) would be somewhat greater.

Our data show that the current relaxations become slower after intracellular acidification with Gly-Leu (pHo = 7.0), with tau 2 increasing from 10 to 28 ms (n = 4; Fig. 3B). This increase in tau 2 was partially reversed when the oocytes were incubated for an additional 10 min in pHo 8.0 bath solution at -50 mV, to allow pHi to recover partially (Fig. 3B). Recovery of tau 2 was further enhanced if oocytes were incubated for 1 h in pHo 8.0 bath solution at +10 mV (data not shown). Measurement of pHi using pH-sensitive microelectrodes showed that the recovery of tau 2 is associated with a partial recovery of pHi (Fig. 3D). The increase in tau 2 is consistent with our model that reducing pHi increases H+ binding to the intracellular transporter surface and thereby shifts the carrier toward an inwardly occluded state (Fig. 6B). In further agreement, the maximum of tau 2 shifted to more negative potentials when pHi was decreased because a more negative Vm is required to dissociate H+.

To rule out the possibility that the changes in the relaxation kinetics of PepT1 are due to accumulation of dipetides in the oocytes, control experiments were performed with Trp-Gly (2 mM). We recently showed that Trp-Gly taken up by PepT1 in oocytes is rapidly hydrolyzed into Trp and Gly (14). Incubation with Trp-Gly had the same effect on PepT1 pre-steady-state kinetics as Gly-Leu (data not shown).

To further test whether the changes in the relaxation kinetics are due to intracellular acidification, we incubated oocytes in 30 mM acetate buffer to reduce pHi. Under non-clamping conditions, application of 30 mM acetate reduced the intracellular pHi by ~0.2 pH units (Fig. 3E) without changing the resting membrane potential, which was around -50 mV (data not shown). The effect of acetate on the relaxation characteristics (Fig. 3C) and on the recovery of pHi after a 20 min incubation in pH 8.0 bath solution at -50 mV was similar to that obtained by incubation with peptide, except that the acetate procedure was accompanied by more pronounced recovery in tau 2 values (Fig. 3C).

The third line of evidence in support of our hypothesis that the relaxation kinetics of PepT1 depend on pHi was derived from co-expression studies of PepT1 and the neuronal and epithelial high affinity glutamate transporter EAAC1 (9, 13) (Fig. 4A). Because EAAC1-mediated uptake of L-glutamate is coupled to the cotransport of one H+ (or alternatively to the countertransport of one OH-) uptake of L-glutamate causes intracellular acidification. Previously, we demonstrated that EAAC1-mediated uptake of glutamate decreased pHi from ~7.6 to 7.5 (9). Because EAAC1 per se does not produce large pre-steady-state currents in response to voltage jumps (9), it can be assumed that co-expression of EAAC1 with PepT1 does not interfere with the pre-steady-state kinetics of PepT1. Thus, any observed changes of the pre-steady-state currents of oocytes expressing both EAAC1 and PepT1 can be mainly attributed to PepT1 expression. Transient currents displayed by these oocytes were analyzed before and after a 10-min incubation with L-glutamate (2 mM, Vh = -100 mV). The pre-steady-state currents recorded at pHo 7.0 in the absence of peptide and L-glutamate were both inwardly and outwardly directed, similar to the symmetrical currents shown in Fig. 3A, top. Pre-incubation with L-glutamate resulted in predominantly inwardly directed pre-steady-state currents at Vh = -50 mV, analogous to the data shown in Fig. 3A, bottom. Analysis of the pre-steady-state currents revealed that the dependence of tau 2 on membrane potential (Fig. 4B) was consistent with those shown in Fig. 3, B and C. Analysis of charge movements at pHo 7.0 (Fig. 5) revealed that V0.5 shifted by -17.6 mV in response to EAAC1-mediated intracellular acidification (from -30.1 ± 6.9 mV (n = 3) to -47.7 ± 1.6 mV (n = 3)), which is in line with our hypothesis that V0.5 roughly follows the electrochemical potential for protons.

Sensitivity of the PepT1 Pre-steady-state Kinetics to pH0 Changes after Intracellular Acidification

When we analyzed the pHo dependence of the transient charge movements before and after intracellular acidification, either by incubation of oocytes in Gly-Leu or acetate, we found nearly the same linear dependence of V0.5 on pHo (Fig. 5). The apparent valence z, moving in response to the voltage steps, was 0.65 ± 0.03 (S.E., n = 7) and did not change significantly with reduction of pHi. The slopes of the pHo dependence of V0.5 for Gly-Leu and acetate-treated oocytes were -49 mV (r2 = 0.99, n = 4) and -60 mV (r2 = 0.99, n = 3) per pHo unit, respectively. The line representing the linear relationship between V0.5 and pHo shifted toward more negative potentials in response to intracellular acidification. This suggests a qualitative equivalence between raising pHo and lowering pHi, in agreement with the hypothesis that V0.5 is proportional to Delta µH, and thus to the pHi - pHo difference. The observed effects of pHi and pHo also are consistent with the tau 2/voltage relationships (Figs. 2A and 3, B and C), where a decrease of the inwardly directed H+ gradient through manipulation of either pHi or pHo results in a shift of the tau 2 maximum toward more negative potentials. Thus, our data indicate that there is a fixed relationship between Delta µH, Vm, and tau 2.


DISCUSSION

Several studies on Na+- and H+-coupled solute transporters indicated that varying the external ion concentration affects their pre-steady-state kinetics, shifting the voltage dependence of the transient currents (1-5, 10). Our data show that pHo affects the pre-steady-state kinetics of rabbit PepT1 in a similar way, with the charge movements dependent on pHo. The shift of the maximal time constant tau 2max toward more negative potentials in response to increasing pHo is in agreement with studies of Hilgemann and coworkers (2, 11) addressing the charge movements of the Na+,K+-pump. These investigators showed that the Vm dependence of the maximal capacitance, which is proportional to tau 2max, shifts toward more negative potentials when the extracellular Na+ concentration is lowered.

A new aspect of our study is the determination of the effect of changes of the intracellular pH on the pre-steady-state kinetics. To our knowledge, the effect of intracellular coupling ion-concentration on the pre-steady-state kinetics has not yet been determined for an ion-coupled solute transporter. In the present investigation, we show that the PepT1-elicited pre-steady-state currents depend on both pHo and pHi. The effect of increasing pHo on the voltage dependence of the transient charge movement was equivalent to that of decreasing pHi. We found that either increasing pHo or decreasing pHi shifts the tau 2max of the pre-steady-state currents toward more negative potentials. Our data, therefore, indicate that an important determinant of the polarity of the pre-steady-state kinetics of PepT1 is the electrochemical potential for protons across the oocyte plasma membrane.

Mechanisms of Charge Movement

An important question is how the H+ electrochemical ion gradient affects the direction and amplitude of the transient charge movements of PepT1. A mechanistic concept of transient charge movements in ion-coupled transporters was proposed by Wright and coworkers for the Na+-coupled glucose transporter SGLT1 (3). This concept was additionally proposed to account for the pre-steady-state currents observed for human PepT1 (10, 18) (Fig. 6A). The investigators proposed that the pre-steady-state charge movements recorded in the absence of peptide substrate were mostly due to reorientation of the empty carrier in the membrane following binding/dissociation of H+ (Fig. 6A, see voltage-sensitive conformational change). They predicted that the empty carrier has a negative charge (Fig. 6A, ominus ) which moves inwardly within the membrane following H+ dissociation and that this movement is associated with a structural change of the empty carrier from an outwardly to an inwardly facing conformation. The negative charge is proposed to represent the center of movable, positive, or negative charges on the carrier. However, the molecular basis of the charge movements has not yet been elucidated, and it is currently unknown which amino acid residues in PepT1 or SGLT1 contribute to charge movement.

Since the above model does not consider the affect of intracellular pH on the pre-steady-state kinetics of PepT1, we have developed a revised model in which the empty carrier has a single H+-binding site that is symmetrically accessible from both sides of the membrane (Fig. 6B). We hypothesize that H+ binding to the extra- or intracellular surface is followed by a conformational change resulting in the formation of outwardly or inwardly facing occluded states, respectively, and that these protonated states cannot cycle between the inside or outside in the absence of peptide. The pre-steady-state currents in response to voltage jumps are predicted to be due to binding or dissociation of H+ within the membrane electric field near the extra- or intracellular transporter surface (steps 2/1 and 4/5) and to reorientation of the empty carrier during the voltage sensitive conformational change (step 6). Increasing the H+ chemical gradient (Delta µH) and/or decreasing Vm (hyperpolarization) are predicted to shift the carrier toward the outwardly occluded state. Similarly, decreasing the H+ chemical gradient (Delta µH) and/or increasing Vm (depolarization) are predicted to shift the carrier toward the inwardly occluded state. Thus, consistent with our findings, the model predicts that the magnitude and polarity of the transient charge movements depend on the H+ electrochemical gradient.

Which Rate Constants of PepT1 Affect the Time Constants of the Transient Currents?

According to our model, the tau 2 values for dissociation/association of H+ at the extracellular surface depend on the rate constants of steps 2, 1, and 6, and those for dissociation/association at the intracellular surface on the rate constants of steps 4, 5, and 6. Since the transient currents occur in the millisecond range (Fig. 2a), whereas binding and dissociation of ions into or out of an "ion well" near the membrane surface more likely occur in the microsecond range (2), steps 1 and 5 are expected to contribute less to the tau 2 values. We therefore propose that the tau 2 values are set by the slow conformational changes (steps 2 and 4) which precede H+ dissociation at the extra or intracellular surface and/or the voltage-sensitive conformational change (step 6).

Symmetry of Extra- and Intracellular Events

We propose that dissociation/association of H+ will predominantly occur at the extracellular surface if pHi of oocytes is not reduced by acidification (Figs. 1 and 2), whereas at elevated pHo and reduced pHi (Figs. 3 and 4), dissociation/association is predicted to be increased at the intracellular surface. According to our model, there will be a pHo/pHi combination for any given Vm in the physiological range that yields an equal population of inwardly and outwardly facing states, resulting in binding/dissociation of H+ at both sides of the membrane. If the voltage-sensitive conformational change (step 6) accounts for most of the charge movements, both intra- and extracellular events can be described by a single Boltzmann distribution. However, if steps 2/1 and 4/5 of the transport cycle are rate-limiting, the Q/Vm relationships probably represent the composite of at least two closely related Boltzmann distributions that exist at the extra- and intracellular surface.

Relationship of tau 2max and Delta µH

An important aspect of our work is that we have extended the pre-steady-state kinetics described by Wright and colleagues (10) by including H+-dissociation/binding events at the intracellular transporter surface. In addition, we show that intracellular acidification reverses the decrease in tau 2max after hyperpolarization (Figs. 3B and 4B versus Fig. 2A). This finding is in favor of the view that an additional step may participate in the overall pre-steady-state cycle. For example, if steps 4/5 become rate determining when decreasing pHi, this would explain the increase in tau 2max at negative potentials. However, to fully understand the molecular basis of the pre-steady-state kinetics of PepT1, further studies addressing the rate constants of individual steps of the transport cycle and their dependences on the H+ electrochemical gradient will be required.

The Normal Transport Cycle of PepT1

PepT1 is predicted to bind H+ first and then peptide in an orderly fashion, consistent with studies of Mackenzie et al. (10) who showed that the apparent maximal transport rate of human PepT1 for Gly-Sar is not significantly attenuated when Ho+ is reduced 10-fold. At lower pHo, we predict that the number of PepT1 molecules that exist in the outwardly facing state is increased and that H+ binding results in a conformational change (Fig. 6B, step 2), leading to the outwardly occluded state and that this state has an increased affinity for peptides. From a physiological perspective, this concept makes sense because the pHo in the unstirred layer near the intestinal brush border membrane is acidic (pH 5.5-6.0), which favors the outwardly occluded state and promotes efficient peptide absorption (21, 24).

The Multi-substrate Single File Model

In some respects, our kinetic model resembles the multi-substrate single file model recently proposed by Lester and coworkers (6) to account for the pre-steady-state kinetics of ion-coupled transporters. The model treats an ion-coupled transporter as a single file of ion binding sites, with ends open to the external and internal solutions. It predicts that, once an ion is within the transporter, it is allowed to move between sites and to the solutions on either end of the transporter. A state of the transporter is defined by the occupancy of multiple binding sites or wells by ions in the permeation pore. The kinetics of the ion-coupled transporter are then reconstructed by possible transitions between different states in response to voltage jumps. Steps 2, 1, 6, 5, and 4 of our model (Fig. 6B) could be considered as sites in the single-file model. However, PepT1 is predicted to have only a single ion binding site that is either inwardly or outwardly facing, depending on intra- and extracellular pH.

PepT1 as a Potential pH Biosensor

Since the magnitude and polarity of the transient currents depend on the proton chemical gradient Delta µH, we examined whether this relationship can be used to estimate changes in pHi by analysis of the pre-steady-state currents of PepT1. The midpoint potential V0.5 can be determined according to Delta (pHi - pHo) = rho  * V0.5, where rho  represents a conversion factor (0.023 pH units/mV) that roughly corresponds to F/RT. rho  is given by the slope of the relationship V0.5 to pHo (Fig. 5). In support of our finding that V0.5 follows Delta µH, the slopes for different pHi values were similar. We estimated the decrease in pHi induced by EAAC1-mediated H+-dependent glutamate uptake by coexpression of EAAC1 and PepT1 (Fig. 5) and compared the result with pHi-measurements obtained using pH microelectrodes. In oocytes injected with both EAAC1 and PepT1, we observed a glutamate-induced shift in V0.5 of -17.6 mV at pH0 = 7.0 (Fig. 5, closed triangles). From the above equation, the observed shift in V0.5 predicts an intracellular acidification of 0.4 pH units. Previous measurements using pH-sensitive microelectrodes gave a shift of ~0.1 pH units (9). The difference may be related to monitoring pHi close to the plasma membrane (PepT1) as opposed to measuring it deep in the cytoplasm (microelectrodes). Thus, our data raise the possibility that PepT1 can be used as a biosensor to monitor pHi changes in close vicinity of the plasma membrane.

Symmetry of the Pre-steady-state Kinetics of Na+-coupled Transporters

A challenging question that remains to be addressed is whether the symmetry of binding of the coupling ion binding to either side of PepT1 also pertains to Na+-coupled transporters. Increasing [Na+]i in oocytes expressing SGLT1 would be expected to have a similar effect as intracelluar acidification on PepT1. The effect of changing [Na+]i on the pre-steady-state kinetics of a Na+-coupled solute transporter have not yet been reported.


FOOTNOTES

*   This work was supported by National Institutes of Health Grant DK43171 (to M. A. H.).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.
§   Supported by a research fellowship from the Deutsche Forschungsgemeinschaft (DFG).
par    To whom correspondence should be addressed: Renal Division, Brigham and Women's Hospital, 75 Francis St., Boston, MA 02115. Tel.: 617-732-5850; Fax: 617-732-6392.
1   The abbreviations used are: GABA, gamma -aminobutyric acid; Omega , ohm; Mes, 4-morpholineethanesulfonic acid.

Acknowledgments

We thank Sela Mager (California Institute of Technology, Pasadena, CA) and Dr. Bryan Mackenzie for stimulating discussions.


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