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J. Biol. Chem., Vol. 281, Issue 39, 28837-28849, September 29, 2006

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Mapping Conformational Changes of a Type IIb Na⁺/P_i Cotransporter by Voltage Clamp Fluorometry^*

From the Institute for Physiology and the Center for Integrative Human Physiology (ZIHP), University of Zurich, CH-8057 Zurich, Switzerland

Received for publication, April 21, 2006 , and in revised form, July 5, 2006.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION CONCLUSIONS REFERENCES

 
The fluorescence of a fluorophore depends on its environment, and if attached to a protein it may report on conformational changes. We have combined two-electrode voltage clamp with simultaneous fluorescence measurements to detect conformational changes in a type IIb Na⁺/P_i cotransporter expressed in Xenopus oocytes. Four novel Cys, labeled with a fluorescent probe, yielded voltage- and substrate-dependent changes in fluorescence (F). Neither Cys substitution nor labeling significantly altered the mutant electrogenic properties. Different F responses to voltage and substrate were recorded at the four sites. S155C, located in an intracellular re-entrant loop in the first half of the protein, and E451C, located in an extracellular re-entrant loop in the second half of the protein, both showed Na⁺, Li⁺, and P_i-dependent F signals. S226C and Q319C, located at opposite ends of a large extracellular loop in the middle of the protein, mainly responded to changes in Na⁺ and Li⁺. Hyperpolarization increased F for S155C and S226C but decreased F for Q319C and E451C. The labeling and F response of S155C, confirmed that the intracellular loop containing Ser-155 is re-entrant as it is accessible from the extracellular milieu. The behavior of S155C and E451C indicates a strong involvement of the two re-entrant loops in conformational changes during the transport cycle. Moreover, the data for S226C and Q319C suggest that also the large extracellular loop is associated with transport function. Finally, the reciprocal voltage dependences of the S155C-E451C and S226C-Q319C pairs suggest reciprocal conformational changes during the transport cycle for their respective local environments.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION CONCLUSIONS REFERENCES

 
The type II sodium-phosphate cotransporters (encoded by the solute carrier SLC34A gene family) are polytopic membrane proteins that mediate thermodynamically coupled transport of inorganic P_i and Na⁺ across the cell membrane. Their physiological role is to facilitate cellular uptake of P_i by coupling it to the transmembrane electrochemical gradient. Three subtypes have been currently identified: NaPi-IIa and IIb are electrogenic and operate with a 3:1 Na⁺: stoichiometry and translocate one net positive charge per transport cycle (for review, see Refs. 1 and 2), whereas NaPi-IIc is electroneutral and operates with a 2:1 Na⁺: stoichiometry (3).

The transport kinetics of electrogenic type II Na⁺/P_i cotransporters have been studied earlier by electrophysiology and uptake assays in the Xenopus oocyte expression system (reviewed in Ref. 1). Recently, we established the method of voltage clamp fluorometry (VCF)³ to gain new insights into putative conformational changes occurring during the transport cycle (4). VCF relies on the property that the fluorescence of a fluorophore is sensitive to its local environment, and thus changes in fluorescence recorded from a fluorophore attached to a particular site in a protein can be interpreted as conformational changes affecting the environment of the fluorophore. VCF was first implemented in the study of gating-related conformational changes in K⁺ channels (5, 6). Since then, VCF has been applied to several membrane transporter systems, including the glucose transporter SGLT1 (7, 8), the glutamate transporter EAAT3 (9), the GABA transporter GAT1 (10), the serotonin transporter SERT (11), and the Na⁺/K⁺- and H⁺/K⁺-ATPases (12, 13).

In our first study (4) we introduced a novel Cys residue in the putative re-entrant loop between transmembrane domains (TMDs) 5-6 (S448C, marked with an open triangle in Fig. 1) of the flounder NaPi-IIb isoform. Labeling S448C with a fluorescent probe allowed us to detect voltage- and substrate-dependent changes in fluorescence (F). Using the new fluorescence data we modified our earlier kinetic model of the electrogenic NaPi-II transport cycle that we derived from the interpretation of steady state and presteady state substrate-dependent currents. In the new model, two Na⁺ ions (instead of one) bind on the external side before a divalent , followed by one more Na⁺ ion. Subsequently the fully loaded carrier translocates to the internal side. After unloading, the empty carrier returns to an outside facing configuration via an electrogenic partial reaction, and each complete forward transport cycle transfers one positive charge to the intracellular medium (14-17).

In the S448C mutant the cotransport function is blocked after labeling, however analysis of current and fluorescence data indicates that the protein is still able to bind substrate. Although the absence of a full cotransport cycle may have facilitated the analysis of the data (the protein could only occupy a subset of possible conformations), it is important to validate and extend those observations to a fully functional transporter. In the present study we mutated ten new residues individually to Cys (see Fig. 1), all of which are located in predicted linker regions that face, or are accessible to, the extracellular milieu. For each mutant, we determined whether we could measure voltage-dependent fluorescence changes (F) after labeling with a fluorophore. We found that four of the novel Cys mutants gave measurable F signals. In contrast to the S448C mutant described above, for the four new mutants, neither the Cys mutation itself nor the subsequent labeling resulted in a significant alteration in their cotransport kinetics, which closely resembled the WT kinetic fingerprint. This finding has allowed us to interpret the voltage and substrate-induced changes in fluorescence as reflecting the associated conformational changes at different sites of the electrogenic type II Na⁺/P_i cotransporter during the complete transport cycle. Our findings therefore offer a first possible glimpse of time-resolved movement of the type II Na⁺/P_i cotransporter.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION CONCLUSIONS REFERENCES

 
Reagents and Solutions—(2-Aminoethyl)methane thiosulfonate hydrobromide (MTSEA) was from Toronto Research Chemicals. 2-((5 (6)-tetramethylrhodamine)carboxylamino)ethyl methanethiosulfonate (MTS-TMR) was from Biotium. All other reagents were from Sigma or Fluka.

The standard experimental solution (ND100) contained (mM): 100 NaCl, 2 KCl, 1.8 CaCl₂, 1 MgCl₂, 10 HEPES, pH 7.4 (adjusted using Tris). In Na⁺ replacement experiments, NaCl was replaced equimolar with choline Cl or LiCl. In the iodide experiments we replaced 50 mM NaCl with 50 mM NaI or sodium glucuronate (the latter serving as a Cl replacement control). Solutions containing the required concentrations of P_i were prepared by adding K₂HPO₄/KH₂PO₄ (pH 7.4). Modified Barth's solution for storing oocytes contained (mM): 88 NaCl, 1 KCl, 0.41 CaCl₂, 0.82 MgSO₄, 2.5 NaHCO₃, 2 Ca(NO₃)₂, 7.5 HEPES, pH 7.5 adjusted with Tris and supplemented with 5 mg/liter doxycyclin.

Oocyte Expression and Molecular Biology—Point mutations were generated in WT flounder NaPi-IIb (GenBank Accession No. AAB16821 [GenBank] ) using QuikChange site-directed mutagenesis (Stratagene) with primers from Microsynth and verification by sequencing (Microsynth). Complementary capped RNA was synthesized using the T3 Message Machine kit (Ambion). Oocytes from Xenopus laevis were prepared as previously described (4), injected with 50 nl of cRNA (0.2 µg/µl), and experiments performed 3-7 days after injection. The oocytes were exposed immediately before fluorescence measurements to 0.4 mM MTS-TMR in ND100 solution for 5 min in the dark.

Conventional Two-electrode Voltage Clamp—The procedure for standard two-electrode voltage clamp has been described in detail previously, as have the protocols (4, 16). Briefly, steady state P_i activation was determined by varying the P_i concentration in the presence of ND100 and subtracting the respective currents in ND100 from those in ND100 + P_i; steady state Na⁺ activation was similarly determined by subtracting the respective responses in NDX from those in NDX + P_i (1 mM), where X is the test Na⁺ concentration (in mM). Steady state P_i-induced currents (I_Pi) were fit with a form of the modified Hill Equation 1,

(Eq. 1)

where [S] is the concentration of the variable substrate (Na⁺ or P_i), I_Pi^max is the maximum electrogenic activity, K_m values the apparent substrate affinity for substrate S; H, the Hill coefficient, and K is a constant that takes account of uncoupled leak effects (17, 18). For P_i activation, H = 1 and Equation 1 reduces to a Michaelian form.

Presteady state relaxations were quantitated by fitting with a two-component exponential function. The faster component, which represents endogenous linear capacitive charging of the oocyte membrane, was subtracted from the total relaxation to yield the NaPi-II-dependent component. This was numerically integrated to obtain the charge Q moved for a step from the holding potential (V_h) to the test potential (V), as previously described (e.g. (17, 18). The Q-V data were fit with a Boltzmann function of the form in Equation 2,

(Eq. 2)

where V_0.5 is the voltage at which the charge is distributed equally between two hypothetical states, z is the apparent valency of an equivalent charge that moves through the whole of the membrane field, Q_max is the total charge available to move, Q_hyp is the charge at the hyperpolarizing limit and is a function of V_h, and e, k, and T have their usual meanings.

Apparatus for Simultaneous Voltage Clamp and Fluorometry—The apparatus for simultaneous VCF has been described in detail previously (4) and consisted of a conventional two-electrode voltage clamp with a laboratory-built fluorescence microscope. The latter comprised a x10 fluorescence objective (CFI S Fluor, 0.5 N.A., 1.2 mm W.D., Nikon) and a filter set (XF33 cube, comprising a 535DF35 excitation filter, 570DRLP dichroic mirror and 605DF50 emission filter; Omega Optical Inc). Emitted light was measured using a silicon photodiode (S1336-18BQ, Hamamatsu) connected directly to the input of an integrating headstage (CV 201, Molecular Devices) and the headstage signal was processed by a patch clamp amplifier (Axopatch 200A, Molecular Devices). The scaled output of the Axopatch 200A was further processed by a differential amplifier/filter unit (LPF-8, Warner Instruments) before digitization.

Experimental Protocols—Voltage-dependent changes in fluorescence (F) were determined using a voltage-step protocol. The membrane voltage was stepped from V_h =-60 mV to test potentials ranging between -200 mV and +200 mV in 40-mV increments for a duration of 100-200 ms (the protocol was adjusted for each NaPi-IIb mutant to obtain saturation of F at extreme potentials, but avoid unnecessary stress to the oocyte), and averaged over 10-15 sweeps. F data were acquired at 20k samples s^-1 and filtered at 70 Hz. The effect of substrate on F was determined by changing the bath solution. To facilitate comparison between conditions and oocytes, one data set was always acquired in ND100 solution for each oocyte. All data were corrected for fluorescence rundown as described previously (4).

View larger version (33K): [in this window] [in a new window]   FIGURE 1. Topology and basic characterization of NaPi-IIb WT and mutants. A, topology model showing the predicted orientation of the TMD and loop regions of NaPi-IIb. Residues substituted with a cysteine are indicated as follows: : significant voltage-dependent changes in fluorescence (F) were observed after labeling with MTS-TMR; : no F was observed after labeling; fl: residue identified in previous study (4) that yields strong F signal but labeling blocks Pi cotransport. B, original recording showing the current response of an oocyte expressing WT NaPi-IIb in ND100 solution (left) and after addition of 1 mM Pi (right). Dotted line indicates holding current at -60 mV, ND100 for both data sets. C, voltage dependence of the Pi-dependent current (measured in unlabeled oocytes) of WT NaPi-IIb and four mutants marked with in A.Pi-dependent currents were obtained by subtracting current traces recorded in ND100 with and without 1 mM Pi and were similar to those shown for the WT in B. D, voltage dependence of the apparent Pi affinity constant (Km(Pi)) of WT and the same four mutants as in C. Means ± S.E., n = 4-5.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION CONCLUSIONS REFERENCES

 
Identification of Sites that Exhibit Voltage-dependent F using Cys Scanning
We introduced novel Cys in each of the predicted external linkers and in the first intracellular loop (predicted to be re-entrant and therefore accessible to the extracellular medium) in NaPi-IIb (Fig. 1A). We characterized each mutant by electrophysiology and compared its behavior to that of the WT. All mutants were functional as evidenced by their robust P_i-induced currents (I_Pi) at -50 mV, 1 mM P_i, which confirmed that the Cys substitution was well tolerated (data not shown). The corresponding current-voltage (I-V) relations were similar to WT (shown in Fig. 1, B and C), with the exception of S520C. We then exposed the mutants to 1 mM MTSEA to see if this procedure would block I_Pi, as we had previously observed with the S448C mutant (4). Apart from S520C (see below), no major changes were seen in the I_Pi of any of the mutants (data not shown).

S520C, located in the fourth extracellular loop, was the only mutant that showed an altered voltage dependence of I_Pi. After MTS treatment the voltage dependence became more like that of the WT (not shown), a phenomenon previously reported for the G134C mutant in rat NaPi-IIa, located in the 1st extracellular loop (18). Thus, we could verify labeling of the S520C mutant from the change in the voltage dependence of I_Pi. Next, we examined whether we could detect voltage-dependent changes in F after incubating with the Cys-reactive fluorescent probe MTS-TMR. Only four mutants (S155C, S226C, Q319C, and E451C, indicated by open circles in Fig. 1A) showed significant F in response to changes in V_h after labeling with MTS-TMR, and only these mutants were studied further. The lack of voltage-dependent F for the other mutants (K110C, Q154C, A156C, T220C, S410C, and S520C) may indicate that these novel Cys were not accessible for labeling. Another explanation is that the attached fluorophore did not sense a change in its environment under the experimental conditions applied (e.g. S520C).

Steady State and Presteady State Kinetics of WT and Mutants
For the four mutants that showed voltage-dependent F, we analyzed steady state and presteady state currents to determine if the transport kinetics had been altered by the mutation or by labeling. Fig. 1C shows the voltage dependence of the P_i-dependent current (I_Pi) in (unlabeled) oocytes expressing WT, S155C, S226C, Q319C or E451C. Each shows qualitatively similar current-voltage (I-V) dependences and they only differ according to the absolute amount of current generated for any given oocyte. Taken together, all constructs yielded approximately similar I_Pi when averaged over batches and individual oocytes; however, there was considerable variation in I_Pi between individual oocytes, most likely because of variations in the expression levels of transporters at the cell membrane. We then measured the apparent P_i affinity (K_m(P_i)) at voltages ranging between -140 and +20 mV for WT and the four mutants. As shown in Fig. 1D, for S226C, Q319C, and E451C, K_m(P_i) was similar to that of the WT. For S155C, K_m(P_i) was increased (i.e. the apparent P_i affinity was decreased) significantly compared with that of the WT, which indicated that the substitution at this site caused a moderate alteration of the cotransport kinetics.

Next, we compared the steady state and presteady state currents before and after labeling with MTS-TMR (WT, S155C, S226C, E451C) or MTSEA (WT, S155C, S226C, Q319C). For the WT, no difference was observed in either I_Pi (not shown) or presteady state current recordings before and after incubating with MTS-TMR (Fig. 2, A-C) or MTSEA (not shown), which is consistent with the assumption that none of the 19 native Cys of the flounder NaPi-IIb protein are accessible with this reagent. Similar results were obtained with MTSEA (not shown). The results agree with previous work, which showed that the WT rat NaPi-IIa isoform could not be labeled by Cys-reactive biotinylating reagents (19) and that WT flounder NaPi-IIb did not show any voltage-dependent F after incubation with MTS-TMR (4). Thus, we can conclude that the WT NaPi-IIb protein is not functionally modified by the Cys-reactive reagents used in this study. Fig. 2C shows the Boltzmann fit parameters obtained from presteady state current recordings acquired over a range of Na⁺ concentrations. The results are similar to those obtained previously (14), but in the present study more Na⁺ concentrations were included. As depicted in Fig. 2C, the predicted maximum amount of charge available for translocation (Q_max) increased with increasing Na⁺. Fitting the Q_max data with the Michaelis-Menten equation (Equation 1, H constrained to 1) yielded an apparent K_m(Na) of 8 ± 1mM for the Na⁺-dependent component of Q_max, compared with 4 ± 2 mM (S155C), 24 ± 3 mM (S226C), 11 ± 3 mM (Q319C), and 24 ± 5 (E451C) (not shown).

In addition to Q_max, the apparent valency (z) and the midpoint of the Boltzmann distribution (V_0.5) were also Na⁺-dependent (Fig. 2C). z varied between 0.3 and 0.6, depending on the Na⁺ concentration. For V_0.5, the most negative value was obtained with 25 mM Na⁺, and increasing or decreasing Na⁺ from this value led to more positive V_0.5 values. The V_0.5 data obtained in the 25-125 mM range were fitted with a straight line to yield a slope of 113 ± 11 mV/log[Na] that was comparable to the values obtained for the mutants (S155C: 104 ± 12, S226C: 91 ± 4, Q319C: 105 ± 8 and E451C: 129 ± 5). The complex Na⁺ dependence of the parameters obtained from the Boltzmann fit most likely results from the contributions of more than two voltage-dependent partial reactions to the overall charge distribution (recall that Equation 2 describes a two-state model; for a detailed discussion, see e.g. Refs. 16 and 17).

For the three mutants S155S, S226C, and Q319C, we documented some minor alterations in the kinetics after labeling. I_Pi was 10-15% reduced after labeling for S155C, Q319C, and E451C in the voltage range -140 to +20 mV. For S226C, labeling resulting in an apparent reduction in the voltage dependence of P_i cotransport, as the slope of the I-V relationship decreased by 50% (not shown). These changes in electrogenic steady state P_i cotransport were paralleled by minor changes in the Na⁺ dependences of the presteady state charge distributions before and after labeling for S155C and S226C, but not for Q319C or E451C (Fig. 2D). In ND0, labeling did not affect V_0.5 for any of the mutants (not shown). However, in the presence of Na⁺, MTS-TMR treatment shifted V_0.5 for S155C to more negative potentials, whereas the opposite effect was seen for S226C (Fig. 2D). This indicated that labeling had introduced a small shift in the preferred occupancy of Na⁺-dependent conformational states for these mutants. On the other hand, the slope V_0.5/log[Na] was not changed for any of the mutants, which suggested that the depth of the hypothetical ion binding well for Na⁺ was unchanged by fluorophore labeling (4, 20, 21). Labeling did not change the total amount of mobile charge (Q_max) nor the apparent K_m(Na) measured by fitting the Q_max data with Equation 1 for any of these four mutants (not shown).

Finally, we investigated the effect of complete replacement of external Na⁺ by Li⁺ on the Q-V distribution of the WT NaPi-IIa protein. Li⁺ does not substitute for Na⁺ as the driving cation for P_i cotransport (not shown), yet evidence for an interaction of Li⁺ ions with the NaPi-II protein was obtained in our previous VCF study (4) using the S448C mutant. Replacing Na⁺ with Li⁺ still resulted in significant voltage-dependent F for this mutant whereas no F was observed in choline replacement. To examine if Li⁺ affected the presteady state kinetics of the WT protein, we recorded presteady state currents in Na⁺ (ND100), choline (ND0), and Li⁺ (LD100) solutions. The Boltzmann fit data are summarized in Table 1. Substituting Na⁺ with Li⁺ or choline reduced the estimated Q_max to the same level (we attribute this residual charge movement to the movement of intrinsic charge of the empty carrier). Also the apparent valency z measured in LD100 was statistically indistinguishable from the ND0 condition, whereas z increased significantly in ND100 solution. In contrast, both Na⁺ and Li⁺ shifted V_0.5 in a statistically significant manner to more positive potentials, compared with choline. These results indicate that Li⁺ ions can also interact with the WT protein and alter its conformational distribution, but without making a substantial contribution to the amount of detectable mobile charge.

View larger version (36K): [in this window] [in a new window]   FIGURE 2. Presteady state current analysis of the effect of introducing single Cys and labeling with MTS-TMR. A, original current recordings of the presteady state current response after a step change in membrane potential from -60 mV to target potentials (-160 to +60 mV in 20-mV increments) obtained from an oocyte expressing WT NaPi-IIb in ND100 (left) and ND0 (right) solution. Dotted lines show the single exponential fitted component corresponding to the presteady state current attributed to NaPi-IIb. B, charge-voltage relationships of WT NaPi-IIb before (left) and after (right) exposure to MTS-TMR. The charge was obtained by integrating the area under the fitted curve in recordings similar to A, acquired at different Na+ concentrations. The data were fit with the Boltzmann equation (Equation 2; solid lines). C, Boltzmann fit parameters obtained for WT NaPi-IIb from data similar to B before (filled squares) and after (empty squares) MTS-TMR treatment. Left, the Qmax data were plotted as a function of Na+ concentration and fitted with the Michaelis-Menten equation with H = 1 and yielded an apparent Km(Na) of 8 ± 1 mM. Mid-left, apparent valency z plotted as a function of the Na+ concentration. Mid-right, The V0.5 data were plotted as a function of the Na+ concentration (left) and then replotted as a function of log[Na] (right). The data points between 25 and 125 mM Na+ were fitted with a linear regression line with the slope 113 ± 11 (control) and 111 ± 5 (MTS-TMR) mV/log[Na]. D, presteady state current analysis of the four mutants before and after exposure to MTS-TMR. The Q-V distributions were very similar to WT and only V0.5/log[Na] is shown for each mutant. The slopes of linear regression lines fitted to the data were as follows (control, MTS-TMR-treated): S155C: 104 ± 12, 101 ± 14; S226C: 91 ± 7, 102 ± 9; Q319C: 105 ± 8, 102 ± 9; E451C: 129 ± 5, 131 ± 4 mV/log[Na]. In C and D we show means ± S.E., n = 3-4.

View larger version (32K): [in this window] [in a new window]   FIGURE 3. Voltage-and substrate-dependent changes in fluorescence (F) recorded in S155C-expressing oocytes. A, original F traces recorded in ND100 (left) and ND0 (right) solution. B, F-V -data were acquired at different Na+ concentrations and fitted with Equation 2 (solid lines). C, F-V -data were acquired at different Li+ concentrations and in 100 mM Li+ with 1 mM Pi. D, F-V-data were acquired in ND100 solution with different amounts of Pi added. E, V0.5 obtained from the fit of Equation 2 to B and C plotted as a function of log[Na] or log[Li]. The data were fit with linear regression lines with slopes of 140 ± 5 mV/log[Na] and 154 ± 4 mV/log[Li]. F, z obtained from the fit of Equation 2 to B and C plotted as a function of [Na+] or [Li+]. G, Fmax obtained from the fit of Equation 2 to D, plotted as a function of Pi and fit with Equation 1 (H constrained to 1). The apparent Km(Pi) from the fit was 99 ± 17 µM. Means ± S.E., n = 4.

View larger version (25K): [in this window] [in a new window]   FIGURE 4. Voltage-and substrate-dependent changes in fluorescence (F) recorded in S226C-expressing oocytes. A, original F traces recorded in ND100 (top) and ND0 (bottom) solution. B, F-V -data were acquired at different Na+ concentrations and in 100 mM Na+ with 1 mM Pi and fitted with Equation 2 (solid lines). Only data obtained in 0, 10, 100 mM Na+ and 100 mM Na+ + 1 mM Pi are shown. C, F-V, data were acquired at different Li+ concentrations (only 0, 10, and 100 mM are shown) and in 100 mM Li+ with 1 mM Pi. D, V0.5 obtained from the fit of Equation 2 to B and C plotted as a function of log[Na] or log[Li]. The data were fit with a linear regression line with the slope 58 ± 8 mV/log[Na] and 59 ± 5 mV/log[Li]. E, z obtained from the fit of Equation 2 to B and C plotted as a function of Na+ or Li+. Means ± S.E., n = 5-6.

The effect of P_i on F appears to have an absolute requirement for Na⁺. In LD100, P_i did not affect any of the Boltzmann fit parameters in a statistically significant manner (see Fig. 3C), which indicated that although Li⁺ may interact with the protein, it does not support P_i binding. Moreover, adding 1 mM P_i to ND0 solution did not alter the F-V distribution, as compared with ND0 alone, which further underscored the dependence of P_i interaction on Na⁺ (not shown).

Mutant S226C
Na⁺ Dependence—After labeling the S226C mutant with MTS-TMR, we observed large voltage-dependent changes in F (total F/F 4-8% in ND100; Fig. 4A). As with the S155C mutant, hyperpolarization decreased F, and saturation was observed at both voltage extremes. We obtained F-V data at different Na⁺ concentrations and fitted these data with Equation 2 (solid lines in Fig. 4B). F_max obtained from the fit was reasonably constant over the entire Na⁺ range (not shown). Compared with S155C, the F-V distribution was shifted to more positive voltages for all Na⁺ concentrations tested: in ND100 V_0.5 was 32 ± 3 mV, compared with -34 ± 1 for S155C, and the shift in V_0.5 toward more negative voltages with reducing Na⁺ was less pronounced than for S155C. The relationship between V_0.5 and log[Na] was linear for Na⁺ concentrations 25 mM, which yielded a slope of 58 ± 8 mV/log[Na] using linear regression (Fig. 4D). Also z was dependent on the Na⁺ concentration (Fig. 4E).

Li⁺ Dependence—The effect of Li⁺ on the F-V distribution was similar to that of Na⁺ (Fig. 4C). The main difference was that the F-V distribution was shifted to more positive voltages compared with the equivalent Na⁺ concentrations, so that at 100 mM Li⁺, V_0.5 was 73 ± 7 mV, compared with 32 ± 3 mV in 100 mM Na⁺. Like Na⁺, in Li⁺ the relationship between V_0.5 and log[Li] was linear for Li⁺ concentrations 25 mM, and the slope 59 ± 5 mV/log[Li] from the linear regression was statistically indistinguishable from that of Na⁺ (58 ± 8 mV/log[Na]) (Fig. 4D). Moreover the Na⁺ and Li⁺-dependences of z were similar (Fig. 4E), which indicated that for this mutant, Li⁺ induces similar changes in the fluorophore environment to Na⁺.

P_i Dependence—To determine whether the F-V distribution was affected by P_i, we measured F in ND100, ND0, and LD100 with 1 mM P_i added in each case (Fig. 4, B and C). In ND100, P_i affected neither F_max nor V_0.5. However, the apparent valency was decreased from 0.45 ± 0.02 to 0.27 ± 0.04. When we added P_i to LD100, we observed a small, but statistically significant decrease in z from 0.42 ± 0.03 to 0.36 ± 0.03. As for ND100, no other Boltzmann fit parameters were significantly affected by adding P_i to LD100. Finally, in ND0 solution, we observed no effect on the F-V distribution after adding P_i. These findings indicate that the S226C site mainly senses conformational changes associated with the empty carrier and the Na⁺ (Li⁺) binding steps that precede P_i binding.

View larger version (24K): [in this window] [in a new window]   FIGURE 5. Voltage-and substrate-dependent changes in fluorescence (F) recorded in Q319C-expressing oocytes. A, original F traces recorded in ND100 (top) and ND0 (bottom) solution. B, F-V -data were acquired at different Na+ concentrations and in 100 mM Na+ with 1 mM Pi and fitted with Equation 2 (solid lines). Only data obtained in 0 and 100 mM Na+ and 100 mM Na+ + 1 mM Pi are shown. C, F-V data were acquired at different Li+ concentrations (only 0 and 100 mM are shown) and in 100 mM Li+ with 1 mM Pi. D, V0.5 obtained from the fit of Equation 2 to B and C plotted as a function of log[Na] or log[Li]. The data were fit with a linear regression line with the slope 55 ± 3 mV/log[Na] and 64 ± 1 mV/log[Li]. E, z obtained from the fit of Equation 2 to B and C plotted as a function of Na+ or Li+. Means ± S.E., n = 4-5.

Li⁺ Dependence—The effect of Li⁺ on the F-V distribution of Q319C (Fig. 5C) was quite similar to that of Na⁺, and thus this mutant phenotype mirrors that of S226C. Like S226C, the F-V distribution of Q319C was shifted to more positive voltages in Li⁺ solutions than in Na⁺. In LD100 V_0.5 was 64 ± 4 mV, compared with only 36 ± 2 mV in ND100. In Li⁺ the relationship between V_0.5 and log[Li] was linear for Li⁺ concentrations 25 mM (Fig. 5D), and the slope 64 ± 1 mV/log[Li] from the linear regression was slightly (and statistically significantly) higher compared with Na⁺ (55 ± 3 mV/log[Na]). The Na⁺ and Li⁺ dependence of z were similar (Fig. 5E), although Li⁺ tended to have a smaller effect on z than Na⁺. Finally, as for Na⁺, F_max was unaffected by varying the Li⁺ concentration (not shown).

P_i Dependence—Adding 1 mM P_i to ND100 produced statistically significant changes in some of the Boltzmann fit parameters for Q319C. We observed a decrease of 14% in F_max, and the apparent valency z decreased from 0.63 ± 0.04 to 0.54 ± 0.05, whereas V_0.5 remained unchanged. In contrast, adding 1 mM P_i to LD100 or ND0 did not change any of the measured parameters significantly. These results show that a fluorophore attached to Q319C partially senses the conformational changes effected by P_i in the presence of Na⁺, and thus this mutant differs in this respect from its counterpart, S226C. Our finding that P_i does not affect the F-V distribution in LD100 or ND0 further supports the notion that P_i interaction requires Na⁺ and neither Li⁺ nor choline can substitute for Na⁺.

Mutant E451C
Na⁺ Dependence—After labeling with MTS-TMR, E451C-expressing oocytes showed a small (total F/F < 1%) but measurable change in F in response to membrane voltage (Fig. 6). As for Q319C, F decreased at depolarizing potentials. F saturated at both hypo-and hyperpolarizing limits for Na⁺ concentrations between 50 and 125 mM and we fit the data with Equation 2 (solid lines in Fig. 6B). V_0.5 was -12 ± 7 mV in ND100, and there was a linear dependence of V_0.5 on log[Na] for Na 50 mM with a slope of 99 ± 2 mV/log[Na] (Fig. 6E). For this Na⁺ concentration range F_max was constant. The apparent valency z decreased with decreasing Na⁺ (Fig. 6F), so that at Na⁺ 25 mM z approached zero, F_max became very large and the F-V distribution could not be reliably fit with Equation 2. We therefore plotted the F-V distribution obtained for Na⁺ concentrations between 0 and 25 mM without offsetting for F_max (inset in Fig. 6B).

View larger version (33K): [in this window] [in a new window]   FIGURE 6. Voltage-and substrate-dependent changes in fluorescence (F) recorded in E451C-expressing oocytes. A, original F traces recorded in ND100 (left) and ND0 (right) solution. B, F-V -data were acquired at different Na+ concentrations and fitted with Equation 2 (solid lines). Only data obtained at 50-125 mM Na+ was reliably fit with Equation 2 and shifted to Fmax. Inset shows F-V data for 0, 10, and 25 mM Na+ without offsetting. C, F-V data were acquired at different Li+ concentrations (only 50, 75, 100, and 100 mM + 1 mM Pi are shown). D, F-V -data were acquired in ND100 solution with different amounts of Pi added. E, V0.5 obtained from the fit of Equation 2 to B and C plotted as a function of log[Na] or log[Li]. The Na+ data were fit with a linear regression line with the slope 99 ± 2 mV/log[Na]. F, z obtained from the fit of Equation 2 to B and C plotted as a function of Na+ or Li+. G, Fmax obtained from the fit of Equation 2 to D, plotted as a function of Pi and fit with Equation 1 (H constrained to 1). The apparent Km(Pi) from the fit was 81 ± 34 µM. Means ± S.E., n = 4-6.

P_i Dependence—The P_i dependence (measured in ND100) of the F-V distribution of E451C is shown in Fig. 6D. F saturated at hyper- and hypopolarizing limits at all P_i concentrations used, and the data were fit with Equation 2 (solid lines in Fig. 6D). The effect of P_i on the F-V distribution closely mirrors that of S155C (Fig. 3G), whereby the effects of voltage on F were opposite. As P_i concentration increased, F_max reported by the fit decreased By plotting the F_max data as a function of P_i (Fig. 6G) and fitting with the Michaelis-Menten equation we obtained a K_m(P_i) of 81 ± 34 µM, which was very similar to the value obtained for S155C (99 ± 17 µM, Fig. 3G). Another similarity to S155C was the finding that both V_0.5 and z were only slightly affected by P_i (not shown).

View larger version (11K): [in this window] [in a new window]   FIGURE 7. Effect of iodide on fluorescence (F). F-V data were acquired in ND100 solution and in solution where 50 mM NaCl was replaced with NaI. Data were fitted with Equation 2 and shifted to Fmax. The fit parameters are given in Table 2. Means ± S.E., n = 5.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION CONCLUSIONS REFERENCES

 
In a previous study we established the methodology of VCF to investigate conformational changes in the type IIb Na/P_i cotransporter (4). We showed that after labeling the protein at a site in the large re-entrant loop in the second half of the protein (S448C; see Fig. 1A) with a fluorophore, we could measure substrate-and voltage-dependent changes in fluorescence. These changes are evidence of conformational changes that occur in the environment sensed by the fluorophore. In this present study we have used VCF to map regions in NaPi-IIb where conformational changes occur during the transport cycle. The new findings serve to deepen our understanding of the partial kinetic steps occurring during transport. We identified four sites which, when mutated to cysteines, could be labeled with a fluorophore and which showed voltage-and substrate-dependent changes in fluorescence. Unlike the previously characterized S448C mutant, whose cotransport activity was blocked after labeling (4), all four mutants identified in the present study remained fully functional after labeling with relatively small changes in their basic kinetics. Thus, the changes in fluorescence associated with each mutant measured under the same experimental conditions could reflect conformational changes that occur in four well-defined locales in the wild-type protein during its transport cycle.

S226C and Q319C—Unlike all other extracellular loop regions in the NaP_i-II protein, the large extracellular glycosylated loop (ECL-2) between putative TMDs 3 and 4 (Fig. 8B) has not previously been subjected to cysteine scanning accessibility studies. Thus, we had no preconceptions about the role (if any) of this large loop in the transport cycle of NaPi-IIb. The mutants S226C and Q319C are located at opposite ends of the loop. Both mutants showed voltage-dependent F in the absence of Na⁺, suggesting that both of them report on conformational changes that occur in response to changes in voltage in the absence of substrate (empty carrier, transition 1-8 in Fig. 8A). Because in our model the empty carrier is assumed to possess one intrinsic negative charge (see Refs. 4, 14, 16), depolarizing potentials are predicted to increase the probability of a conformation in which the substrate binding sites face inward (state 8). Thus interpreting the F-V data for S226C and Q319C in Figs. 4 and 5 suggests that state 8 (empty carrier with inward-facing binding sites) is associated with high fluorescence for S226C and low fluorescence for Q319C.

Because presteady state charge movements are associated with the empty carrier transition for all electrogenic NaPi-II isoforms (14, 16, 17), we can compare the data obtained for the charge-voltage (Q-V) and fluorescence-voltage (F-V) distributions. A comparison of the Boltzmann fit parameters obtained for both methods from experiments done in the absence of Na (ND0), showed that the midpoint (V_0.5) for the F-V data were shifted toward more positive potentials, compared with the Q-V data: from the F-V data we obtained 33 ± 33 mV for S226C and 15 ± 5 mV for Q319C, whereas from the Q-V distribution, we obtained -45 ± 3 mV and -54 ± 4 mV, respectively. If both parameters reported on the same hypothetical empty carrier transition (1-8), we would expect the Boltzmann fit parameters to be the same. This discrepancy is not a consequence of labeling⁴ the transporter with MTS-TMR, since as shown in Fig. 2D, the change in V_0.5 for the empty carrier after labeling is marginal. Instead, the discrepancy most likely arises because the Q-V distribution reports on the global distribution of charge in the membrane electrical field, whereas F reports on a local event.

When we compare the effect of substrate on S226C and Q319C, we see that the two mutants behave in a similar manner. Adding Na⁺ did not significantly change F_max in either mutant but it did affect the midpoint of the F-V distribution (V_0.5) and its steepness (z). In both mutants V_0.5 was shifted to more positive potentials with increasing Na⁺ with a slope of 55-60 mV/log[Na]. This is significantly shallower than the 110 mV/log[Na] measured from the Q-V distribution. When we substituted Na⁺ with Li⁺we observed that Li⁺ shifted V_0.5 to even more positive potentials than Na⁺. Because Li⁺ also shifts V_0.5 of the Q-V distribution measured on the WT protein, these results provide strong evidence that Li⁺ is able to interact with the protein. However, unlike Na⁺, Li⁺ did not increase the maximum amount of charge available for translocation (Q_max) above that observed in ND0. This supports the conclusion reached in our preceding study (4) that Li⁺ is able to interact with the transporter, but that the binding of Li⁺ is voltage-independent.

S155C and E451C—S155C lies in a predicted intracellular re-entrant loop (ICL-1) between TMDs 2-3 (see Fig. 8B). We postulated that this residue could be accessed from the extracellular medium, because in the rat NaPi-IIa isoform, the residue at the equivalent position (Ser-183), when mutated to Cys could be modified by externally applied MTS reagents.⁵ When labeled with MTS-TMR, S155C showed a robust F signal (thus confirming that it is accessible to the external medium), whereas both its neighbors (Q154C and A156C) were silent. It is likely that these latter mutants are inaccessible for labeling, because the equivalent of A156C in rat NaPi-IIa (A184C) was also inaccessible.⁶

E451C lies in the predicted extracellular re-entrant loop (ECL-3) between TMDs 5-6 (Fig. 8B). Interestingly, there is considerable intrasequence homology between ECL-3 and ICL-1, where S155C lies. Previous cysteine scanning work (19, 23, 24, 25) has shown that both regions are very important for transport function. Significantly, E451C lies very close to S448C, which also gives a strong F signal after labeling, but whose transport function is blocked by labeling (4). Unfortunately, a lack of saturation precluded a Boltzmann analysis of the fluorescence signal from S448C, but the data were consistent with P_i causing a profound reduction in F_max when applied in Na⁺ solution. S155C and E451C both respond to P_i by decreasing F_max with a K_i of 90-100 µM, which is similar to the K_i value of 100 µM previously reported for S448C (4). Marked P_i-induced reduction of F_max is thus a common feature of S155C, E451C and S448C, and sets them apart from S226C and Q319C. While the latter appear to be limited to sensing only transitions 8-1-2a(b) (see Fig. 8A), S155C and E451C sense at least 8-1-2a-2b-3, or possibly the whole transport cycle. Taken together, the results point to a similar and significant involvement of these two re-entrant loops in the transport process of NaPi-IIb.

The midpoint voltages (V_0.5)ofthe F-V distribution of S155C and E451C were both shifted to more negative potentials, compared with V_0.5 measured from the Q-V distribution, at all Na⁺ concentrations used (in ND100 the shift was -45 mV for S155C and -10 mV for E451C, unfortunately in ND0 the F-V data could not be reliably fit with a Boltzmann function). This finding contrasts with the behavior of S226C and Q319C, the F-V distributions of which were shifted to more positive potentials than that of the Q-V distribution. At this stage we can only speculate that these differences arise because of the localized nature of the F signals measured for each site.

Conformational Topology Map—Using the information obtained in this study on the response of mobile elements in the NaPi-II protein we can begin to construct a topology map that shows conformational changes and their relationship to substrate binding and translocation. Fig. 8A shows a kinetic scheme for the transport cycle of electrogenic Na/P_i-II transporters, which is based on results from earlier electrophysiology and fluorescence studies (4, 14, 16, 17). On the extracellular side, binding of two Na⁺ ions (transitions 1-2a-2b) precede the binding of one (2b-3), after which one more Na⁺ ion can bind (3-4). The fully loaded carrier then translocates the bound substrates to the intracellular side in an electroneutral step (4-5) and releases the bound substrates (5-6-7-8). The empty carrier then returns to an outward-facing configuration in an electrogenic step (8-1). Voltage dependence was assigned to the empty carrier transition (8-1) based on presteady state current analysis, where substantial charge movement can be detected in the absence of external substrate (14, 16, 17). The presence of external Na⁺ ions increased the amount of detectable charge movement, and therefore we assigned voltage dependence to the Na⁺ binding step preceding P_i binding. Recently we showed that Li⁺ ions are able to interact with the protein in the absence of P_i (4). Whereas the Na⁺ interaction was cooperative, suggesting the binding of more than one Na⁺ to the protein in the absence of P_i, the Li⁺ interaction showed no cooperativity and we postulate that Li⁺ interacts with only one Na⁺ binding site. Because Li⁺ did not contribute any charge movement in addition to that measured for the empty carrier, we hypothesize that the voltage dependence of the first Na⁺/Li⁺ binding step is minimal (for a detailed discussion of this model, see Ref. 4).

View larger version (44K): [in this window] [in a new window]   FIGURE 8. Modeling the conformational changes occuring during the transport cycle of NaPi-II. A, kinetic scheme for the transport cycle of NaPi-II. Transitions that are influenced by voltage are indicated by blue arrows and include transition of the empty carrier (1 8) and the second Na+ binding (2a 2b). No information is available on the internal substrate interaction transitions, but they are depicted to mirror those taking place on the external side and are indicated with gray arrows. B, topology model showing the transmembrane domains and associated loops. The position of the four mutants from which significant F could be measured after labeling with MTS-TMR are indicated. Different colors were used to distinguish the two halves of the protein. C, cartoon schematic of conformational changes that could cause the substrate-and voltage-dependent changes in fluorescence recorded for the four mutants. The two halves of the protein are depicted with the same colors as in B, and the states are numbered according to the model in A. State 1 (outward-facing) predominates at extreme hyperpolarizing potentials in the absence of substrate and is associated with low fluorescence for S155C and S226C and high fluorescence for Q319C and E451C. Binding of two Na+ ions exposes the binding sites for and the third Na+ ion (state 2b). After binding of and the third Na+, the transporter adopts a conformation where the fluorescence of S155C and E451C is not suppressed by extreme potentials, i.e. hyperpolarizing potentials for S155C (state 4) or depolarizing potentials for E451C (state 5). After releasing the substrates on the intracellular side, the transporter reverts to a conformation (state 8) where maximal changes in fluorescence are induced by extreme potentials for all mutants. State 8 represents the inward-facing conformation of the empty carrier that predominates at extreme depolarizing potentials. The assignment of the binding sites for each substrate to a specific half of the protein is arbitrary.

State 1 represents the outward-facing conformation of the empty carrier. Because the empty carrier is assumed to carry one negative charge, this conformation predominates at very negative potentials. In this state, S155C and S226C both show minimal fluorescence, whereas fluorescence is maximal for Q319C and E451C. At very positive potentials the inward-facing configuration predominates (state 8), and the fluorescence intensities for the four sites are reversed.

The transition 1-2b represents binding of the first two Na⁺ ions. This step is associated with a shift in the midpoint voltage (V_0.5) to more positive potentials and an increase in the slope (z) of the F-V relationships for all four sites (see Figs. 3, 4, 5, 6). The shift in V_0.5 indicates that at a given voltage, an increase in external Na⁺ increases the probability of the protein adopting an outward-facing configuration, whereas the increase in z indicates that an increase occurs in the apparent valency of the charges that move in the electrical field, presumably contributed by the Na⁺ ions. For S226C and Q319C, z was less dependent on Na⁺ compared with S155C and E451C, which suggested that the voltage-dependent fluorescence changes reported by the former pair do not strongly reflect Na⁺-induced conformational changes. Note that although the F-V data were fitted with a single Boltzmann, which implies a model where only two states are possible, the underlying processes certainly are more complex and this is exemplified by the dependence of z on [Na]. The change in the Boltzmann fit parameters documented for the four sites therefore arise from changes in the occupancies of several possible conformational states that all contribute to the overall F-V distribution. Transition 2b-4 represents binding of one divalent P_i and the last Na⁺ ion to their respective binding sites that were exposed after binding of the first two Na⁺ ions. In the case of S226C and Q319C, adding P_i caused only minor changes in the F-V distribution recorded at the two sites. This indicates that conformational changes relating to P_i binding and substrate translocation do not significantly perturb the environment of these two sites. Whether or not the transporter undergoes the full transport cycle has only minor effects on the fluorescence recorded at these sites. For simplicity, the cartoons show that all outward-facing conformations decrease the fluorescence of S226C, and all inward-facing conformations decrease fluorescence of Q319C.

In contrast, for S155C and E451C, application of P_i (in the presence of Na⁺) caused a concentration-dependent reduction in F_max. This implies that in the presence of both substrates, the protein adopts conformations (states 4-5) where the fluorescence can no longer be effectively decreased by extreme hyper- or depolarizing potentials, respectively. Because the protein now is able to undergo a full transport cycle, we do not know which conformational states contribute the most to these fluorescence measurements, because the measured fluorescence arises from all possible states weighted by their respective occupancy probabilities and fluorescence intensities. We have drawn the cartoons to show that after binding of P_i and the third Na⁺ ion, the conformation of the protein is altered so that the fluorescence of S155C and E451C does not decrease at hyper- and depolarizing potentials, respectively. The data do not, however, allow us to identify the contribution to the fluorescence of individual states when both substrates are present.

	CONCLUSIONS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION CONCLUSIONS REFERENCES

 
We have identified four sites in the flounder Na/P_i-IIb protein which, when labeled with a fluorescent probe, give rise to voltage-and substrate-dependent fluorescence signals. Mutating the residues to cysteines and subsequent labeling with a Cys-reactive fluorescent probe caused only minor changes in the transport kinetics of the protein, indicating that the fluorescence reported on conformational changes of a minimally perturbed protein. The results show that at least a part of the large glycosylated loop between TMDs 3 and 4 is associated with conformational changes, and that these are mainly related to the transition of the empty carrier and the first Na⁺ binding step(s). In contrast, the two re-entrant loops located between TMDs 2-3 and 5-6 participate in conformational changes that additionally are influenced by P_i binding and/or substrate translocation. The results represent a first step in mapping time-resolved conformational changes of type II Na/P_i cotransporters.

	FOOTNOTES

 
^* This work was supported in part by the Gebert Rüf Foundation and the Swiss National Science Foundation (to H. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence may be addressed: Institute of Physiology, University of Zurich-Irchel, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland. Tel.: 41-44-635-5059; Fax: 41-44-635-5615; E-mail: leilav{at}physiol.unizh.ch. ² To whom correspondence may be addressed. E-mail: IForster{at}access.unizh.ch.

³ The abbreviations used are: VCF, voltage clamp fluorometry; WT, wild type; ANOVA, analysis of variance; TMD, transmembrane domain; MTSEA, (2-aminoethyl)methane thiosulfonate hydrobromide; MTS-TMR, 2-((5 (6)-tetramethylrhodamine)carboxylamino)ethyl methanethiosulfonate.

⁴ We do not know if all transporters in the membrane are labeled with the reagent, or if significant amounts of unlabeled protein remains. This is of no consequence for the fluorescence measurement, because F is only measured from labeled transporters and thus represents a homogenous population. However, when measuring presteady state currents, both labeled and unlabeled cotransporters contribute to the measurements, and since for both S155C and S226C labeling tended to shift the V_0.5 values of the Q-V distribution towards those of the F-V distribution, the question rises whether the remaining difference results from contamination of the Q-V distribution with unlabeled protein. However, because we observed qualitatively similar results using MTSEA, which has a higher reactivity than MTS-TMR and therefore could be expected to react with a larger number of transporters, we assume that the contribution of unlabeled transporters to the Q-V distribution is negligible. Furthermore, the change in the Boltzmann parameters determined from presteady state currents before and after labeling with MTSEA was much smaller (Fig. 2) than the differences we documented between the Q-V and F-V data.

⁵ Radanovic, T., Gisler, S. M., Biber, J., and Murer, H. (2006) J. Membr. Biol., in press.

⁶ T. Radanovic, unpublished observation.

	ACKNOWLEDGMENTS

 
We thank Eva Hänsenberger for preparing oocytes.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION CONCLUSIONS REFERENCES

 

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