I. Cysteine Scanning Mutagenesis
Address correspondence to Ian C. Forster, Physiologisches Institut, Universität Zürich-Irchel, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland. Fax: 41-1-635 5715; email: IForster{at}access.unizh.ch
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ABSTRACT |
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Key Words: mutagenesis site directed electrophysiology phosphate transport proteins electrogenic Xenopus laevis
K. Kohler's present address is Laboratory of Morphogenesis and Cell Signaling, UMR144, Institut Curie, Paris, France.
Abbreviations used in this paper: DTT, dithiothreitol; MTS, methanethiosulfonate; MTSEA, 2-aminoethyl MTS hydrobromide; MTSES, sodium (2-sulfonatoethyl) MTS; MTSET, 2-(trimethylammonium)ethyl MTS bromide; NaPi-IIa, type IIa Na+/Pi cotransporter; PFA, phosphonoformic acid; SCAM, substituted cysteine accessibility method; WT, wild type.
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INTRODUCTION |
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The predicted topology of NaPi-IIa, derived from studies on the rat isoform, suggests a protein with eight transmembrane domains, a large extracellular loop with two N-glycosylation sites and intracellular NH2 and COOH termini (Magagnin et al., 1993; Murer et al., 2000
) (Fig. 1 A). Antibody accessibility studies (Lambert et al., 1999b
) and cysteine scanning approaches (Lambert et al., 1999a
, 2000
; Kohler et al., 2002a
,b
) also support this model. Using SCAM we have identified sites in the putative third extracellular linker (ECL-3) (Lambert et al., 1999a
, 2001
) and the putative first intracellular linker (ICL-1) (Kohler et al., 2002a
,b
) that when modified by methanethiosulfonate (MTS) reagents result in block of cotransport mode activity. Given that NaPi-IIa is a functional monomer (Kohler et al., 2000
), and taking into account our previous SCAM findings, we currently postulate that these two topologically opposed linker regions associate with one another to constitute the transport pathway (Kohler et al., 2002a
,b
). To examine if other external linker regions may also define NaPi-IIa transport properties, we performed SCAM on sites in the first and fourth predicted linker regions, designated ECL-1 and ECL-4, respectively (Fig. 1 A). As shown in Fig. 1 B, these regions are very well conserved among several isoforms of the SLC34A family and therefore we might expect them to play common functional roles.
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MATERIALS AND METHODS |
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Reagents and Solutions
All standard reagents were obtained from either Sigma-Aldrich or Fluka. Methanethiosulfonate (MTS) reagents were obtained from Toronto Research Chemicals (Downsview). The solution compositions (in mM) were as follows. (a) Oocyte incubation (modified Barth's solution): NaCl (88), KCl (1), CaCl2 (0.41), MgSO4 (0.82), NaHCO3 (2.5), Ca(NO3)2, HEPES (7.5), adjusted to pH 7.6 with TRIS and supplemented with antibiotics (10 mg/l gentamycin, streptomycin). (b) Control superfusate (ND100): NaCl (100); KCl (2); CaCl2 (1.8); MgCl2 (1); HEPES-TRIS (10) at pH 7.4. Solutions with intermediate Na+ concentrations were prepared by mixing ND0 and ND100 in the appropriate proportions. (c) Substrate test solutions: inorganic phosphate (Pi) was added to the control superfusate from 1 M K2HPO4 and KH2PO4 stocks that were mixed to give the required pH (7.4 or 6.2). Phosphonoformic acid (PFA) was added to ND100 from frozen 100 mM stock (in H2O) to yield a final concentration of 1 mM. (d) MTS reagents, 2-aminoethyl MTS hydrobromide (MTSEA), 2-(trimethylammonium)ethyl MTS bromide (MTSET), and sodium (2-sulfonatoethyl) MTS (MTSES) were prepared from dry stock in DMSO to give 1 M stock solutions. These were stored at 20°C until required and added to ice-cold ND100 solution. The final concentration of DMSO did not exceed 0.1%, and DMSO alone at this concentration was confirmed not to alter the kinetic characteristics of the expressed constructs.
Immunoblotting of Oocyte Homogenates: Western Blotting
Yolk-free homogenates were prepared 3 d after injection of cRNA. Pools of eight oocytes were lysed together with 160 µl homogenization buffer (1% eluent [Calbiochem] in 100 mM NaCl, 20 mM Tris/HCl, pH 7.6) by pipetting the oocytes up and down (Turk et al., 1996). To pellet the yolk proteins, samples were centrifuged at 16,000 g for 3 min at 22°C. 10 µl supernatant was mixed with 10 µl 2x loading buffer (4% SDS, 2 mM EDTA, 20% glycerol, 0.19 M Tris/HCl, pH 6.8, 2 mg/ml bromphenol blue, 200 mM dithiothreitol [DTT]), denatured for 2 min at 95°C and separated on a 10% SDS-PAGE gel. Separated proteins were transferred onto a nitrocellulose membrane (Schleicher & Schuell). The membrane was then processed according to standard procedures (Sambrook et al., 1989
) using a rabbit polyclonal antibody raised against a synthetic peptide from the NH2 termini of the rat NaPi-IIa cotransporter (dilution 1:2,000). The specificity of the antibody has been demonstrated previously (Custer et al., 1994
). Immunoreactive proteins were detected with a swine anti-rabbit Ig horseradish peroxidaselinked F(ab')2 secondary antibody (dilution 1:5,000) (Amersham Biosciences) and visualized with a chemiluminescence system (Pierce Chemical Co.).
Streptavidin Precipitation of MTSEA-biotinylated Protein
Groups of eight oocytes per NaPi-IIa mutant were incubated for 10 min in 1 mM MTSEA-Biotin, prepared as above. Oocytes expressing the S460C or the WT protein were taken as positive and negative control, respectively. Biotin-streptavidin precipitation was performed as previously described (Lambert et al., 1999a). In brief, after homogenization in 160 µl of homogenization buffer and centrifugation (see immunoblot of oocyte homogenates), a sample for Western blotting was taken. The rest of the supernatant (
120 µl) was incubated for 2 h with 60 µl ImmunoPure immobilized streptavidin beads (Pierce Chemical Co.) on a rotator. After five washing steps with homogenization buffer, precipitated proteins were eluted with 2x loading buffer (including DTT) at 95°C for 5 min. Samples were loaded on a 10% SDS gel and immunoblotted after protein separation.
Functional Assays and Data Analysis
Radiolabeled Pi Uptake.
This procedure has been described in detail elsewhere (Werner et al., 1990). 32Pi uptake was measured 3 d after injection in water-injected (control) and cRNA-injected oocytes (n
10).
Electrophysiology.
The standard two-electrode voltage clamp technique was used as previously described (Forster et al., 1998). The voltage clamp was a laboratory-built system with membrane current measured using a virtual ground bath electrode and with active series resistance compensation to reduce clamp errors. Oocytes were mounted in a small recording chamber (100 µl volume) and continuously superfused (5 ml/min) with test solutions precooled to
20°C. Data were acquired online using Digidata 1200 hardware and compatible pClamp8 software (Axon Instruments, Inc.). Recorded currents were prefiltered at a bandwidth less than twice the sampling rate (typically 20 Hz for fixed voltage recording and 200 Hz for IV determination).
Steady-state Current-Voltage (IV) Relations.
These were determined by using a voltage staircase from 120 mV to +40 mV with 100-ms-long steps, as previously described (Forster et al., 1998), and subtracting the response in the absence of substrate (Pi or PFA) from that in the presence of substrate. Only single sweeps were used.
Incubation with MTS Reagents and Determination of Rate of Modification.
Freshly prepared MTSEA/ET/ES (see above) was delivered to the oocyte chamber using a stainless steel cannula (inner diameter 0.3 mm) positioned near the cell and fed by gravity. Reagents were always applied in the presence of ND100 at a holding potential Vh = 50 mV. Incubation was followed by a 1-min washout period before applying Pi (1 mM) as the test assay. A further 1-min washout period was allowed before reincubation at the next concentration of MTS reagent to ensure complete removal of substrate and return of response to a steady-state holding current.
Cys Modification Reaction Rate.
The effective second order reaction rates were determined by fitting a single decaying exponential to the peak Pi-dependent current after a cumulative exposure time t, (IPit) determined following each successive MTS exposure:
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All curve fitting was performed using GraphPad Prism version 3.02/4.02 for Windows (GraphPad Software).
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RESULTS |
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As shown in Fig. 2 B, mutants displayed three types of behavior, depending on their response to 1 mM Pi before and after MTSEA exposure: (1) WT-like (see Fig. 2 B, inset 1) in which little change in activity occurred, found for five mutants in ECL-1 (G130C, V132C, D135C, K138C, and D139C) and one in ECL-4 (S532C); (2) one mutant in ECL-1 (G134C) that showed increased activity (Fig. 2 B, inset 2); and (3) those in which a loss of activity occurred, like F137C (Fig. 2 B, inset 3) and others in ECL-1 (I136C and N140C) and ECL-4 (M533C, A534C, and A538C). In addition to cotransport mode (IPi), the leak mode was assayed electrophysiologically by measuring the response to the blocker PFA (1 mM) (Forster et al., 1998) under the same experimental conditions. At Vh = 50 mV, all the functional mutants showed a reduced holding current during PFA application (IPFA), which indicated that the leak mode was still viable. Two mutants in ECL-4 (G535C and W536C) gave 32Pi uptake significantly greater than H2O-injected cells (Fig. 2 A); however, the magnitude of the Pi-dependent currents was typically
15 nA using oocytes from three donor frogs. This low electrogenic activity and the absence of any detectable effect of MTSEA on 32Pi uptake led to the exclusion of these mutants from further investigation.
The invariance of IPi before and after MTSEA exposure (G130C, V132C, D135C, K138C, and D139C in ECL1; S532C in ECL-4), could indicate that (a) the mutated sites were functionally unimportant, (b) other characteristics were modified that were not identified under the assay conditions, or (c) the sites were not accessible from the external medium by MTSEA. To test the latter possibility, we incubated mutant-expressing oocytes in MTSEA-Biotin and performed a streptavidin precipitation with immunodetection using an antibody raised against the NH2 terminus of the NaPi-IIa protein. We used the mutant S460C in ECL-3 (Fig. 1 A), which we have previously shown to be accessible by MTSEA (Lambert et al., 1999a), and WT-expressing oocytes as positive and negative controls, respectively. As shown in Fig. 3, except for D135C (ECL-1) and W536C and Q537C (ECL-4), MTSEA-biotin was able to bind to all mutants, which confirmed the accessibility of these linkers from the external medium.
Rate of MTS Modification Reveals Differences in Apparent Accessibility
We estimated the rate of the Cys modification reaction for mutants that showed a significant change of cotransport function after MTS exposure under standard assay conditions, by measuring IPi after successive exposures to a fixed concentration of MTS reagent. The MTS reagent concentration was chosen initially by trial and error so that a 2-min exposure resulted in an intermediate change of activity, typically in the range 3060% of the initial response. The test concentration was found to vary over two orders of magnitude, depending on the mutation site (Table I). Fig. 4 A shows a representative record from an oocyte that expressed mutant F137C (ECL-1) during repeated exposure to 10 µM MTSET. After each exposure and washout, the response to Pi was tested and the holding current was allowed to recover to the initial baseline before the next exposure. After normalization, these data were fit with a single exponential function (Eq. 1). Under the assumption that the MTS reagent was always in excess, we could estimate the effective second order reaction rate (k*) (Table I). Impermeant (MTSET) and semi-permeant (MTSEA) reagents were used to confirm the sidedness of the reaction, i.e., to confirm that modification occurred from the external medium in the present study. Fig. 4 (B and C) illustrates the behavior of selected mutants from ECL-1 and ECL-4, respectively.
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We previously reported that oocytes that expressed the flanking mutations in ECL-4 (S532C and A538C) did not show a significant loss of transport function when exposed to MTSEA at 100 µM (Lambert et al., 1999a). However, in the present study, at 10-fold greater MTSEA concentration, significant loss of function was observed for A538C, but still not for S532C (Fig. 2 B). Additional electrophysiological assays (unpublished data) on mutant S532C showed
20% loss of response to 1 mM Pi (Vh = 50 mV) when exposed to 1 mM MTSEA for 10 min and
40% after 25 min (unpublished data; n = 3). Because of its low reactivity compared with the other mutants in ECL-4, this site was considered only marginally accessible. At 1 mM concentration, it is possible that MTSEA was also present intracellularly, due to its reported membrane permeability (e.g., Holmgren et al., 1996
), and therefore the loss of cotransport function may have been due to cytosolic action on other, internally accessible cysteines. However, exposure to 1 mM of MTSET also led to a comparable loss of activity for A538C, albeit with slower reaction rates (Fig. 4 C; Table I). This confirmed that the novel cysteines were accessible from the extracellular medium, in agreement with their predicted topology and the surface biotinylation assay.
Substrate Activation Unaffected by Cys Substitution and Cys Modification
The finite activity that remained at the completion of the MTS modification reactions suggested that kinetic properties of the mutated cotransporter were sensitive to cys modification. The most likely explanation would be a reduction (or increase, in the case of G134C) of the apparent substrate affinities at 50 mV. We therefore screened all the functional mutants to compare their substrate activation properties before and after a 5-min application of 1 mM MTSEA. To detect large deviations from the WT behavior of the apparent affinities for Pi (KmPi) and Na+ (KmNa), we applied a two-point assay, because in the case of poorly expressing mutants, a complete dose response could be difficult to obtain due to contamination from endogenous currents at low substrate concentrations. We have previously applied these assays to identify a mutant in the first intracellular linker region that showed significantly decreased apparent substrate affinities compared with the WT (Kohler et al., 2002a).
Pi activation was assayed by comparing the ratio of the responses to 0.1 mM Pi and 1 mM Pi for superfusion in ND100, termed the Pi activation index. Similarly, a Na+ activation index, given by the ratio of the responses to 1 mM Pi for superfusion in ND50 and ND100, was used to quantitate the Na+ activation. We confirmed the sensitivity of using this assay with simulations in which we assumed the Pi and Na+ activation characteristics could be described by the modified Hill equation. For Pi activation (Fig. 5 A, left), the typical range of the index for the WT, based on assays performed in this study as well others (Lambert et al., 2000; Kohler et al., 2002a
), should allow detection of changes in KmPi outside the range 0.05 < KmPi < 0.12 mM. For the Na+ activation (Fig. 5 A, right), we would conservatively expect to detect changes in KmNa outside the range 40 < KmNa < 60 mM. For example, if in the case of mutant G137C, the 60% decrease in the electrogenic response to 1 mM Pi after MTSEA exposure were due only to an increase in the apparent KmPi from the typical WT value of 0.06 mM to 1.65 mM, the Pi activation index would decrease from
0.66 to 0.15, based on a simple Michaelian model for Pi activation and the same maximum transport rate ± MTSEA.
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Cys Substitution and Cys Modification Alter Voltage Dependency
Further insight into the mechanism underlying the changes in transport activity after cys modification was obtained from the currentvoltage (IV) relationships for Pi-dependent current. These were determined for superfusion with ND100 in response to the application of 1 mM Pi over voltage window 120 V
+20 mV, in which contamination by endogenous currents was negligible. IV data for four mutants that showed significantly changed activity after MTSEA treatment (1 mM, 35 min), G134C, I136C, F137C (ECL-1), and M533C (ECL-4), are shown in Fig. 6 A. Similar IV data was also obtained using the positively charged MTSET and the negatively charged MTSES (unpublished data). To account for differences in expression levels between oocytes that expressed the same mutant, IV data were normalized to the magnitude of IPi at 100 mV (1 mM Pi) before MTSEA exposure for WT, I136C, F137C and M533C. In the case of G134C, data were normalized to the magnitude of IPi after MTSEA exposure to aid comparison with the WT data. For the WT, MTSEA, MTSET, MTSES treatment at concentrations up to 10 mM resulted in insignificant changes to IPi over the voltage window shown (unpublished data).
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The negative slopes for G134C MTS and M533C + MTS were inconsistent with the expected behavior of an electrogenic secondary-active transport system in which one net positive charge is transported per cycle (Forster et al., 1998). However, we could reconcile this apparent anomaly when we took account of an error introduced by the subtraction procedure used to obtain IPi. In these assays, we eliminated oocyte endogenous currents by subtracting the holding current in ND100 from the current in ND100 + Pi. However, if the leak and cotransport modes are assumed to be mutually exclusive (Kohler et al., 2002b
), this procedure would underestimate the true cotransport mode current by an amount equal to the leak, if we assume that it is fully suppressed in the presence of 1 mM Pi. The leak mode activity was estimated by recording the change in holding current when superfusing in 1 mM PFA (IPFA). Fig. 6 B shows the Pi-dependent and leak currents for a representative oocyte that expressed M533C (B) and G134C (C) before and after incubation in 1 mM MTSEA for 3 min. The effect of MTS exposure on the leak current was minimal over the voltage range 120 < V < 0. For V > 0 mV, we found that endogenous Cl currents could also be induced by PFA in noninjected oocytes, and these data were therefore considered unreliable. When IPi was corrected for the leak error by adding IPFA at each potential, the leak-corrected IPi data now showed a positive slope with voltage-independent rate-limiting behavior at hyperpolarizing potentials for M533C + MTS and G134C MTS.
The unique behavior of the G134C steady-state IV data before and after MTS exposure can also account for the apparent anomaly between the 32Pi uptake assay and the electrophysiological assay at Vh = 50 mV for this mutant. As shown in Fig. 6 C there is a crossover of the leak-corrected G134C IV data before and after MTS exposure at approximately 40 mV for this oocyte. Under voltage clamp conditions for Vh < 50 mV, we would therefore predict an increased cotransport activity after MTS exposure. However, for the 32Pi assay conditions used in this study, the membrane potential (Vm) was undefined. Therefore, externally applied Pi would induce a net inward positive charge movement (3 Na+ + 1 HPO42 per cycle) that would depolarize the oocyte membrane. If the shift in Vm were more positive than the crossover potential, a reduced uptake would be expected, since 32Pi uptake and net translocated charge are tightly coupled for electrogenic type II Na+/Pi cotransporters (Forster et al., 1999).
To test this hypothesis, we measured the change in membrane potential (Vm) with the voltage clamp loop open before and after MTS exposure. For the representative G134C-expressing oocyte in Fig. 6 C, application of 1 mM Pi induced a change in Vm from 28 mV to +5 mV, whereas after MTS exposure, Vm changed from 35 to 10 mV. For a given cell impedance, the Pi-dependent change in Vm is proportional to the net charge translocated, which is a function of the number of transporters in the membrane (assumed constant) and the transporter turnover rate. It follows from these data that we would expect a reduced 32Pi uptake after MTS exposure, as we observed. We found that this result was also consistent with the corresponding currents at the Vm reached during Pi application (MTS, 56 nA; +MTS, 42 nA), which we found by interpolation of the leak-corrected IV data of Fig. 6 C. The reduced depolarizing shift in Vm after MTS exposure was confirmed for a batch (n = 9) of G134C-expressing oocytes, under the same conditions as for the tracer flux assay. The change in Vm due to Pi superfusion was +31 ± 1 mV for MTS, and this decreased to +19 ± 1 mV for +MTS. These findings serve to underscore the importance of defining the membrane potential in transport assays involving electrogenic membrane proteins.
From the IV data of Fig. 6 A, it was obvious that the effect of cys substitution at Gly-134 and modification of Cys-136, Cys-137C, and Cys-533 was to reduce the transport activity induced by 1 mM Pi over a wide voltage range. By normalizing the leak-corrected IV data to V = 0, the relative effect of hyperpolarizing membrane potentials on the cotransport activity before and after MTS exposure was determined (Fig. 7 A). These data showed that cys substitution at Ile-136 and Met-533 resulted in constructs with a voltage dependency that was indistinguishable from the WT. Cys modification at these sites increased (I136C + MTS) and decreased (M533 + MTS), respectively, the response to changes in membrane potential in the hyperpolarizing direction relative to the WT. Cys substitution at Phe-137 gave an increased voltage dependency compared with the WT that was further augmented after MTS exposure, like I136C + MTS. Finally, cys substitution at Gly-134 gave a reduced voltage dependency compared with the WT that was comparable to M533C + MTS. After MTS exposure, the voltage dependency of G134C was similar to that of I136C + MTS.
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DISCUSSION |
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To interpret our data, which is based on the introduction of novel Cys residues in the WT backbone, we made a tacit assumption that the target Cys residue was the novel Cys itself and not one or more of the 12 native Cys residues. Ideally, SCAM should be performed on a cys-less backbone to avoid ambiguities that could arise if the introduction of a novel Cys exposes a previously inaccessible and functionally important native Cys (Kaback et al., 2001; Kamdar et al., 2001
). Unfortunately, a cys-reduced construct of NaPi-IIa shows significantly lower transport activity compared with the WT (Kohler et al., 2003
) and this would exacerbate detailed kinetic analyses. Nevertheless, three pieces of evidence support the validity of the above assumption: (1) when removed singly, no native Cys appears to be critical for viable cotransport function (Lambert et al., 2000
); (2) up to eight Cys can be removed, while retaining transport function (Kohler et al., 2003
), the remaining four being involved in two disulfide bridges (Lambert et al., 2000
; Kohler et al., 2003
); and (3) introduction of a Cys at site 460, previously found to be functionally important for the WT backbone (Lambert et al., 1999b
), shows the same behavior in the cys-reduced NaPi-IIa (Kohler et al., 2003
).
Accessibility and Membrane Topology
Although we used MTSEA as the standard probe reagent, exposure to impermeant MTSET gave qualitatively similar results. This confirmed that the sites were accessible from the external medium only. Small differences in reactivity between MTSEA and MTSET were observed in the rates of modification (i.e., the rate of change of the electrogenic activity) at different sites in ECL-1 and ECL- 4 (Table I). The faster reaction rates of MTSET for most mutants most likely reflect the higher reactivity of this reagent with small thiols compared with MTSEA (Karlin and Akabas, 1998). For G134C and A538C, MTSEA was more reactive than MTSET, perhaps because the greater bulk of MTSET restricts its access to these sites. As summarized in Fig. 8, the effective second order reaction rate constants lay within the range of values that we have previously reported for Cys mutants in ECL-3 (Lambert et al., 2001
) and establish conclusively that these three regions are accessible from the external aqueous environment. Rates of modification for extracellularly accessible sites in other transport proteins such as the excitatory amino acid transporter (EAAT1) (e.g., Leighton et al., 2002
), the mitochondrial citrate transporter (Ma et al., 2004
), and the serotonin transporter (SERT) (Chen et al., 1997
) have been reported to lie in the same range as we found for NaPi-IIa. In the present study, the modification rates for mutants in ECL-1 were consistently faster than those in ECL-4. This would suggest that ECL-4 was less accessible from the extracellular medium. At one site in ECL-1 (Asp-135) and one site in ECL-4 (Trp-536) we were unable to detect the novel cysteines using the biotinylation assay, which indicated that these cysteines were inaccessible, either because of the bulk of MTSEA-Biotin or the specific folding of the linkers at these sites.
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Modulation of Steady-state Voltage-dependent Kinetics
In a previous SCAM study, a cluster of mutants was found in ECL-3, most of which showed full suppression of cotransport mode activity after MTS exposure (Lambert et al., 2001). These constructs yielded characteristically similar Pi and PFA responses at the end of the modification reaction, from which we concluded that Pi could interact with NaPi-IIa and thereby inhibit the leak mode, but the cotransport mode was suppressed. This led us to postulate that these sites are associated with the transport pathway itself (Lambert et al., 1999a
, 2001
; Kohler et al., 2002a
,b
). In the present study, all the MTS-sensitive mutants, except A538C, showed a Pi-dependent electrogenic response at 50 mV that asymptoted to a nonzero level, at the end of the modification reaction. This behavior suggested that they may not be directly associated with the transport pathway or the substrate recognition site as, in the latter case, kinetic analysis of these mutants under voltage clamp conditions revealed that apparent substrate affinities appeared unchanged, either from cys substitution or cys modification. On the other hand, cysteine engineering at sites in ECL-1 and ECL-4 resulted in significantly altered voltage-dependent activity compared with the WT, depending on the site of cys substitution or cys modification.
Mutants could be broadly classified according to whether there was increased, decreased, or unchanged activity compared with the WT for hyperpolarizing membrane potentials (0 to 100 mV) (Fig. 7 B). The finding that most mutants deviated from WT behavior suggested that the amino acid residues in ECL-1 and ECL-4 were critical determinants of the NaPi-IIa voltage-dependent kinetics. Several "hot spots" emerged from these assays, for which the voltage-dependent activity of the corresponding mutants deviated significantly from the WT, either due to cys substitution alone, cys modification, or both. For example, cys substitution at Ile-136 increased the voltage-dependent activity, whereas at the neighboring Phe-137, cys substitution was well tolerated. However, cys modification at both sites led to a significantly increased voltage-dependent activity. Interestingly, cys substitution at Gly-130 and Ile-136 led to the same increase in activity. This was mimicked by cys modification of Cys-134, intermediate in the amino acid sequence in ECL-1, and by cys substitution at Ala-534 and Ala-538 in ECL-4. Such similar behavior indicated that engineering at sites in each linker had induced the same voltage-sensitive conformational changes in the protein. Complementary behavior was also documented, in which the opposite effects on voltage dependency were found for cys substitution at Gly-134 in ECL-1 and Met-533 in ECL-4. G134C-MTS and M533C + MTS both exhibited a weakly voltage-dependent activity, whereas that of G134C + MTS exceeded the WT, and the voltage dependency of M533C MTS was indistinguishable from the WT. Interestingly, cys substitution at the Ser-532 resulted in the same weak voltage-dependent activity as shown by M533C + MTS with little change in activity after MTS exposure. This suggested that the conformational change induced by modification of Cys-533 in ECL-4 was the same as that made by introducing a cysteine at the preceding site (Ser-532), or at Gly-134 in ECL-1. The novel complementary behavior of G134C and M533C is the subject of a more detailed kinetic analysis in the accompanying study (Ehnes et al., 2004).
In addition to relative changes in voltage-dependent activity, the IV behavior revealed that for G134C MTS and M533C + MTS, the maximum transport rate at the hyperpolarizing limit was also affected. In each case, the leak-corrected IV curves showed rate-limiting behavior in the same voltage range where the WT, G134C + MTS, and M533C MTS voltage-dependent activity was still approximately linear. The overall behavior of these mutants suggested that the cys substitution and cys modification at these sites had (a) slowed one or more voltage-independent rate constants in the transport cycle and (b) induced a depolarizing shift in voltage-dependent transitions associated with either the empty carrier, first Na+ binding transition, or both, in accordance with the current alternating access model for NaPi-IIa (Forster et al., 2002).
Are sites in ECL-1 and ECL-4 part of the voltage-sensing mechanism of NaPi-IIa? Three pieces of evidence suggest this is not the case. First, one might expect mobile charged or polar residues to constitute the voltage sensor and their removal would have a dramatic effect on the electrogenic behavior of the protein. Although ECL-4 has no charged residues, three sites in ECL-1 (D-135, K-138, and D-139) could be potential candidates, but when replaced by cysteines, the resulting mutants were still electrogenic (Fig. 7 B), albeit with only small deviations in voltage dependency from the WT. Similarly, substitution of the polar Ser-532 (ECL-4) with a nonpolar Cys weakened the voltage dependency (Fig. 7 B). These findings suggest that the particular charged or polar residues may certainly interact with the putative voltage-dependent elements to modulate the voltage dependency of transport, but the intrinsic voltage sensitivity of the protein remains intact. In another electrogenic cotransporter, the Na+-coupled glucose transporter (SGLT-1), polar residues have been shown to modulate charge movements and alter empty carrier kinetics without affecting the overall apparent valency of the cotransporter (Panayotova-Heiermann et al., 1994). Second, one member of the SLC34 family, the renal type IIc isoform, is electroneutral (Segawa et al., 2002
; unpublished data). NaPi-IIc has a high homology with the electrogenic type IIa Na+/Pi cotransporter, with both charged residues in ECL-1 being conserved (Fig. 1 B). Third, the same MTS-induced changes to the voltage dependency occurred using either positively or negatively charged MTS reagents. This suggests that the charge introduced by these reagents either lies outside the transmembrane field or does not interact with the intrinsic voltage-sensing elements.
In summary, the outermost external linkers of the NH2- and COOH-terminal halves of NaPi-IIa contain sites that when occupied by Cys residues, are localized in an externally accessible, hydrophilic environment, which is consistent with hydropathy predictions. The native residues in these linkers are not absolutely essential for Na+-coupled Pi cotransport function; however, cys substitution or covalent modification of the novel cysteines by externally applied MTS reagents affected the voltage-dependent kinetics of the mutant transporters. As changes in membrane potential are expected to induce major conformational changes in the native protein during the transport cycle (e.g., Loo et al., 1998), the findings reported here suggest that linkers ECL-1 and ECL-4 contain specific sites that may interact directly or indirectly with mobile, voltage-sensitive elements of the NaPi-IIa protein.
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ACKNOWLEDGMENTS |
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This work was supported by grants to H. Murer from the Swiss National Science Foundation (31-46523), the Hartmann Müller-Stiftung (Zurich), the Olga Mayenfisch-Stiftung (Zurich), and the Union Bank of Switzerland (Zurich) (Bu 704/7-1).
Lawrence G. Palmer served as editor.
Submitted: 12 March 2004
Accepted: 14 September 2004
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REFERENCES |
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