Institute of Physiology, University of Zurich, Zurich CH-8057, Switzerland
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ABSTRACT |
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Intrasequence comparison of the type IIa Na+-Pi cotransport protein revealed two regions with high similarity in the first intracellular (ICL-1) and third extracellular (ECL-3) loops. Because the ECL-3 loop contains functionally important sites that have been identified by cysteine scanning, we applied this method to corresponding sites in the ICL-1 loop. The accessibility of novel cysteines by methanethiosulfonate reagents was assayed electrophysiologically. Mutants N199C and V202C were fully inhibited after methanethiosulfonate ethylammonium exposure, whereas other mutants showed marginal reductions in cotransport function. None showed significant functional loss after exposure to impermeant methanethiosulfonate ethyltrimethylammonium, which suggested a sidedness of Cys modification. Compared with the wild-type (WT), mutant A203C showed altered Na+ leak kinetics, whereas N199C exhibited decreased apparent substrate affinities. To delineate the role of residue N199 in conferring substrate affinity, other mutations at this site were made. Only two mutants yielded significant 32Pi uptake and inward Pi-induced currents with decreased Pi affinity; for the others, Pi application suppressed only the Na+ leak. We suggest that ICL-1 and ECL-3 sites contribute to the transport pathway and that site N199 is implicated in defining the transport mode.
electrophysiology; secondary structure; type IIa sodium-phosphate cotransporter
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INTRODUCTION |
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THE REABSORPTION OF
PI in the renal proximal tubule is achieved by a
secondary, active, electrogenic Na+-coupled cotransport
system that is mediated by the type IIa Na+-Pi
cotransport protein (NaPi-IIa) located at the brush-border membrane
(21). As expressed in Xenopus laevis oocytes,
the kinetic properties of this protein have been described in detail by
study of the 32Pi uptake or measurement of the
Pi-induced inward current under voltage clamp. This current
can be explained in terms of a 3:1 Na+/HPO
The rat NaPi-IIa isoform is a 637-amino acid glycoprotein with a
glycosylated molecular mass of 90-100 kDa (8, 20,
21). The topological model predicted from hydrophobicity data
(20) and confirmed by epitope studies (18)
exhibits a secondary structure with eight transmembrane domains, a
large extracellular loop with two N-glycosylation sites
(8), and intracellular NH2 and COOH termini
(Fig. 1B). Intrasequence
comparison of the protein revealed that part of the first intracellular
loop of the NH2-terminal half (ICL-1) shows a high degree
of similarity to part of the third extracellular (ECL-3) in the
COOH-terminal half (15). To identify regions of functional
importance (12), we used the substituted-cysteine
accessibility method (SCAM). We could show that cysteine substitutions
in the putative ECL-3 loop yield functional constructs that are readily
inhibited by external application of methanethiosulfonate (MTS)
reagents. This led to the proposal that part of the ECL-3 region
contributes to both the cotransport mode and slippage mode pathways of
NaPi-IIa (16, 17). Based on the intrasequence similarity
between the ICL-1 and ECL-3 regions, we hypothesized that functionally
important residues should also exist in the NH2-terminal
region.
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We therefore mutated eight amino acids, one by one, within the identified stretch in the ICL-1 site to a cysteine. These corresponded to sites already identified in the ECL-3 site to yield transporters that were of functional importance based on kinetic properties before and after MTS treatment (17). After the mutations were expressed in Xenopus laevis oocytes, the cotransport function of the mutants was assayed electrophysiologically before and after exposure to MTS reagents (as a means of identifying functionally important sites). Two mutants (N199C and V202C) showed a complete loss of cotransport function after incubation in MTS-ethylammonium (MTSEA), which suggested that these sites were accessible and functionally important; the others were only partially or insignificantly inhibited after incubation. Incubation with impermeant MTS-ethyltrimethylammonium (MTSET) did not invoke a significant inhibition of the Pi-induced current (IPi) for any of the eight mutants. Furthermore, kinetic characterization of the mutants revealed two with significantly altered steady-state kinetics compared with the wild-type (WT). Mutant A203C showed an increased transport rate for the Na+ leak, and mutant N199C exhibited decreased apparent substrate affinities. Further amino acid substitutions at site 199 resulted in mutants that displayed either WT-like cotransport function (with reduced substrate affinity) or slippage mode only. This confirmed that this site is critical for establishing the transport mode of NaPi-IIa.
Taken together with our recent report (14) that documents NaPi-IIa to be a functional monomer, these new findings extend our previous observations (16, 17) and are consistent with NaPi-IIa having a single transport pathway that involves associated parts of the ECL-3 and ICL-1 loops.
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EXPERIMENTAL PROCEDURES |
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Reagents and chemicals. All restriction enzymes were obtained from Pharmacia Biotech or Biofinex. Oligonucleotide primers were obtained from Microsynth (Balgach, Switzerland), and a mutagenesis kit was purchased from Stratagene. MTS reagents were obtained from Toronto Research Chemicals (Toronto, Canada). Other reagents were obtained from Fluka (Buchs, Switzerland).
Molecular biology. Mutations were introduced in accordance with the instructions in the manual accompanying the QuikChange site-directed mutagenesis kit (Stratagene) as previously described (16). The sequence was verified by sequencing. All constructs were cloned in pSport1 (GIBCO-BRL). The in vitro synthesis and capping of cRNAs were performed according to instructions in the Ambion MEGAscript TM T7 kit manual (16).
Solutions. The solutions for the electrophysiological assays were composed as follows. Control superfusate ND100 contained (in mM) 100 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, and 5 HEPES adjusted to pH 7.4 with Tris. Control superfusate ND0 composition was as for ND100 but with N-methyl-D-glucamine used to replace Na+, and solutions were adjusted to pH 7.4 with HCl. Solutions with intermediate Na+ concentrations were prepared by mixing ND0 and ND100 in the appropriate proportions. For the substrate test solutions, Pi was added to ND100 from a 1 M K2HPO4/KH2PO4 stock that was preadjusted to pH 7.4, and the Na+-Pi cotransport inhibitor PFA was added to ND100 from frozen stock (in H2O) to yield a final concentration of 3 mM. MTSEA and MTSET were prepared in DMSO, frozen in aliquots at 1 M and 10 M, and freshly diluted in ND100 from the stock for each oocyte immediately before use. The final concentration of DMSO did not exceed 0.2%. At this concentration, the kinetic characteristics of the expressed constructs were unaltered.
Xenopus laevis oocyte expression. The procedures for oocyte preparation and cRNA injection have been described in detail elsewhere (29). Oocytes were injected with either 50 nl of water or 50 nl of water containing 10 ng of cRNA. Oocytes were incubated in modified Barth's solution and the experiments were performed 3-4 days after injection. All pooled data were generated with oocytes from at least two different donor frogs.
Functional assays and data analysis. The procedure used for the 32Pi-uptake assay has been described in detail elsewhere (29). 32Pi uptake was measured 3 days after injection of both water- and cRNA-injected oocytes (n = 8).
The standard two-electrode voltage-clamp technique was used as previously described (4). The steady-state response of an oocyte to Pi and PFA was always measured at a holding potential (Vh) of
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(1) |
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(2) |
Immunoblots of oocyte homogenates. Western blots were made from yolk-free homogenates of oocytes prepared 3 days after injection (H2O or cRNA) as previously described (28) and stained with rabbit polyclonal antibodies raised against synthetic peptides from the NH2 terminus of NaPi-IIa (2).
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RESULTS |
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Identification of similar regions and expression of mutants in oocytes. Figure 1A shows the result of an intrasequence comparison of the NaPi-IIa cotransporter protein. This revealed two stretches of amino acids from 414 to 464 in the ECL-3 site of the COOH-terminal half and from 160 to 210 in the ICL-1 of the NH2-terminal half that have a high degree of similarity (80%). The location of these stretches is indicated on the current topological model for NaPi-IIa (Fig. 1B). As indicated in Fig. 1C, the aligned amino acid sequences include two regions of five and six residues, respectively, that show exact identity. We applied SCAM to eight selected sites in the ICL-1 region (Fig. 1C) that were chosen to correspond to those sites identified in the ECL-3 site as being functionally important within the corresponding stretch in the ECL-3 site (17).
Cys mutants were expressed in Xenopus oocytes and characterized in terms of expression (via Western blot analysis) and transport function by measuring the IPi under voltage clamp. As shown in Fig. 2A, all eight mutants were expressed in comparable amounts and with the same expression pattern as the WT NaPi-IIa protein. All constructs were expressed at the membrane as evidenced by IPi >10-fold higher than the endogenous response (data not shown). Electrogenic responses varied between 40 and 80% of the WT activity found in oocytes from the same donor frog (data not shown). Mutant N199C was notable for displaying IPis that were typically only 20% of the WT response. Such variation in activity could be due to the induction of altered kinetics by Cys mutagenesis (see Kinetic characterization of Cys mutants) and/or reduced surface expression due to improper membrane targeting. Because our primary interest in this study was construct function, the latter effect was not investigated in detail here.
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Effect of MTS reagents on transport function of
mutants.
The NaPi-IIa cotransporter exhibits two transport modes that can be
assayed by electrophysiology: a Na+-dependent leakage or
slippage mode in the absence of external Pi
(stoichiometry = 1 Na+) that is inhibited by the
competitive inhibitor PFA and a cotransport mode (assumed
Na+/HPOIPFA, where IPFA is the
PFA-induced current. To quantify the cotransport mode activity, we
assumed that the Na+ leak is uncoupled from the cotransport
mode. As the IPi was measured relative to the holding current, this must be adjusted by
IPFA to obtain the true cotransport current
relative to the zero-transport level [i.e., the cotransport activity
is then given by IPi + IPFA as previously described
(17)].
Kinetic characterization of Cys mutants.
As previously reported (16, 17), Cys mutagenesis alone can
lead to altered transport kinetics for NaPi-IIa. We therefore investigated the slippage and cotransport mode characteristics of the
new mutants. First, the Na+ leak
(IPFA) was determined for each mutant and
quantitated relative to the cotransport mode current as a slippage
index (17):
IPFA/(IPFA + IPi). IPFA
and IPi were determined using the
standard test concentrations (1 mM Pi and 3 mM PFA) at
Vh =
50 mV. All mutants showed a WT-like slippage index except N199C, which showed little detectable
Na+ leak and A203C, which showed a fivefold larger index
(Fig. 3A; see also Fig.
2B). These findings indicated that Cys substitution at sites
199 and 203 had led to differentially altered kinetics for the slippage
mode. Second, for the cotransport mode, we assessed the behavior of the
mutants using the indices for Pi and Na+
activation (17). These indices were obtained from the
ratio of IPi at 0.1 and 1 mM
Pi (100 mM Na+) for Pi activation
and the ratio of IPi at 50 and 100 mM Na+ (1 mM Pi) for Na+ activation
at Vh =
50 mV. All mutants except N199C
showed Pi and Na+ activation indices that were
within the expected WT tolerance (Fig. 3B).
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Slippage mode behavior of mutant A203C.
The current scheme for NaPi-IIa slippage mode kinetics involves an
empty carrier-voltage-dependent transition, binding and debinding of
one Na+ ion, and an electroneutral translocation by
Na+ (4, 16). To identify which of these
transitions could be influenced by cysteine-203, we characterized the
steady-state behavior of A203C in the slippage mode.
Na+-induced currents were measured at different
Na+ concentrations in the absence of Pi
relative to the response in 0 mM Na+. As shown in Fig.
4A, A203C showed an increase
in Na+ leak at all concentrations over and above the WT
response. Endogenous effects accounted for <10% of these currents as
confirmed with noninjected control oocytes (data not shown). These data
could be described analytically using a Michaelis-Menten relationship, which was in agreement with our earlier finding (4) and
also confirmed that Cys modification had not altered the stoichiometry of the Na+ interaction with the transporter. The fit
indicated that the predicted apparent affinity for Na+
(K
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(3) |
Cotransport mode behavior of mutant N199C.
The deviation from WT behavior of the activation indices for mutant
N199C suggested alterations in the respective substrate affinities. We
therefore generated dose-response curves for this mutant for
Pi activation (at 100 mM Na+) and
Na+ activation (at 1 mM Pi) as shown in Fig. 4,
C and D, respectively. Unlike the WT, the
Pi activation for N199C at 100 mM Na+
(Vh = 50 mV) did not saturate even at 4 mM Pi, although the fit using the Hill equation indicated a
Michaelian relationship (predicted Hill coefficient,
nH = 0.8). The estimated apparent affinity
constant for Pi
(K
Mutations at site 199 lead to functional and dysfunctional mutants.
To explore further the role of site 199 in conferring substrate
affinity, we made other amino acid substitutions at this site by
including conservative and nonconservative substitutions with different
side-chain lengths and/or charges. The six mutant constructs (N199A,
N199D, N199Q, N199R, N199H, and N199T) were assayed for expression in
Western blot analysis of whole oocyte lysate (Fig. 5A). This confirmed that all
mutants were expressed with the same molecular weight and similar
protein amounts as the WT transporter.
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DISCUSSION |
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We have identified two sites, 199 and 202, in the putative ICL-1 of the NaPi-IIa protein that are accessible to the Cys-modifying reagent MTSEA when the native residues are replaced by cysteines. Cys modification with externally applied MTSEA led to full suppression of the cotransport function. In contrast, the membrane-impermeant reagent MTSET, which is of comparable size and is positively charged like MTSEA (11, 12), did not significantly alter cotransport function. In agreement with other evidence, this suggests that MTSEA can permeate the lipid bilayer (9, 12) and that Cys residues substituted at sites 199 and 202 were accessed from the cytosol. It is also possible that MTSEA could access these sites via the lipid phase; however, the intact oocyte preparation does not allow us to easily distinguish this case from direct cytosolic access. Additional indirect evidence in support of this sidedness of Cys accessibility is that, unlike our previous cysteine-scanning studies of ECL-3 regions, in which concentrations of MTS reagents in the micromolar range were generally sufficient to alter cotransport (16, 17), it was necessary to use concentrations in the millimolar range to effect the same change of function. Furthermore, the estimated reaction-rate constants were three orders of magnitude slower than we have previously reported for Cys mutants in ECL-3 loops (17). These slower rates may reflect the influences of transmembrane diffusion rate and Cys scavenging in the oocyte cytosol as the rate-limiting steps in the overall MTSEA-Cys reaction kinetics. Exposure to MTSEA for mutants with Cys residues substituted at sites after 202 led to marginal loss of cotransport function. This suggests that either the corresponding cysteines were even less accessible or, if modification did occur, these sites are not functionally important.
Residue at site 199 is a critical determinant of transport mode. A significant finding in this study was that one site, 199, was a strong determinant of steady-state substrate-activation kinetics and the transport mode. Substitution of the native Asp with Cys, Ala, or Thr resulted in constructs that displayed inward IPis, which are indicative of intact cotransport function (and were confirmed by 32Pi uptake) albeit with a significantly reduced apparent substrate affinity. Extending the size of the native Asp199 (e.g., with an additional ---CH2 group, as for Gln) or substituting charged residues (such as His, Arg, or Glu) led to transporters that only functioned in the slippage mode: Pi no longer induced an inward current, and IPi was then identical to IPFA. The presence of a detectable Na+ leak confirmed that the interaction of Na+ with the empty carrier still occurred with these constructs in accordance with the current kinetic scheme for NaPi-IIa (4). Moreover, this behavior was identical to that observed after Cys modification by MTSEA, which supports the notion that size and/or neutrality of this residue was critical for definition of cotransport mode integrity. That all of the mutants at site 199 gave an electrogenic response to Pi (either inward cotransport current or suppression of slippage) suggests that these mutations did not fundamentally alter the Pi recognition site, and therefore the binding affinity for Pi might not have been affected. Our documented increased apparent affinities for three of the substitutions might then be attributed to modified kinetics of subsequent transitions in the transport cycle.
In the NaPi-IIa kinetic scheme, the ordered substrate-binding sequence is Na+-Pi(HPOCys substitutions modify slippage mode properties. Two mutants showed contrasting slippage indices that resulted from the Cys substitution. For mutant A203C, the Na+-leak turnover rate increased, whereas a Cys substitution at site 199 resulted in a mutant with a significantly smaller index compared with the WT. A lower slippage rate for mutant N199C would also be consistent with the lower apparent substrate affinities documented; however, we were unable to further characterize the slippage mode for this mutant because of the limited resolution of the Na+-leak currents. Nevertheless, these findings point to the functional importance of these sites in determining (directly or indirectly) the Na+ leak. Whereas mutant A203C was only marginally modified by MTSEA exposure, which suggests that this site was not readily accessible to the aqueous environment, the cotransport function of N199C was fully inhibited. Interestingly, when a Cys was substituted between sites 199 and 203 (V202C), full inhibition of cotransport after MTSEA exposure occurred, and this mutant displayed normal slippage behavior. This underscores the different roles that neighboring residues can play in conferring specific transport properties.
Structure-function implications. The present challenges of structure-function studies on multisubstrate transport systems include identifying the structural elements that contribute to the transport pathway(s) and distinguishing between multiple [as proposed for the bacterial lac permease (10)] and common pathways (e.g., Refs. 26, 27) for the co- and driven-substrate translocations. What are the implications of our new findings with respect to NaPi-IIa?
Our present findings do not allow us to make a distinction between separate and common slippage/cotransport pathways. It is tempting to speculate that the altered slippage mode behavior through Cys substitution at sites 199 and 203 might indicate that these sites are directly associated with the Na+-leak pathway. However, MTSEA exposure for all constructs left the slippage mode unchanged, independently of whether the cotransport function was suppressed. The robustness of the Na+ leak for all constructs tested in ICL-1 contrasts with our finding that MTS modification of some sites in ECL-3 led to transporters with a significantly increased Na+ leak as well as a fully suppressed cotransport mode (17) after external application of MTS reagents. We therefore conclude that sites in ICL-1 that directly contribute to the Na+-leak pathway still remain to be identified. On the other hand, our present findings together with previous kinetic and structure-function studies do allow us to speculate on the identity of the Pi-translocation pathway. First, given that NaPi-IIa is a functional monomer (14) and that one HPO
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ACKNOWLEDGEMENTS |
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The authors acknowledge financial support provided to H. Murer from the Swiss National Science Foundation (Grant 31-46523), the Fridericus Stiftung (Vaduz, FL-9490) Hartmann Müller-Stiftung (Zurich), the Olga Mayenfisch-Stiftung (Zurich), and the Union Bank of Switzerland (Zurich, Bu 704/7-1).
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FOOTNOTES |
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Address for reprint requests and other correspondence: I. C. Forster, Physiologisches Institut, Universität Zürich-Irchel, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland (E-mail: IForster{at}access.unizh.ch).
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.
First published November 13, 2001;10.1152/ajprenal.00282.2001
Received 13 September 2001; accepted in final form 5 November 2001.
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