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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS
1Institute of General Physiology and Biological Chemistry "G. Esposito," School of Pharmacy, University of Milan, Milan; 2Department of Structural and Functional Biology and Center for Neurosciences, University of Insubria, Varese, Italy; and 3Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut
Submitted 11 May 2007 ; accepted in final form 9 July 2007
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ABSTRACT |
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residue interaction; oxidants; tertiary structure
Like the highly homologous protein CAATCH1, KAAT1 has a unique cation selectivity, being activated by K+ and Li+ in addition to Na+ (3, 7, 10, 29, 41). Because of these properties, KAAT1 and CAATCH1 can be useful for investigating structure/function relationships of ion binding sites within the NSS superfamily, since sequence comparisons and site-directed mutagenesis studies may identify the structural differences that determine the particular features of their ion dependence.
In the initial analysis of KAAT1 structure and function, it was possible to establish the role of a number of amino acid residues. Arg76 was found to be essential for KAAT1 activity and involved in the interaction with the inhibitor phenylglyoxal (8), and Glu59 appeared to be involved in the three-dimensional organization of the helices constituting the KAAT1 permeation pathway (30). KAAT1 was also mutated to introduce residues that are conserved in the NSS superfamily but not in KAAT1 (23) in an attempt to reverse its ability to utilize K+ in addition to Na+. The resulting multiple mutant was not functional, but the work did show the significance of Tyr147 in KAAT1 cation and substrate selectivity.
Concentrating on KAAT1- and CAATCH1-specific negative-charged transmembrane residues, we have recently identified Asp338 (located in TM VII) as a residue that contributes to K+ activation and amino acid coupling. The mutation of Asp338 to glutamate abolished the substrate-induced K+ current, whereas leucine was still transported, albeit with altered kinetic properties, in the presence of Na+. (24). The role performed by Asp338 suggests that it may be located in the cation binding site of the cotransporter; furthermore, it is plausible that it interacts with other residues to allow KAAT1 activity. In the present study, we extended this approach to obtain structural information about the KAAT1 amino acid cotransporter.
The NSS family of transporters contains many members from prokaryotic species, including the bacterial tryptophan transporter TnaT, the first prokaryotic transporter identified in this family (2), and LeuTAa, whose high-resolution structure is currently the best model for understanding the architecture of this transporter family (41). These transporters retain the Na+ dependence that characterizes NSS transporters in animals and provide a useful model system for testing hypotheses relating to structure and function in this family.
Our group (24) recently showed that Asp338, a specific residue in TM7 of KAAT1 and the related lepidopteran transporter CAATCH1, is essential for K+-driven amino acid transport by KAAT1. In other members of the NSS family, it is extremely rare for acidic residues to be present at this position. Furthermore, in KAAT1 and CAATCH1, a lysine is found in TM2 at position 102 (KAAT1), but basic amino acids are even rarer at the corresponding position of other NSS family transporters. The two published predicted secondary structures (7, 23) show that KAAT1 has five basic residues located in transmembrane domains: Arg76, Lys102, Arg242, Lys386, and His461. The roles of Arg76 and Arg242 have been previously studied (8), and we have found that the mutation of His461 does not alter KAAT1 function (unpublished observations); of Lys102 and Lys386, we selected Lys102 because it is present in only 3% of the 250 NSS proteins considered in the sequence alignment (see extract in Fig. 1), and its transmembrane localization (TM2) is confirmed by 8 of 10 hydrophobicity algorithms considered as well as in the structure of LeuTAa from Aquifex aeolicus (41). Moreover, in the LeuTAa crystal structure, TM2 and TM7 are in close proximity. The corresponding positions in LeuTAa (Ile56 and Asn286) were found within 10 Å of each other in this structure.
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MATERIALS AND METHODS |
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Oocyte expression of KAAT1 WT and mutated transporter. The plasmid vector p-SPORT1 bearing the wild-type (WT) or mutant KAAT1 cDNA was linearized by means of Not1 digestion, in vitro capped, and transcribed using T7 RNA polymerase (Stratagene). The oocytes were harvested and selected as previously described (30) and then injected with 12.5 ng of synthesized cRNA dissolved in 50 nl of RNase-free water with the use of a manual microinjection system (Drummond); the controls were noninjected oocytes. Before the experiment, the oocytes were incubated at 16°C for 3 days in Barth's solution supplemented with 50 µg/ml gentamicin sulfate and 2.5 mM sodium pyruvate.
Transport experiments. To measure leucine uptake, groups of 10–14 oocytes were incubated for 60 min in 120 µl of the uptake solution (100 mM NaCl, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, and 10 mM HEPES-Tris, pH 8) with 500 kBq/ml (5.62 TBq/mmol) of [3H]leucine (GE Healthcare Europe; specific activity 5.74 TBq/mmol, 1 pmol corresponding to 149 cpm), rinsed in ice-cold wash solution (100 mM choline chloride, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, and 10 mM HEPES-Tris, pH 8), and dissolved in 250 µl of 10% SDS for liquid scintillation counting. The data show KAAT1-mediated transport, plotted as the difference between the mean uptake measured in cRNA-injected oocytes and that observed in noninjected oocytes.
To measure tryptophan uptake, Escherichia coli CY15212 cells transformed with a plasmid bearing the TnaT gene under control of a constitutive promoter (2) were grown overnight from a 15% glycerol stock in Luria-Bertani broth in the presence of ampicillin (100 µg/ml) at 37°C with shaking. Before the experiment, the culture was diluted 100-fold into the same medium, and the incubation was continued until the culture reached an absorbance at 600 nm (A600) of 0.8. The cells were then washed three times with ice-cold M9 minimal medium buffer (88.4 mM NaH2PO4, 21.6 mM KH2PO4, 8.4 mM NaCl, and 18.3 mM NH4Cl) including 20 mM glucose at 4°C. Two hundred microliters of the cell suspension in M9 medium was loaded into each of the 5-ml polystyrene tubes at room temperature, and uptake was initiated by adding 20 µl of L-[5-3H]tryptophan (American Radiolabeled Chemicals, St. Louis, MO) to the cell suspension. Uptake was terminated by filtering the cell suspension through the glass microfiber filters (934-AH; Whatman, Clifton, NJ) that were presoaked in 0.1% polyethyleneimine, followed by washing the cells twice with ice-cold M9 medium. The filters were then prepared for liquid scintillation counting by adding Opti-Fluor (PerkinElmer Life Sciences, Boston, MA). Nonspecific uptake is defined as uptake in the untransformed CY15212 cells. An aliquot of 25 µl of cell suspension was used to measure the protein concentration and normalize uptake data.
Kinetics. The kinetic experiments were performed by incubating oocytes for 5 min in the uptake solutions. In the leucine kinetic experiments, [3H]leucine concentrations ranged from 5 µM to 2 mM (1,100–2,950 KBq/ml; 5.73–4.09 TBq/mmol). In the Na+ activation experiments, Na+ concentration ranged from 0 to 200 mM and NaCl was replaced by choline chloride to keep osmolarity constant; the concentration of [3H]leucine was 1 mM (4.78 TBq/mmol). The kinetic parameters were calculated using an iterative, multiparameter, nonlinear regression program (SigmaPlot; SPSS, Chicago, IL). For tryptophan uptake saturation kinetics, uptake assays were carried out as reported in Transport experiments with multiple concentrations of L-[5-3H]tryptophan ranging from 20 nM to 4 µM. Nonlinear regression fits of experimental and calculated data were performed with Origin (OriginLab, Northampton, MA), which uses the Marquardt-Levenberg nonlinear least-squares curve-fitting algorithm. The statistical analysis given was from multiple experiments.
Electrophysiology.
A two-microelectrode voltage clamp was used to perform the electrophysiological experiments (GeneClamp 500B; Axon Instruments, Union City, CA). The reference electrodes were connected to the experimental oocyte chamber via agar bridges (3% agar in 3 M KCl) to minimize the effects of chloride on junction potential. Borosilicate electrodes with a tip resistance of 0.5–2 M
were filled with 3 M KCl. The holding potential was kept at –60 mV, and the typical protocol consisted of 200-ms voltage pulses spanning the range from –160 to +20 mV in 20-mV steps. Four pulses were averaged at each potential; the signals were filtered at 1 kHz and sampled at 2 kHz. pCLAMP 8 software (Axon Instruments) was used for the experimental protocols, data acquisition, and analyses; the data were prepared using Origin 5.0 software (formerly Microcal). The composition of the external control solution was (in mM) 98 NaCl, 1 MgCl2, 1.8 CaCl2, and 5 HEPES; in the other solutions, NaCl was replaced by KCl, LiCl, or tetramethylammonium chloride (TMA-Cl). The pH was adjusted to 7.6 by adding the corresponding hydroxide for each alkali ion and TMAOH for TMA+ solution. Amino acids (leucine, threonine, and proline; 500 µM) were added to induce transport-associated currents, which were estimated by subtracting the traces in the absence of amino acid from those in its presence under each experimental condition. All experiments were performed at room temperature.
Oxidation/reduction studies. For the oxidation experiments, the oocytes were preincubated for 5 min in 0.2 mM Cu(II) (1,10-phenanthroline)3 (CuPh) in wash solution at pH 7.5 before the uptake assay. The CuPh stock solution (150 mM) was freshly prepared for each experiment by mixing 60 µl of 250 mM CuSO4 and 40 µl of 1.25 M 1,10-phenanthroline dissolved in 1:1 water-ethanol. For DTT treatment, oocytes, after incubation with CuPh, were treated for 5 min with freshly prepared 12 mM DTT and then washed and incubated for the uptake measurements.
Homology modeling. Figure 11 was prepared using DeepView/Swiss Protein Data Bank viewer downloaded from http://www.expasy.org/spdbv/. Homology modeling of KAAT1 putative Na1 site was made on the basis of LeuTAa structure (accession code 2A65).
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RESULTS |
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22% of the WT amino acid accumulation (Fig. 4A). Kinetic analysis of the V56K/N264D double mutant demonstrated that the Km for substrate (tryptophan) was elevated 11-fold and Vmax was reduced 14-fold (Fig. 4B), whereas transport by the TnaT single mutants was not detected. Just as a neutralizing mutation of Lys102 was able to correct the transport defects caused by mutation of Asp338 in KAAT1, the deleterious effect of a charged residue at either of the corresponding positions of TnaT was partially corrected by the presence of an oppositely charged residue at the other position.
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The behavior of the K102V mutant was also altered in the presence of K+. For threonine and proline, inward currents in this mutant were larger than those of the WT. Moreover, they showed a less pronounced curvature (Fig. 7, Bb and Bc). Once again, leucine (0.5 mM) gave rise to an inverted U-shaped current-voltage (I–V) relationship, with currents smaller than those of threonine or even proline (at least at the most negative potentials; Fig. 7Ba). The I–V relationships of the WT and K102V shown in Fig. 7Ba cross each other at potentials between –50 and –60 mV.
The two insets in Fig. 7 provide more detail concerning the behavior of WT and the K102V mutant over the range of potentials at which uptake is presumably measured (11): i.e., from –40 to +20 mV, considering the native membrane potential of the oocyte, and the depolarizing effects of the inward current and of 150 mM external K+. It can be clearly seen that in Na+, the K102V mutant gave rise to smaller currents in this voltage range compared with the WT, whereas in K+, the mutant carried larger currents, because the I–V curves cross below –40 mV in K+ but around +20 mV in Na+. All of these electrophysiological characteristics are in line with the uptake data (Figs. 2 and 3). These results, therefore, suggest that although the lysine-to-valine mutation modified the rates of KAAT1-mediated transport, there is no reason to suspect substantial changes in the coupling between charge and substrate.
Comparison of the voltage dependence of the K102V mutant in the presence of either Na+ or K+ reveals an important aspect of cation selectivity in this mutant. Despite differences in the amplitudes of transport and leakage currents, all substrates displayed similar voltage dependences regardless of whether the driver cation was Na+ or K+. This result suggests that K102V is less able than WT to distinguish between Na+ and K+.
Because the K102V mutation restored the transport activity of the D338E transporter close to that of the WT, we also analyzed the electrophysiology of K102V/D338E. Figure 8 shows that the currents elicited by the addition of leucine and threonine were abolished in presence of K+ (Ba and Bb) and strongly reduced, compared with the WT, in the presence of Na+ (Aa and Ab). No current was elicited by proline in the presence of either Na+ or K+ (Ac and Bc).
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Because the transport-associated currents were altered in both the single K102V and the double K102V/D338E mutants, and because the single mutant K102V showed altered voltage dependence in presence of K+, we also analyzed their uncoupled leakage currents to clarify the selectivity order for cations. These uncoupled currents (i.e., transmembrane charge movement induced by the presence of cations in the absence of substrate) are shown in Fig. 9. The behavior of WT KAAT1 has been previously described (3) and is reported in this study as a control, together with the currents recorded from noninjected oocytes and from oocytes expressing the D338E mutant (24). As shown previously, WT KAAT1 carried a much higher leakage current in Li+ than in Na+ or K+. This cation selectivity order was also observed in the D338E mutant. However, Fig. 9Bshows that the cation selectivity of K102V and K102V/D338E was altered. In both mutants containing the K102V mutation, all cations elicited uncoupled currents of similar magnitudes (means are not significantly different, Student's t-test, P level 0.05), supporting the idea that the Lys102 is important for the recognition of cations.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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One method for identifying a functional interaction between two residues in a protein is second-site suppressor analysis. In this approach, a functional change, such as inactivation, induced by mutation of one residue is corrected or reversed by mutating a second residue. In this work, we show that mutation of Lys102 corrected some but not all effects of the previously described D338E mutation and also partially reversed the transport defect in a D338N mutant. Moreover, the same second-site suppression occurred in a distantly related member of the same family, TnaT from S. thermophilum. Mutating either Val56 (corresponding to Lys102) to lysine or Asn264 (corresponding to Asp338) to aspartate completely inactivated transport by TnaT, but the double mutant V56K/N254D regained significant activity.
The functional interaction implied by these findings could result from a direct interaction between the two residues, or it could occur indirectly. In thiol cross-linking assays, the proximity of two residues can be detected by their substitution with cysteine and the creation of a disulfide bond under oxidative conditions (16). Direct interaction between Asp338 and Lys102 is strongly suggested by the results of cross-linking experiments reported presently. In these experiments, conditions favoring disulfide formation inhibited a mutant of KAAT1 with cysteines at 102 and 338 but not either single cysteine mutant (Fig. 10). This apparent cross-linking by disulfide formation in the presence of an oxidant, as well as subsequent reversal by conditions that reduce disulfides, supports the direct interaction between these two residues.
Our group (24) previously showed that mutation of Asp338 in KAAT1 to glutamate inactivated K+-driven substrate accumulation and the corresponding currents. As an asparagine residue, this position is thought to contribute to coordination of a Na+ ion in LeuTAa and other transporters in the NSS family (41). The loss of K+-dependent transport phenomena in D338E could result from a decreased ability of glutamate at that position to effectively coordinate a K+ ion. The role of Lys102 may be to interact with Asp338 to maximize its ability to coordinate both Na+ and K+ for coupling to substrate transport. This hypothesis is consistent with the effect of the K102V mutation, which alters the relative effectiveness of Na+ and K+, decreasing the effectiveness of Na+ (Figs. 2 and 4) and increasing that of K+ (Fig. 3). This proposal would also explain why of all the known NSS transporters, only KAAT1 and CAATCH1, which are unique in their ability to couple transport to K+, contain aspartate and lysine at these two positions. Because of the distance of Lys102 from the substrate and cation binding site predicted by the LeuTAa structure, it is unlikely that this residue directly interacts with bound substrate or cations, but rather that it exerts its effect by influencing Asp338. This interpretation is consistent with the observation that reversing Lys102 and Asp338 ablates transport. This K102D/D338K double mutant retains the potential for these two residues to interact but cannot utilize the aspartate carboxyl group for coordination of a cation.
In the context of the bacterial tryptophan transporter TnaT, mutation of the corresponding residues Val56 to lysine or Asn264 to aspartate as in KAAT1 are not compatible with functional expression of the protein. However, the two mutations together partially restore transport activity. Also in KAAT1, the D338N mutant contains only Lys102 of the two proposed interacting charged residues. Neutralizing this residue in the K102V/D338N double mutant increased transport activity, consistent with the deleterious effect of an unpaired charge in TM2. The lack of suitable reagents prevents us from determining whether the nonfunctional TnaT mutants are expressed as inactive proteins or are misfolded and degraded, but in either case, the recovery seen with the double mutant V56K/N264D supports the interaction between these two positions.
An electrophysiological analysis of these mutants provides some insight into the role played by Lys102 and its interaction with Asp338. Although it is not easy to explain the complex behavior of leucine-induced currents in the K102V mutant (Fig. 7), we may speculate about their origin. Unlike neurotransmitter transporters that are highly selective for a particular transported substrate, neutral amino acid transporters such as KAAT1 have broader substrate selectivity. The varied structures of amino acids transported by KAAT1 lead not only to different Km values of the transporter for its substrates but also to qualitatively different behavior. For example, the I–V curves for KAAT1, particularly in K+, vary depending on the transported substrate (Figs. 7 and 8). This is likely due to interaction between substrates and cations as in the leucine-Na+ interaction in LeuTAa (41). Na+ binding in LeuTAa is also stabilized partly by Asn286, which corresponds to Asp338 in KAAT1. As stated above, the opposite charges carried by Lys102 and Asp338 may interact, stabilizing a cation in the binding pocket and facilitating cation-substrate coupling during the transport cycle, whereas the mutation of lysine to valine abolishes this ionic interaction and may therefore alter the three-dimensional organization of the binding pocket, disrupting ion selectivity and coupling. The fact that KAAT1 mutants with valine replacing Lys102 seem unable to discriminate between cotransported cations in measurements of uncoupled leak currents (Fig. 9) supports this hypothesis. It is possible that the interaction with Lys102 is essential for maintaining Asp338 in the correct position to interact with Na+, K+, or Li+ and to discriminate among them.
The K102V/D338E mutant had greater transport capacity in 100 mM Na+ (at least for leucine, Fig. 6) than the WT, but this was not coupled with a corresponding increase in transport-associated current (Fig. 8). The reduction in transport current suggests that the double mutation uncoupled transport and allowed substrates to cross the membrane without concomitant cation flux. The mutation, therefore, removed the central feature of the mechanism that allows the cation electrochemical gradient to drive amino acid uptake by cotransport. Because we have not determined how much of the substrate-associated current represents cotransported cation, it is possible that most of the measured current represents uncoupled cation movements. This consequence is likely due to the D338E mutation, which also lacks transport currents, as previously described (24), thus confirming the fundamental role of this residue in the coupling mechanism. Electrophysiological analysis of the double mutant (K102V/D338E) showed that its transport currents were indistinguishable from those of the single D338E mutant (24). The recovery of leucine uptake in K102V/D338E therefore indicates that the K102V mutation restored the ability of D338E to transport amino acids in the presence of Na+ or K+ (Figs. 2 and 3) but not to couple their fluxes (Fig. 8). It is worth noting that the mutated transporter (which acts as a high-capacity uniporter) was still activated by sodium even if the cation was poorly transported (Figs. 6 and 8). As previously suggested (24), these results are consistent with the possibility that Na+ plays a dual role as the (transported) driver ion and the (nontransported) activator of the protein. Two Na+ binding sites have been found in each monomer crystal of the leucine transporter LeuTAa (41). Asp338 corresponds to Asn286 of the prokaryotic transporter, which contributes to the Na1 binding site. Since it is well conserved, Yamashita et al. (41) suggest that it may be the binding site for the cotransported Na+ ion common to all NSS members. In addition, some NSS transporters with stoichiometries of 2:1 or 3:1 must possess additional sites such as the less well conserved Na2. Simultaneous measurements of ionic currents and leucine uptake have shown that the ratio of moles of transferred charge to moles of transported leucine for KAAT1 WT is 2.75 (4), which is compatible with the presence of multiple Na+ interacting sites.
The comparative kinetic analysis of D338E and K102V/D338E activity revealed that the uptake recovery observed for the double mutant was related to a shift in apparent affinity for both leucine and Na+ (Figs. 4 and 5) toward WT values. Because substrate and Na+ form intimate ionic interactions in the LeuTAa structure, the possibility exists that, also for KAAT1, changes in substrate Km may result from altered Na+ binding.
Our findings indicate that Lys102 and Asp338 directly interact, because the presence of the oxidant CuPh led to specific inhibition of the K102C/D338C mutant, which was largely reversed by treatment with the reducing agent DTT (Fig. 10). CuPh treatment has previously been successfully used to demonstrate the proximity of engineered cysteine pairs in other cotransporters (5, 18, 40). For CuPh to indicate a relationship between two cysteine residues, the two must be close enough to form a disulfide bond, and that bond must make a detectable difference in transporter function or structure. For example, if the distance between the two residues varies due to essential conformational changes during the transport process, their linkage in a disulfide bond may affect transporter activity. Disulfide bond formation does not occur efficiently if the cysteinyl sulfur atoms are much more than 8 Å apart (6). On the basis of the atomic coordinates of LeuTAa (Protein Data Bank code 2A65), despite the relatively low sequence identity (22%) with KAAT1, we have generated a homology model of the Na1 binding site of KAAT1 (Fig. 11) in which the distance between Lys102 and Asp338 side chains appears to be 12.30 Å, and if both residues are substituted with cysteine, the distance increases to 13.36 Å. Thus these residues may be close, but not quite close enough, unless the two positions are closer in KAAT1 than in the LeuTAa crystal or conformational changes bring the two closer. The observed inhibitory effect of a covalent bond between residues in positions 102 and 338 suggests that the distance between these residues may increase in some step of the transport cycle. The proposed electrostatic interaction between Lys102 and Asp338 may not be preserved through the transport cycle. In fact, the double mutant K102V/D338E, with only one charge, is functional. Another possibility, however, is that the distance between the two residues remains constant during transport but that the disulfide bond alters the relative relationship between positions, leading to inhibition.
Our analysis indicates that KAAT1 TM2, through Lys102, has a specific interaction with TM7. In the bacterial leucine transporter LeuTAa, the residue corresponding to Asp338 of KAAT1 (Asn286) forms part of the Na1 binding site. From its apparent interaction with Asp338, we now conclude that Lys102 indirectly contributes to the cation binding site and permeation pathway of KAAT1. Sen et al (34) have recently shown that a pincer-like structure allows DAT TM7 to interact with TM1 and TM6 at sites critical for cotransporter function.
Thus, although a crystal structure of KAAT1 would be necessary to delineate the precise three-dimensional organization of this cotransporter, the biochemical approach used in this study allowed us to obtain the first specific structural information about KAAT1 and has shown that the LeuTAa structure provides a useful reference point for these investigations.
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ACKNOWLEDGMENTS |
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Present address of A. Soragna: Institute of General Physiology and Biological Chemistry "G. Esposito," School of Pharmacy, University of Milan, Via Trentacoste 2, 20134 Milan, Italy.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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)-, K102V (
)-, D338E (
)-, and K102V/D338E (
)-mediated leucine uptake as a function of external leucine concentration and in the presence of 100 mM NaCl. Each point of the curves is the mean ± SE of 10–14 oocytes in a representative experiment of at least 3 independent experiments. Inset: highlighting of amino acid uptake measured in the range of leucine concentration between 0 and 0.2 mM. Error bars smaller than the symbols are not shown.
