1Institute of General Physiology and Biological Chemistry, University of Milano, 20134 Milano; and 2Department of Structural and Functional Biology, University of Insubria, 21100 Varese, Italy
Submitted 29 July 2002 ; accepted in final form 10 May 2003
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ABSTRACT |
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amino acid transport; structure/function; amino acid modifiers; Manduca sexta
These morphological and physiological features seem to be shared, with only minor variations, by other transporters in lepidopteran larvae (16, 24, 31, 37). Another transporter named CAATCH1 has recently been cloned from the midgut of Manduca sexta larvae. This transporter has a 90% identity with KAAT1 (12) but seems to have some interesting functional differences, suggesting that it mediates an amino acid flux not energetically coupled to the cationic movements (34, 41).
Information from sequence analysis and site-directed mutagenesis may lead to the identification of specific amino acid residues that are important for the various functions of the cotransporter. This approach has allowed identifying the arginine 76 as a residue critical for KAAT1 activity (10).
Recently, Liu et al. (25) showed that the KAAT1 mutant Y147F has increased transport activity and altered substrate selectivity.
This work addresses the role of glutamate 59. By site-directed mutagenesis, we have mutated this conserved residue and the transporter has been expressed in the plasma membrane of Xenopus oocytes. E59G KAAT1 activity is deeply modified as highlighted by amino acid fluxes and current measurements. These functional changes and the different sensitivity of wild-type (WT) and E59G KAAT1 to sulfhydryl reagent N-ethylmaleimide (NEM) and to the arginine modifier phenylglyoxal (PGO) indicate that glutamate 59 is important for the three-dimensional organization of the transporter.
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MATERIALS AND METHODS |
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Oocyte expression of WT and mutated KAAT1. For uptake and electrophysiological experiments, p-SPORT1 plasmid containing WT or mutant KAAT1 cDNA and pAMV plasmid containing WT rGAT-1 cDNA were Not1 digested, in vitro capped, and transcribed using T7 RNA polymerase (Stratagene).
Oocytes were isolated from mature Xenopus laevis female frogs and manually defoliculated after treatment with 1 mg/ml collagenase A (Boehringer Mannheim) in the Ca2+-free buffer OR II (82.5 mM NaCl, 2 mM KCl, 1 mM MgCl2, 5 mM HEPES/Tris, pH 7.5) for 2030 min at room temperature. Healthy oocytes were then selected and maintained at 17°C in Barth's medium [88 mM NaCl, 1 mM KCl, 0.82 mM MgSO4, 0.41 mM CaCl2, 0.33 mM Ca(NO3)2, 2.4 mM NaHCO3, 10 mM HEPES/Tris, pH 7.5] supplemented with 2.5 mM pyruvic acid and 50 mg/l gentamycin sulfate. The day after, oocytes were injected with 50 nl of the synthesized cRNA (12.5 ng/oocytes) by using a manual Drummond injection system. Noninjected oocytes were considered as control.
Transport experiments. Amino acid uptake was measured 3 days after
injection. Groups of 810 oocytes were incubated in 100 µl of uptake
solution (100 mM NaCl, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2,
10 mM HEPES-Tris, pH 8) with 0.1 mM [3H]leucine,
[3H]phenylalanine, [3H]proline, or
[3H]glutamic acid (all from Amersham Pharmacia Biotech, 37 MBq/ml)
for 60 min. For rGAT-1-expressing oocytes, the uptake solution had the same
composition, but the pH was 7.6 and included 0.1 mM -[3H]
aminobutiryc acid. Oocytes were then washed in ice-cold wash solution (100 mM
choline chloride, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10
mM HEPES-Tris, pH 8) and dissolved in 250 µl of 10% SDS for liquid
scintillation counting. Experiments in which the expression level of WT KAAT1
and WT rGAT-1 was <5 times above background were discarded.
Several figures report the KAAT1-mediated or the GAT-1-mediated transport that represents the difference between the mean uptake measured in cRNA-injected oocytes and the mean uptake observed in noninjected oocytes.
In methanethiosulfonate (MTS) and NEM inhibition experiments, oocytes were preincubated for 30 min with 2.5 mM 2-aminoethyl methanethiosulfonate (MTSEA), 10 mM MTSES (sodium [2-sulfonatoethyl] methanethiosulfonate), 1 mM MTSET ([2-(trimethylammonium)ethyl] methanethiosulfonate hydrobromid), or 1 mM NEM in Barth's solution, in the presence or in the absence of Na+ and leucine. In some experiments, substrates were added before MTS reagents. In the absence of Na+, NaCl was replaced by choline chloride. Oocytes were subsequently washed with the wash solution and incubated for transport measurement. In PGO inhibition experiments, oocytes were incubated in the above indicated uptake solution containing 7.5 mM PGO.
Kinetics. Na+ activation experiments and leucine kinetics were performed on WT, E59G-KAAT1, and water-injected oocytes. In Na+ activation experiments, Na+ concentrations ranged from 0 to 100 mM, and NaCl was osmotically replaced by choline chloride. L-[3H]leucine concentration was 0.1 mM. In leucine kinetics, L-[3H]leucine concentration ranged from 25 to 1,000 µM (3,7007,400 kBq/ml).
Kinetic parameters were calculated by using a multiparameter, iterative, nonlinear regression program (SigmaPlot, Jandel, CA).
Electrophysiology. A two microelectrode voltage-clamp was used to perform electrophysiological experiments (Gene-clamp, Axon, Union City, CA). 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; signals were filtered at 1 kHz and sampled at 2 kHz. Experimental protocols, data acquisition, and analysis were done using the pCLAMP 8 software (Axon). Pre-steady-state currents were isolated by separation of the fast and slow component of the current relaxation by double exponential fitting, as described elsewhere (13). The external solution was composed as follows (in mM) 98 NaCl or TMACl, 1.8 CaCl2, 1 MgCl2, and 5 HEPES-free acid, at pH 7.6. Leucine (1 mM) was added to induce transport-associated current.
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RESULTS |
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Kinetics. Na+ activation has been further investigated measuring 0.1 mM leucine uptake as a function of Na+ concentration (Fig. 4). E59G KAAT1 shows a reduced Na+ activation, with an increase of KNa+50 from 6.4 ± 2 to 47 ± 4 mM, and a Vmax reduction from 137 ± 12 to 46 ± 3 pmol · oocyte1 · 60 min1. In the absence of Na+, leucine uptake by the mutant is virtually zero, which indicates that leucine uptake is still Na+ dependent. This dependence can also be observed at high leucine concentration (1 mM) (see Fig. 4, inset). Under this condition, leucine uptake by the mutant is not different from that measured in control oocytes not expressing the transporter. These results exclude the possibility that the mutant could transport leucine as a uniporter with low affinity for the amino acid. The small WT KAAT1-mediated leucine uptake in the absence of Na+ (Fig. 4, inset) can be explained by a local concentration of Na+, extruded by the Na+ K+-ATPase, able to activate the WT-transporter characterized by a high apparent affinity for Na+. The same effect cannot be observed in the mutant because it has a reduced apparent affinity for sodium. The physiological meaning of KAAT1 high apparent affinity for Na+ has been discussed previously (36, 38).
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Leucine kinetics performed at 100 mM NaCl show that E59G KAAT1 has a reduced apparent affinity for the substrate and a Vmax value, which is about 50% of the WT (Fig. 5).
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This Vmax reduction should not be a consequence of the modified sodium apparent affinity (KNa+50: 47 mM); in other words, at 100 mM NaCl, E59G KAAT1 should be almost saturated.
Electrophysiology. We measured pre-steady-state (transient), uncoupled, and coupled currents in WT and E59G KAAT1 (Fig. 6). In the oocytes expressing WT KAAT1, the typical Na+-dependent transient and uncoupled currents can be observed. As described previously (6, 7), pre-steady-state and uncoupled currents are particularly evident in the presence of Na+, although they can also be seen in the TMA+ solution. In the oocytes expressing E59G KAAT1, large pre-steady-state and uncoupled currents are visible in the presence of either Na+ or TMA+ and no significant changes occur between the two conditions. In addition, and contrary to the behavior of the WT, no steady-state current increment nor disappearance of the transients is observed in the E59G mutant upon the addition of 1 mM leucine (see also Fig. 7F).
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To quantitatively analyze these results, in a previous work on KAAT1 (6) we isolated the transient and uncoupled currents by subtraction of the traces obtained in TMA+ from those obtained in the other conditions. However, the presence of large pre-steady-state and uncoupled currents in the E59G mutated transporter, even in TMA+ solution (Fig. 6), prevented the use of that procedure. Therefore, we separated the pre-steady-state currents from the capacitive transients by fitting double exponential functions to the current relaxations (14, 18).
A summary of the results of the electrophysiological experiments is shown in Fig. 7, where average data from several oocytes are plotted, illustrating the different characteristics displayed by the pre-steady-state, uncoupled, and transport-associated currents in the WT and mutated transporters. The results from the WT transporter show the characteristic bell-shaped and sigmoidal curves, respectively, for the relaxation time constant and charge vs. voltage curve in the presence of Na+ and absence of organic substrate (Fig. 7, A and B, open circles) (6, 7). These two parameters are strongly reduced when Na+ is replaced by TMA+ (Fig. 7, A and B, open squares) or when leucine is added in the presence of Na+ (Fig. 7, A and B, open triangles). These last findings are slightly different from those obtained with the TMA+ subtraction procedure and may indicate that TMA+ itself may partially replace Na+ in contributing the pre-steady-state currents.
Indeed, using the exponential peeling method, we obtained very similar findings in another transporter, the renal GABA-betaine transporter BGT-1 (13).
In the E59G mutant, however, the properties of the pre-steady-state current are different: the bell-shaped time constant vs. voltage and the sigmoidal charge vs. voltage curves are less steep (Fig. 7, D and E, open circles) and, more importantly, replacement of Na+ with TMA+ (Fig. 7, D and E, open squares), or addition of leucine to the Na+ solution (Fig. 7, D and E, open triangles) do not cause the significant changes seen in the WT.
Other differences between WT and mutated transporters concern the uncoupled and transport-associated currents. The voltage-current (I-V) curves of Fig. 7C show the progressive increase in steady-state current upon replacing TMA+ (squares) with Na+ (circles) and adding to this last solution 1 mM leucine (triangles). The same kind of plot, in the case of the E59G transporter (Fig. 7F), shows no significant change in the steady-state currents in the three conditions.
Therefore, these experiments indicate that although pre-steady-state and steady-state currents are still present in the E59G mutant, they are not significantly affected by the addition of organic substrate.
Activity recovery. Glutamate 59 has been mutated into aspartate, which is the amino acid most frequently found in this position among the members of the family, and into arginine, leucine, and cysteine. Only the substitution of glutamate with a negatively charged or polar side-chain amino acid induced a transport recovery. As shown in Fig. 8, E59D and E59C mutants show, respectively, a 70 and 100% transport activity compared with the native transporter.
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MTS effect. The transport sensitivity to sulfhydryl-modifying reagents that vary in size, charge, and membrane permeability can give some insight on membrane protein topology (2, 15, 19, 39). We tested the effect of impermeant (MTSES and MTSET) and permeant (MTSEA) sulfhydryl reagents on WT and E59C KAAT1 to investigate the localization of glutamate 59. As a control, we performed the same experiment also on GAT-1 that is known to be inhibited by MTSES and MTSET (4, 11, 21, 43). These reagents do not affect the transport in WT and E59C KAAT1, whereas cause a large inhibition on GAT-1 in the same conditions (Fig. 9). Figure 10 shows that whereas WT and E59G KAAT1 are 22 and 39% inhibited, respectively, the mutant E59C is 70% inhibited by 2.5 mM MTSEA. Most probably, the major determinant of the increased sensitivity of E59C KAAT1 to sulfhydryl modification is the presence of cysteine in position 59. Besides, MTSEA effect is not influenced by the presence of Na+ or leucine and substrates do not protect from MTSEA inhibition even if they are added before MTSEA treatment (not shown). Taken together, these results indicate that residue 59 is not accessible from the extracellular side and is not located at the binding sites for substrates.
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NEM effect. Figure 11 shows the effect of the highly permeant sulfhydryl reagent NEM on WT and E59G KAAT1. NEM exerts a 49% inhibition on the WT protein, and this effect is prevented by the presence of leucine. This result indicates that the reagent binds cysteine residue(s) accessible only in the absence of the amino acid. As expected, NEM exerts a strong inhibition (95%) on the E59C mutant (not shown) and surprisingly also on the mutant E59G (82%), which has the same cysteine residues as the native protein. Leucine does not prevent NEM inhibition in the mutant (Fig. 11), indicating that NEM effect is not only quantitatively but also qualitatively different in WT vs. E59G KAAT1. Despite the membrane permeability, NEM and MTSEA do not exert the same inhibition on WT and E59G KAAT1. Similar results have been recently observed in the glutamate transporter EAAT1 (39). This could be due to NEM and MTSEA different steric hindrance and reactivity (2, 15, 19, 39).
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PGO effect. PGO binds covalently arginine residues and inhibits amino acid uptake mediated by KAAT1 (10). Figure 12 shows that PGO exerts a significantly higher inhibition in the mutant E59G than in the WT protein. Because arginine residues are the same in the two transporters, this effect is consistent with a modified accessibility of arginine residues to PGO in E59G KAAT1.
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DISCUSSION |
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In this context, the analysis of the different functional features of native and mutated transporters may give some insight on the molecular mechanisms involved, and in some cases may allow the dissection of different protein functions.
The high identity among several transporters in the region including the first putative transmembrane domain (Fig. 1) suggests that it may be involved in common general aspects of transport activity.
The monoamine transporters have an aspartate residue in TM1 crucial for transport activity. In DAT, aspartate 79 has been implicated in the binding of the substrate dopamine and the cocaine analog CFT. We suggested that the carboxylic acid of the aspartic residue may interact with dopamine's amine and recognize tropane nitrogen for cocaine binding (22).
The TM1 domain of rat SERT contributes to the permeation pathway of the transporter: the mutant D98E showed a decreased 5HT transport capacity, a selective loss of antagonist potencies, and a perturbed ion dependence, as well as modified substrate-induced currents (1).
Pantanowitz and coworkers
(30) have shown that among the
arginine residues present in the GAT-1 membrane domains, only arginine 69 is
essential for activity. They suggested its involvement in ion binding through
the interaction with electrons of the adjacent tryptophan 68, whose
presence also appears crucial for GAT-1 activity
(23,
27).
Bennet and coworkers (4) found that arginine 44 and tryptophan 47 in GAT-1 are critical for transport activity and that, if arginine 44 is replaced by lysine or histidine, only the exchange activity is recovered, which suggests that the mutant is impaired in the reorientation of the unloaded transporter.
The results presented here show that amino acid transport activity of KAAT1 is reduced in the mutant E59G, whereas the substrate and ionic selectivity are little affected (Figs. 2 and 3). Some functional features of the transporter appear deeply modified and seem due to impaired functionality of the transporter rather than to altered cell surface targeting of the protein (Fig. 7, B and E). It is worth noting that the reduced leucine uptake and Vmax could be explained with a reduced surface expression of the transporter, but not the different NEM and PGO inhibition patterns, the reduced leucine and Na+ apparent affinity, nor the large pre-steady-state and uncoupled currents.
The sodium dependence is changed in a complex way. Large pre-steady-state and uncoupled currents are measured in the absence of leucine, and no current increase is observed upon addition of the transported amino acid (Figs. 6 and 7). However, the residual amino acid transport induced by the mutant is still sodium dependent because in the absence of sodium no leucine uptake was measured (Fig. 4).
The apparent discrepancy of a sodium-dependent leucine flux and the absence of leucine-induced current increase may be explained as follows: the mutation causes a modification in the permeation pathway(s) of the cotransporter, producing an enlarged and less specific selectivity filter for cations. This would allow not only Na+ and K+ but even TMA+ to contribute to the generation of transient pre-steady-state and to uncoupled currents, explaining the results shown in Figs. 6 and 7. The transporter still recognizes the amino acid substrates even if with lower apparent affinity (Fig. 5); however, the high conductive state of the mutated protein is not significantly influenced by the presence of the amino acid. In other words, the already high uncoupled Na+ flux in absence of amino acid is not increased when the amino acid is present and it is dragged along. Therefore, no leucine-induced increase in current is observed, nor do the transient currents disappear despite the fact that amino acid uptake can be detected. A similar explanation has been already invoked to explain the lithium-coupled leucine uptake in KAAT1 (8). However, the amino acid flux is still sodium dependent. Na+ is possibly required on the extracellular side of the membrane only as an activator that facilitates the binding of the organic substrate. In this case, sodium would not be a driver but a ligand able to induce the binding and permeation of the amino acid.
The presence of large pre-steady-state currents in the mutant E59G and the similarity in the quantity of intramembrane charge movements observed in the WT and E59G KAAT1 (Fig. 7, B and E) suggest a quantitatively comparable level of expression.
The high sensitivity of the mutant E59C to membrane permeant sulfhydryl reagents (MTSEA and NEM) and the insensitivity to impermeant sulfhydryl reagents (MTSES and MTSET) confirm the intracellular localization of residue 59. The lack of sodium and leucine protection of MTSEA inhibition suggests that E59 is not located in the permeation pathway.
It is worth noting that in GAT1 and in most members of the family (5, 11, 21, 43), the conserved Cys 74 (GAT1 numbering), located in the first extracellular loop, is the only cysteine accessible to impermeant sulfhydryl reagents. This residue is lacking in KAAT1, and this probably explains the insensitivity to MTSES and MTSET reagents.
The effect of the highly permeant sulfhydryl reagent NEM shown in Fig. 11 is consistent with a tertiary structure modification of the E59G mutant. In fact, 1 mM NEM causes a 49% inhibition of leucine uptake in the native transporter, and this effect is prevented by the presence of leucine, whereas the reagent causes a much higher (82%) inhibition in the mutant E59G, not prevented by the organic substrate. The different inhibition pattern exerted on proteins having the same cysteine residues, WT and E59GKAAT1, can be explained only by a modified accessibility of the target residues due to a conformational change of the protein.
PGO effect supports this hypothesis: in the mutant E59G, not only cysteine but also arginine residues become more accessible to the modifying reagents.
The activity recovery observed in E59D and E59C KAAT1 suggests a role of the negative charge in this position. In fact, the reactivity of E59C mutant toward NEM and MTSEA indicates that the introduced cysteine may be present in thiolate form (S) because it has been demonstrated that the intrinsic reactivity of the thiol (SH) is over 5 x 1.010-fold less than the thiolate (S) and makes a negligible contribution to the reactivity of thiols toward NEM and MTS reagents (2, 19).
In conclusion, the complex pattern of functional changes observed in E59G KAAT1 and the effects of sulfhydryl and arginine modifiers indicate that glutamate 59 plays a critical role in the three-dimensional organization of the transporter possibly shared by other members of the family.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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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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