Glutamate 59 is critical for transport function of the amino acid cotransporter KAAT1

V. Franca Sacchi,1 Michela Castagna,1 Stefania A. Mari,1 Carla Perego,1 Elena Bossi,2 and Antonio Peres2

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


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
KAAT1 is a neutral amino acid transporter activated by K+ or by Na+ (9). The protein shows significant homology with members of the Na+/Cl-dependent neurotransmitter transporter super family. E59G KAAT1, expressed in Xenopus oocytes, exhibited a reduced leucine uptake [20–30% of wild-type (WT)], and kinetic analysis indicated that the loss of activity was due to reduction of Vmax and apparent affinity for substrates. Electrophysiological analysis revealed that E59G KAAT1 has presteady-state and uncoupled currents larger than WT but no leucine-induced currents. Site-directed mutagenesis analysis showed the requirement of a negative charge in position 59 of KAAT1. The analysis of permeant and impermeant methanethiosulfonate reagent effects confirmed the intracellular localization of glutamate 59. Because the 2-aminoethyl methanethiosulfonate hydrobromid inhibition was not prevented by the presence of Na+ or leucine, we concluded that E59 is not directly involved in the binding of substrates. N-ethylmaleimide inhibition was qualitatively and quantitatively different in the two transporters, WT and E59G KAAT1, having the same cysteine residues. This indicates an altered accessibility of native cysteine residues due to a modified spatial organization of E59G KAAT1. The arginine modifier phenylglyoxal effect supports this hypothesis: not only cysteine but also arginine residues become more accessible to the modifying reagents in the mutant E59G. In conclusion, the results presented indicate that glutamate 59 plays a critical role in the three-dimensional organization of KAAT1.

amino acid transport; structure/function; amino acid modifiers; Manduca sexta


KAAT1 IS AN AMINO ACID COTRANSPORTER with low cation selectivity, as it can use either sodium or potassium as driver cation. Sequence analysis indicates that this 634-amino acid protein belongs to the super family of Na+/Cl-dependent neurotransmitter transporters, which includes several amino acid transporters (9, 29). This homology was at first unexpected because KAAT1 was cloned from the intestinal epithelium of Manduca sexta larva (Lepidoptera), but it is also supported by functional results. A goblet cell surrounded by absorptive columnar cells is the functional unit that characterizes the midgut of Manduca sexta larva. An electrogenic proton pump localized in the goblet cell generates a high electrical potential difference, which energizes the amino acid uptake mediated by KAAT1 in the brush border of columnar cells. The proton pump also energizes a K+/2H+ exchanger that determines the alkalinization of the lumen and causes potassium secretion (17). As a consequence of the intestinal secretion and of the diet of these larvae, the lumen has a very high K+ concentration, whereas sodium is very low (36, 38). In this particular ionic environment, KAAT1 mediates the K+-coupled uptake of {alpha}-amino acids, preferentially in the anionic form, with an uncharged side chain (33, 42). However, KAAT1 can also mediate a Na+-coupled amino acid transport, and Na+ can activate the amino acid transport at low concentration (37). Therefore, it is possible to study the amino acid transport mediated by KAAT1, expressed in Xenopus laevis oocytes and driven by Na+ gradient. KAAT1 is Cl dependent and, like the other members of the Na+/Cl-dependent neurotransmitter transporter super family (27, 28), shows different modes of action. It mediates amino acid uptake and exhibits amino acid-elicited currents in the presence of either potassium or sodium (6). In the absence of organic substrate, it can mediate cation fluxes, which generate the so-called uncoupled currents (6, 7). Moreover, the predicted secondary structure of KAAT1 is similar to that proposed for the GABA transporter GAT-1. KAAT1 has 12 transmembrane domains, intracellular NH2 and COOH termini, and a large extracellular loop between transmembrane domains III and IV (9).

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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Site-directed mutagenesis. The mutants E59G-, E59R-, E59L-, E59D-, and E59C-KAAT1 were synthesized by PCR using the QuickChange site-directed mutagenesis kit (Stratagene) and the following primers: E59G: 5'-gtatggtccaacaacatcgaattcttgatgtcctgcatcg-3'; E59R: 5'-gtatggtccaacaacatccgattcttgatgtcctgcatcg-3'; E59L: 5'-gtatggtccaacaacatcctattcttgatgtcctgcatcg-3'; E59D: 5'-gtatggtccaacaacatcgacttcttgatgtcctgcatcg-3'; E59C: 5'-gtatggtatggtccaacaacatctgcttcttgatgtcctgcatcg-3'. DNA sequencing confirmed the mutations.

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 20–30 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 8–10 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 {gamma}-[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 (3,700–7,400 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.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Amino acid uptake. The NH2-terminal region between residues 51 and 76 is highly conserved in a number of proteins belonging to the super family of Na+/Cl-dependent neurotransmitter transporters (Fig. 1), and in particular, this amino acid stretch is identical in KAAT1 and CAATCH1. Furthermore, in rGAT-1, another member of the family, cytoplasmatic amino acids preceding residue 51 are not conserved and not necessary for function (3, 4, 26). Because ions and charged molecules are transported, charged amino acids in the membrane domain should be important for transport activity (20). In this region of KAAT1, the only conserved negative residue is glutamate 59, located, according to Kyte-Doolittle hydropathy model, in the cytoplasm close to the presumed first transmembrane domain (TM1), as shown in Fig. 1. Because it has been suggested that TM1 may be involved in general transport function (1, 4, 2123, 27, 30), we mutated this residue and analyzed the properties of the transporter expressed in X. laevis oocytes. The first studied mutant was E59G KAAT1, in which glutamate was substituted with a residue void of charge and side chain which, as shown in Fig. 1, is the most frequently found amino acid in prokaryotic transporters. Figure 2 shows 0.1 mM leucine, phenylalanine, proline, and glutamate uptakes in X. laevis oocytes expressing WT or E59G KAAT1 in the presence of 100 mM NaCl. The transport activity of the mutant is reduced to 20–30% compared with the native protein, whereas the substrate selectivity is qualitatively similar with the exception of proline that is not transported by the mutant. Glutamate is not a substrate for WT and E59G transporters. Furthermore, some inhibition experiments (not shown) confirmed that the model amino acid {alpha}-methyl-amino isobutyric acid (MeAIB) is not a substrate for these two proteins. The most unusual feature of the neutral amino acid cotransporter KAAT1 is its low cation selectivity (6, 7, 9, 37): although K+ is the driver cation in vivo, Na+ and to some extent Li+ can also activate the amino acid uptake. Figure 3 shows that this selectivity pattern is not changed in the mutant and that the two proteins are chloride dependent. Figure 3, inset shows that a high extracellular K+ concentration (150 mM), despite the obvious effect on the membrane potential, can activate leucine uptake in WT and E59G KAAT1.



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Fig. 1. Putative KAAT1 topology with outlined alignment of KAAT1 NH2 terminus with members of the Na+/Cl-dependent neurotransmitter transporter super family. Glu59 is indicated by a black square.

 


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Fig. 2. Amino acid uptake mediated by wild-type (WT) and E59G KAAT1. Uptakes of 0.1 mM [3H]leucine, [3H]phenylalanine, [3H]proline, and [3H]glutamate were measured in the presence of 100 mM NaCl. Bars represent KAAT1-mediated amino acid uptakes and are the means ± SE of groups of 8–10 oocytes in a representative experiment.

 


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Fig. 3. WT and E59G KAAT1 ionic selectivity. Bars represent WT and E59G KAAT1-mediated 0.1 mM leucine uptake, measured in the presence of 100 mM NaCl, LiCl, and Na-gluconate. Inset: 0.1 mM leucine uptakes in the presence of 150 mM KCl measured in noninjected oocytes (vertical line bars) and in KAAT1-injected oocytes (square bars). Empty bars represent KAAT1-mediated uptake. Data are means ± SE of groups of 8–10 oocytes in a representative experiment.

 

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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Fig. 4. Na+ activation of 0.1 mM leucine uptake. Curves show WT ({bullet}) or E59G KAAT1-mediated ({circ}) leucine uptake, measured at different external Na+ concentrations. NaCl was osmotically replaced by choline chloride. Inset: uptake of 1 mM leucine at 0 mM NaCl (100 mM choline chloride) in noninjected oocytes (vertical line bars) and in WT- or E59G KAAT1-injected oocytes (square bars); empty bars represent KAAT1-mediated transport. Data are means ± SE of groups of 8–10 oocytes in a representative experiment.

 

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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Fig. 5. Kinetics of leucine uptake. Curves show WT- ({bullet}) or E59G KAAT1-mediated ({circ}) leucine uptake as a function of external leucine concentration and in the presence of 100 mM NaCl. Each point is the mean ± SE of a group of 8–10 oocytes in a representative experiment.

 

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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Fig. 6. Pre-steady-state, uncoupled, and coupled current in WT (middle) and E59G KAAT1 (right); at left currents from an uninjected oocyte. A, B, and C show uncorrected current traces in response to voltage pulses to –140, –100, –20, and +20 mV from a holding potential of –60 mV, in the solutions indicated at left. In the 2 lower rows, the Na+ minus TMA (D) and the Na+ +leucine minus TMA+ (E) records are shown to illustrate the transient and uncoupled currents and the coupled currents. In the oocyte-expressing E59G (right), large transient and steady currents are visible in the presence of TMA+, and no significant changes occur in the other 2 solutions, as shown in the 2 lower panels. For comparison, currents records obtained in the corresponding conditions from a noninjected oocyte are shown at left.

 


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Fig. 7. Analysis of pre-steady-state, uncoupled, and coupled currents in WT (top) and E59G KAAT1 (bottom). A and D: results of fitting of the transients using the slow exponential isolation method: open symbols are the relaxation time constants of the presteady-state current in the indicated solutions; filled squares are the time constant of the fast exponential (pooled from all conditions), indistinguishable from those derived by single exponential fitting in noninjected oocytes. B and E: corresponding Q/V curves obtained from integration of the peeled-off slow exponentials in the indicated conditions. C and F: uncorrected steady-state currents in the indicated solutions. Data represent mean values ± SE from 3 oocytes expressing WT KAAT1 and 6 oocytes expressing E59G KAAT1. Oocytes were pooled from 3 frogs.

 

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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Fig. 8. Activity recovery. Uptake of 0.1 mM leucine in the presence of 100 mM NaCl, mediated by WT, E59G, E59R, E59L, E59D, and E59C KAAT1. Data are expressed as percentages of the transport measured in WT KAAT1. Bars represent means ± SE measured in groups of 8–10 oocytes in 3 independent experiments.

 

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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Fig. 9. MTSES and MTSET effect on WT KAAT1-, E59C KAAT1-, and WT GAT1-mediated uptake. Oocytes were preincubated for 30 min with 10 mM MTSES or 1 mM MTSET in Barth's solution. Bars represent inhibition of 0.1 mM leucine uptake (WT and E59C KAAT1) or 0.1 mM GABA uptake (WT GAT1) expressed as percentages of the respective control measured in the absence of MTS and are means ± SE of 2 independent experiments.

 


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Fig. 10. MTSEA inhibition of WT-, E59G-, and E59C KAAT1-mediated leucine uptake. Oocytes were preincubated for 30 min with 2.5 mM MTSEA in Barth's solution. Bars represent inhibition of 0.1 mM leucine uptake expressed as percentages of the respective control in the absence of MTSEA and are means ± SE of 5 independent experiments. *Significantly different (P < 0.1); **significantly different (P < 0.002).

 

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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Fig. 11. N-ethylmaleimide (NEM) inhibition of WT- and E59G KAAT1-mediated 0.1 mM leucine uptake. Oocytes were preincubated 30 min with 1 mM NEM in Barth's solution in the absence or in the presence of 1 mM leucine. Bars represent inhibition of leucine uptake expressed as percentages of the respective control in the absence of NEM and are means ± SE of 4 independent experiments.

 

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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Fig. 12. Phenylglyoxal (PGO) inhibition of WT- and E59G KAAT1-mediated 0.1 mM leucine uptake. Uptake was measured in the presence of 7.5 mM PGO. Bars represent inhibition of leucine uptake expressed as percentages of the respective control in the absence of PGO and are means ± SE of 2 independent experiments.

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the last 10 years, many members of the Na+/Cl-dependent neurotransmitter transporter family have been cloned and investigated. These studies have highlighted that transport proteins are more complex than expected because not only do they mediate organic substrate and coupled ionic fluxes but they also exhibit channel-like conducting states (35, 40). The structural determinants for these functional properties are largely undefined and may involve separate or common permeation pathways.

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 {pi} 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.


    DISCLOSURES
 
This work was supported by MIUR grant COFIN 2001.


    ACKNOWLEDGMENTS
 
We thank Daniela Raciti, Luca Beltrame, and Gabriele Meloni for assistance during the experiments.


    FOOTNOTES
 

Address for reprint requests and other correspondence: V. Franca Sacchi, Institute of General Physiology and Biological Chemistry, Via Trentacoste 2, 20134 Milano, Italy (E-mail: Franca.Sacchi{at}unimi.it).

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.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
1. Barker El Moore KR, Rakhshan F, and Blakely RD. Transmembrane domain I contributes to the permeation pathway for serotonin and ions in the serotonin transporter. J Neurosci 19: 4705–4717, 1999.[Abstract/Free Full Text]

2. Bednar RA. Reactivity and pH dependence of thiol conjugation to N-ethylmaleimide: detection of a conformational change in calchone isomerase. Biochemistry 29: 3684–3690, 1990.[ISI][Medline]

3. Bendahan A and Kanner BI. Identification of domains of a cloned rat brain GABA transporter which are not required for its functional expression. FEBS Lett 318: 41–44, 1993.[ISI][Medline]

4. Bennet ER, Hailking S, and Kanner BI. Mutation of arginine 44 of GAT-1, a (Na+ + Cl)-coupled {gamma}-amino butyric acid transporter from rat brain, impairs net flux but not exchange. J Biol Chem 275: 34106–34113, 2000.[Abstract/Free Full Text]

5. Bennet ER and Kanner BI. The membrane topology of GAT-1, a (Na+ + Cl)-coupled gamma-aminobutyric acid transporter from rat brain. J Biol Chem 272: 1203–1210, 1997.[Abstract/Free Full Text]

6. Bossi E, Centinaio E, Castagna M, Giovannardi S, Vincenti S, Sacchi VF, and Peres A. Ion permeation and binding at the lepidopteran amino acid transporter KAAT1 expressed in Xenopus oocytes. J Physiol 513: 729–742, 1999.

7. Bossi E, Sacchi VF, and Peres A. Ionic selectivity of the coupled and uncoupled currents carried by the amino acid transporter KAAT1. Pflügers Arch 438: 788–796, 1999.[ISI][Medline]

8. Bossi E, Vincenti S, Sacchi VF, and Peres A. Simultaneous measurements of ionic currents and leucine uptake at the amino acid cotransporter KAAT1 expressed in Xenopus laevis oocytes. Biochim Biophys Acta 1495: 34–39, 2000.[ISI][Medline]

9. Castagna M, Shayakul C, Trotti D, Sacchi VF, Harvey WR, and Hediger MA. Cloning and characterization of a potassium-coupled amino acid transporter. Proc Natl Acad Sci USA 95: 5395–5400, 1998.[Abstract/Free Full Text]

10. Castagna M, Vincenti S, Marciani P, and Sacchi VF. Inhibition of the lepidopteran amino acid cotransporter KAAT1 by phenylglyoxal: role of arginine 76. Insect Mol Biol 11: 283–289, 2002.[ISI][Medline]

11. Chen JG, Liu-Chen S, and Rudnick G. External cysteine residues in the serotonin transporter. Biochemistry 36: 1479–1486, 1997.[ISI][Medline]

12. Feldman DH, Harvey WR, and Stevens BR. A novel electrogenic amino acid transporter is activated by K+ or Na+, is alkaline pH-dependent, and is Cl-independent. J Biol Chem 275: 24518–24526, 2000.[Abstract/Free Full Text]

13. Forlani G, Bossi E, Perego C, Giovannardi S, and Peres A. Three kinds of currents in the canine betaine-GABA transporter BGT-1 expressed in Xenopus laevis oocytes. Biochim Biophys Acta Research 1538: 172–180, 2001.[ISI]

14. Forster IC, Biber J, and Murer H. Proton-sensitive transitions of renal type II Na+-coupled phosphate cotransporter kinetics. Biophys J 79: 215–230, 2000.[Abstract/Free Full Text]

15. Frillingos S, Sahin-toth M, Wu J, and Kaback HR. Cys scanning mutagenesis: a novel approach to structure-function relationship in polytopic membrane proteins. FASEB J 12: 1281–1299, 1998.[Abstract/Free Full Text]

16. Giordana B, Leonardi MG, Casartelli M, Consonni P, and Parenti P. K+-neutral amino acid symport of Bombyx mori larval midgut: a system operative in extreme conditions. Am J Physiol Regul Integr Comp Physiol 274: R1361–R1371, 1998.[Abstract/Free Full Text]

17. Harvey RW and Wieczorek H. Energization of animal plasma membranes by chemiosmotic H+V-ATPases. J Exp Biol 200: 203–216, 1997.[Abstract/Free Full Text]

18. Hazama A, Loo DDF, and Wright EM. Presteady-state currents of the rabbit Na+/glucose cotransporter (SGLT1). J Membr Biol 155: 175–186, 1997.[ISI][Medline]

19. Javitch JA. Probing structure of neurotransmitter transporters by substituted-cysteine accessibility method. Methods Enzymol 296: 331–346, 1998.[Medline]

20. Kanner BI. Sodium-coupled neurotransmitter transport: structure, function and regulation. J Exp Biol 196: 237–249, 1994.[Abstract/Free Full Text]

21. Kanner BI. Transmembrane domain I of the {gamma}-aminobutyric acid transporter GAT-1 plays a crucial role in the transition between cation leak and transport modes. J Biol Chem 278: 3705–3712, 2003.[Abstract/Free Full Text]

22. Kitayama S, Shimada S, Hongxia X, Markham L, Donovan DM, and Uhl GR. Dopamine transporter site-directed mutations differentially alter substrate transport and cocaine binding. Proc Natl Acad Sci USA 89: 7782–7785, 1992.[Abstract]

23. Kleinberger-Doron N and Kanner BI. Identification of tryptophan residues critical for the function and targeting of the {gamma}-amino butyric acid transporter (Subtype A). J Biol Chem 269: 3063–3067, 1994.[Abstract/Free Full Text]

24. Leonardi MG, Casartelli P, Parenti P, and Giordana B. Evidence for a low affinity, high-capacity uniport for amino acids in Bombix mori larval midgut. Am J Physiol Regul Integr Comp Physiol 274: R1372–R1375, 1998.[Abstract/Free Full Text]

25. Liu Z, Stevens BR, Feldman DH, Hediger MA, and Harvey WR. K+ amino acid transporter KAAT1 mutant Y147F has increased transport activity and altered substrate selectivity. J Exp Biol 206: 245–254, 2003.[Abstract/Free Full Text]

26. Mabjeesh NJ and Kanner BI. Neither amino or carboxyl termini are required for function of the sodium- and chloride-coupled {gamma}-amino butyric acid transporter from rat brain. J Biol Chem 267: 2563–2568, 1992.[Abstract/Free Full Text]

27. Mager S, Kleinberger-Doron N, Keshet GI, Davidson N, Kanner BI, and Lester HA. Ion binding and permeation at the GABA transporter GAT-1. J Neurosci 16: 5405–5414, 1996.[Abstract/Free Full Text]

28. Mager S, Min C, Henry DJ, Chavkin C, Hoffman BJ, Davidson N, and Lester HA. Conducting states of a mammalian serotonin transporter. Neuron 12: 845–859, 1994.[ISI][Medline]

29. Palacin M, Estevez R, Bertran J, and Zorzano A. Molecular biology of mammalian plasma membrane amino acid transporters. Physiol Rev 78: 969–1054, 1998.[Abstract/Free Full Text]

30. Pantanowitz S, Bendahan A, and Kanner BI. Only one of the charged amino acids located in the transmembrane {alpha}-helices of the {gamma}-amino butyric acid transporter (subtype A) is essential for its activity. J Biol Chem 268: 3222–3225, 1993.[Abstract/Free Full Text]

31. Parthasarathy R, Xie T, Wolfersberger MG, and Harvey WR. Substrate structure and amino acid K+-symport in brush border membrane vesicles from larval Manduca sexta midgut. J Exp Biol 197: 237–250, 1994.[Abstract/Free Full Text]

33. Peres A and Bossi E. pH effects on the uncoupled, coupled and presteady-state currents at the transporter KAAT1 expressed in Xenopus laevis oocytes. J Physiol 525: 83–89, 2000.[Abstract/Free Full Text]

34. Quick M and Stevens BR. Amino acid transporter CAATCH1 is also an amino acid-gated cation channel. J Biol Chem 276: 33413–33418, 2001.[Abstract/Free Full Text]

35. Rudnick G. Bioenergetics of neurotransmitter transport. J Bioenerg Biomembr 30: 173–185, 1998.[ISI][Medline]

36. Sacchi VF, Castagna M, Trotti D, Shayakul C, and Hediger MA. Neutral amino acid absorption in the midgut of lepidopteran larvae. Adv in Insect Physiol 28: 168–184, 2001.[ISI]

37. Sacchi VF, Parenti P, Perego C, and Giordana B. Interaction between Na+ and the K+-dependent amino acid transport in midgut brush border membrane vesicles from Phylosamia cynthia larvae. J Insect Physiol 40: 69–74, 1994.[ISI]

38. Sacchi VF and Wolfersberger MG. Amino acid absorption. In: The Biology of the Insect Midgut, edited by Lehane MJ and Billingley PF. London: Chapman and All, 1996.

39. Seal RP, Leighton BH, and Amara SG. A model for the topology of excitatory amino acid transporters determined by the extracellular accessibility of substituted cysteines. Neuron 25: 695–706, 2000.[ISI][Medline]

40. Sonders MS and Amara SG. Channels in transporters. Curr Opin Neurobiol 6: 294–302, 1996.[ISI][Medline]

41. Stevens BR, Feldman DH, Liu Z, and Harvey WR. Conserved tyrosine-147 plays a critical role in the ligand-gated current of the epithelial cation/amino acid transporter/channel CAATCH1. J Exp Biol 205: 2545–2553, 2002.[ISI][Medline]

42. Vincenti S, Castagna M, Peres A, and Sacchi VF. Substrate selectivity and pH dependence of KAAT1 expressed in Xenopus oocyte. J Membr Biol 174: 213–224, 2000.[ISI][Medline]

43. Wang JB, Moriaki A, and Uhl GR. Dopamine transporter cysteine mutants: second extracellular loop cysteines are required for transporter expression. J Neurochem 64: 1416–1419, 1995.[ISI][Medline]





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