1Laboratory of Cellular and Molecular Physiology, Department of Structural and Functional Biology and Center for Neurosciences, University of Insubria, 21100 Varese; and 2Institute of General Physiology and Biological Chemistry, University of Milan, 20134 Milan, Italy
Submitted 13 January 2004 ; accepted in final form 9 May 2004
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ABSTRACT |
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structure and function; Manduca sexta
Clearly, these distinct functional differences, together with the high degree of identity between KAAT1 and CAATCH1, represent a favorable situation in which to attempt the construction of chimeric transporters between these two proteins, with the aim being the identification of structural regions involved in substrate recognition.
To develop a strategy for the construction of the chimeras, we considered two aspects. First, we took into account the schematizations emerging from the literature suggesting that the transporter sequences may be subdivided in regions with specialized, though nonexclusive, functional roles (10, 17, 20): 1) the amino-terminal region, up to the fourth putative transmembrane segment, which should include ion dependence and the permeation pathway, and 2) the central and carboxy-terminal regions, responsible for the recognition of organic substrates and possibly involved in the interaction with inhibitors. Second, we considered the restriction maps of the two proteins: because of the high homology of the two sequences, it was easy to find common restriction sites in appropriate positions to construct chimeric proteins without the necessity of introducing point mutations. Obviously, it is very likely that different parts of the sequence might contribute to the substrate specificity; it has been suggested that the three extracellular loops IV, V, and VI might form a "pocket" to which the substrate may bind (27). Studies performed on other proteins of the family suggest a role for the central domains of the protein, a region where KAAT1 and CAATCH1 differ for only 21 amino acids, with 15 conservative and 4 semiconservative substitutions; considering their position in the hypothetical tertiary structure and their chemical characteristics, only about half of them appear to be adequate to play a role in substrate recognition: G188S, G190N, E200Q, D286Q, W288A, A298S, A313P, L332W, S370A, and L416F (where the first letter is the amino acid in KAAT1 and the last is the amino acid in CAATCH1). Therefore, we decided to focus our attention from the external loop II to transmembrane domain VIII, where differences are present in significant number. As a consequence of these considerations, we constructed chimeras between KAAT1 and CAATCH1 in which three or four transmembrane domains were substituted in either the amino- or the carboxy-terminal regions and constructed others in which the five central domains of the protein were exchanged. The chimeras were expressed in Xenopus oocytes, and their functional characteristics were studied with electrophysiological measurements and radioactive uptake assays.
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MATERIALS AND METHODS |
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Using two restriction sites that were in the same position in CAATCH1 and KAAT1, NcoI (499) and PmlI (1276), and the NotI site in the multiple cloning site of pAMV-PA, we constructed four chimeric proteins having [with reference to the putative 12-transmembrane domain topology suggested by hydrophobicity profiles (6, 9)] the first three transmembrane domains of KAAT1 and the last nine transmembrane domains of CAATCH1 (K3C9), or vice versa (C3K9), or the first three and last four transmembrane domains of KAAT1 and the central five transmembrane domains of CAATCH1 (K3C5K4), or vice versa, as shown in Fig. 2. The correct construction of the cDNAs was controlled by restriction analysis and complete sequencing (MWG-Biotech).
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Xenopus laevis frogs were anesthetized in a 0.10% (wt/vol) solution of MS222 (tricaine methansulfonate) in tap water; portions of ovary were removed through a small incision on the abdomen that was subsequently sutured, after which the animal was returned to water. The oocytes were treated with 1 mg/ml collagenase (type IA; Sigma) in Ca2+-free ND-96 for at least 1 h at 18°C. Healthy-looking stages V and VI oocytes were collected and injected with 12.5 ng of the selected cRNA in 50 nl of water, using a manual microinjection system (Drummond). The oocytes were incubated at 18°C for 34 days in NDE solution (ND-96 solution: 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, and 5 mM HEPES supplemented with 50 µg/ml gentamicin and 2.5 mM Na-pyruvate at pH 7.6), before electrophysiological studies.
Electrophysiology and data analysis. A two-microelectrode voltage clamp was used to perform electrophysiological experiments (GeneClamp; Axon Instruments, 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 140 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 analyses were done using the pCLAMP 8 software (Axon Instruments).
Solutions. The external control solution had the following composition (in mM): 98 NaCl, 1 MgCl2, 1.8 CaCl2, and 5 HEPES; in the other solutions, NaCl was replaced by KCl 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. When chloride was replaced, gluconate salts were used. Amino acids (leucine, threonine, proline, and methionine; 500 µM) were added to induce transport-associated currents. Solutions were superfused by gravity onto the oocyte by a pipette tip placed very close (12 mm) to the cell.
Uptake experiments. Amino acid uptake was measured 3 days after injection. Groups of 1012 oocytes were incubated in 100 µl of uptake solution (100 mM NaCl, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, and 10 mM HEPES-Tris, pH 8) with 0.5 mM [3H]leucine, [3H]proline, or [3H]threonine (37 MBq/ml; Amersham Pharmacia Biotech) for 60 min, and then oocytes were washed 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. In K+ experiments, NaCl was replaced by 150 mM KCl. The higher osmolarity did not influence uptake (not shown). Experiments in which the expression level of KAAT1 or CAATCH1 was less than five times above background were discarded. The data reported in the figures show KAAT1-, CAATCH1-, and chimera-mediated transport representing the difference between the mean uptake measured in cRNA-injected oocytes and the mean uptake measured in noninjected oocytes.
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RESULTS |
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Leak current and Cldependence. A conspicuous "leak" or "uncoupled" current has been observed in both KAAT1- and CAATCH1-expressing oocytes (1, 2, 9, 21), and it has been attributed to a channel-like ionic flow through the transporter when the organic substrate is absent. The presence of this characteristic has been incorporated into the name of CAATCH1: the "CH" stands for "channel." We compared the properties of the leak current in the two transporters to verify its usefulness in distinguishing the behavior of our chimeras. The leak current was operatively determined as the difference between current level in the presence of Na+ or K+ and current level in TMA+ solution (1, 2). The results, shown in Fig. 3A, suggest that the amplitude and selectivity of the uncoupled current in the two transporters do not differ sufficiently to represent a useful criterion for discrimination.
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Transport-associated current in the presence of either Na+ or K+. The ability of KAAT1 and CAATCH1 to handle different neutral amino acids in the presence of either Na+ or K+ was initially checked by measuring the transport-associated currents at a holding potential of 60 mV and applying Na+ or K+ solutions containing 500 µM of one of the four amino acids (leucine, threonine, methionine, or proline). The results in Fig. 4 show that the published observations (9) are confirmed and extended. In particular, in CAATCH1 leucine does not give rise to inward transport-associated currents in either Na+ or K+, whereas methionine is able to generate currents when the transporter is bathed in K+ but not in Na+; conversely, in CAATCH1, proline is able to elicit currents only in the presence of Na+. In KAAT1, all four amino acids support inward currents in the presence of Na+, whereas in the presence of K+, proline does not.
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Current-voltage relationships. To obtain a more complete description of the behavior of the chimeras, we extended this analysis to a larger voltage range and to include other amino acids as well. Figures 6 and 7 show the current-voltage relationships derived from experiments in the two ionic conditions with different amino acids. The results in the presence of Na+ (Fig. 6) confirm that only those constructs having the KAAT1 central domains are able to generate an inward current when leucine is added, whereas in CAATCH1 and CAATCH1-like chimeras, leucine consistently causes an outwardly directed difference current for potentials negative to 40 mV. Furthermore, proline and threonine remain the preferred amino acids in all constructs, with the potency order gradually changing from a slight prevalence of threonine at all voltages in KAAT1 to a prevalence of proline at low voltages (in C3K9) and to a more marked prevalence of proline at all voltages in all other constructs and in CAATCH1. When the main ion present is K+ (Fig. 7), leucine is the amino acid producing the largest currents at all potentials in the constructs having the KAAT1-derived central region, although threonine also generates similar currents in C3K5C4. However, threonine definitely becomes the most potent substrate in the constructs having the CAATCH1-derived central region, which also shows some ability of proline to induce inward current at negative potentials.
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The structural composition of the chimeras and the transport activities shown in Fig. 9 therefore confirm that in both cotransporters, the amino-terminal region up to the first three transmembrane domains and the last four transmembrane domains are not involved in the amino acid discrimination. Consequently, the middle region is sufficient to confer substrate selectivity in both KAAT1 and CAATCH1.
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DISCUSSION |
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This also is true for proteins of relatively distant organisms, such as, for instance, the human (hSERT) and Manduca sexta serotonin transporters (MasSERT): MasSERT is comparatively less sensitive to cocaine than most members of the monoamine transporter subfamily, and this feature can be exploited to investigate drug selectivity. Studies on chimeras from these transporters suggest that transmembrane domains 1 and 2 affect substrate transport, possibly changing the transporter conformations (24). The two lepidopteran transporters used in our work, KAAT1 and CAATCH1, are 90% identical, yet they exhibit characteristic substrate selectivity sequences that may be exploited to identify the domains involved in substrate recognition.
Before the results obtained using our chimeras are discussed, it is perhaps worthwhile to expend a few words on the behavior of the wild-type transporters. KAAT1 and CAATCH1 are exceptional among ion-coupled cotransporters in being able to function using the K+ gradient in addition to the Na+ gradient (6, 9), and we know that this property is related to the specific ionic conditions of the larval intestine, which is rich in K+ and poor in Na+ (22). However, Na+ and K+ are not completely equivalent in both proteins: the current-voltage relationships are more strongly inwardly rectifying when the carrier ion is K+ than when it is Na+ (see Figs. 6 and 7; Ref. 1, 6). In KAAT1, the current-voltage curve in the presence of K+ is negatively shifted compared with that in Na+, and this corresponds to the more negative position of the intramembrane charge movement measured in the absence of organic substrate; this difference is likely to have a physiological meaning, considering the very negative value of the apical membrane potential (8).
The qualitative correspondence of the voltage dependence of pre-steady-state and transport-associated currents in both Na+ and K+ (1) is in agreement with a similar correlation observed in the rat GABA transporter rGAT1, and in that case, it has been explained with a kinetic model in which ionic interaction and charge translocation must precede the binding of the organic substrate. This might constitute the basis for the understanding of another interesting observation in KAAT1 and CAATCH1: namely, the fact that for each transporter, the substrate selectivity order changes depending on whether K+ or Na+ is the main bathing ion. Figures 6 and 7 show that whereas proline generates the largest currents in KAAT1 in the presence of Na+, this amino acid is the least effective in the presence of K+. Analogously, in CAATCH1, whereas proline is the preferred amino acid in the presence of Na+, the largest current in the presence of K+ is produced by threonine.
In our interpretation, the picture that emerges from these observations and considerations is one in which Na+ or K+ may first interact, each with its own voltage dependence characteristics, with the transporters, creating a structural moiety to which each substrate amino acid may bind and trigger the subsequent transport steps with its own peculiar kinetics. In this view, for instance, the moiety created by the complex Na+-KAAT1 may be highly favorable for the interaction with proline, whereas that originated by the K+-KAAT1 complex might be much less so. In summary, the overall turnover rate of the transporters might depend on the kind of ternary complex formed by the 1) transporter, 2) ion(s), and 3) organic substrate.
Leak current and its block. The leak current properties in the absence of organic substrates appear to be so similar in KAAT1 and CAATCH1 that they cannot be exploited in structure-function studies performed in chimeric constructs between the two transporters. The Cl independence of the proline-induced current, as reported for CAATCH1 (9), is also present in KAAT1 (Fig. 3B), whereas the Cl dependence of leucine transport, as exhibited by KAAT1 (6), cannot be studied in CAATCH1. Again, the Cl dependence of the two transporters does not appear to be a useful test for discriminating purposes.
Although present in many cotransporters, leak currents are particularly conspicuous in CAATCH1 and KAAT1. We have speculated that this may be so to favor absorption of Na+ from the K+-rich intestinal lumen (1). The reduction of the membrane current by an organic substrate, which may even be accompanied by transport (3, 21), would represent a slowing down of the turnover rate, instead of its increase. In other words, there may be a basal cycling rate of the transporter, in the absence of organic substrate, that may be either increased or decreased, depending on the kind of interacting amino acid and on the ionic conditions. The interaction of methionine and proline, first described for CAATCH1 (9) but also observed in the present study in KAAT1 (Fig. 8), is very interesting in this respect. In both transporters, proline is able to increase the basal turnover rate; methionine instead accelerates the turnover rate in KAAT1 (to a lesser degree compared with proline) but slows it in CAATCH1.
The current-voltage relationships shown in Figs. 6 and 7 under leucine block are rather intriguing: the apparently outward current induced by leucine in the presence of Na+ is slightly larger in the chimeras with the central CAATCH1 domains compared with CAATCH1; in the presence of K+, leucine may induce an inward current at potentials more positive than 80 mV, with a positive shift in the zero current potential compared with Na+. However, these observations are affected by some uncertainties, because the leucine block of the leak current is not complete (Figs. 4 and 5), the voltage dependence of the leak current is shifted to more negative voltages in the presence of K+ anyway (1), and, furthermore, the contribution of the current carried by the oocyte endogenous channels (especially in K+ solution) is not known. Given these reservations, we prefer not to derive any deductions from these kinds of observations.
Determinants of substrate selectivity. Our results from the chimera studies show that the central transmembrane domains, from IV to VIII, strongly determine the selectivity order for the recognition of organic substrates (Figs. 5, 6, 7, and 9). The 63 different amino acids (10%) are variably distributed along the entire sequences of KAAT1 and CAATCH1: 8 (1.3%) of the different amino acids are in the amino-terminal region (from the beginning to the NcoI site), with 5 conservative substitutions; 21 (3.4%) are in the central region (between NcoI and the PmlI sites), with 15 conservative and 4 semiconservative changes; and finally, 34 (5.3%) are located in the carboxy-terminal region (from PmlI to the end), with 18 conservative and 6 semiconservative changes. The eight amino acids located in the first three transmembrane domains do not appear to play any role in substrate selectivity; this finding is not surprising, because the amino terminus is the most conserved region in these two proteins and also in the whole Na+/Cl-dependent transporter family, which suggests that this region is involved in general transport functions shared by all members of the family. Some quantitative differences in the degree of efficacy of the various amino acids (see Figs. 6 and 7) may indicate that the carboxy-terminal region, where the two transporters show a high variability, may nevertheless give only a very small contribution to the selectivity characteristics. Therefore, the remaining 21 different residues located between transmembrane domains IV and VIII appear to be the best candidates for a functional role in the structures responsible for substrate recognition.
It has already been suggested (25) that the region including domains IV, V, and VI may form a substrate binding pocket in the related transporter rGAT1; recently, the fourth extracellular loop has also been involved in substrate interaction (27). Working on KAAT1, our group has also shown that aspartate 338, presumably located in the transmembrane domain VII, is implicated in cation selectivity and in the coupling between cation and amino acid fluxes (16). The present data seem to extend the role of this region in the functioning of K+-coupled lepidopteran transporters. In the region encompassed by transmembrane domains IV to VIII, KAAT1 and CAATCH1 differ for only 21 residues, which will possibly be mutated individually and in combinations to further restrict the definition of the structural determinants of the substrate specificity of these transporters.
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GRANTS |
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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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