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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS
Laboratory of Cellular and Molecular Physiology, Department of Structural and Functional Biology, and Center for Neurosciences, University of Insubria, Varese, Italy
Submitted 1 September 2006 ; accepted in final form 22 November 2006
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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transport; oligomerization; Slc6; fret
NSS members have been demonstrated with different approaches to form constitutive oligomers at the plasma membrane of living cells (6, 36, 37, 39, 40). Recent evidence (48) from the high-resolution crystal structure of a bacterial member of the family, the leucine transporter LeuTAa (accession no. NP_214423) from Aquifex aeolicus, confirmed that transporters belonging to this family form oligomers. These data indicate that the dimer interface is formed by EL2, TM9, and TM12. The relevance of the last transmembrane domains is further supported by experiments on SERT and GAT1, which indicate that TM12, together with TM11, participates in multimerization (15, 22). Starting from these observations, it is now important to understand the role of this quaternary arrangement and to find the possible determinants involved in the contact sites that stabilize the multimeric structure in the different members of the family.
Although it is reasonably established that NSS members form oligomeric complexes, no conclusive information are available about their functional role. Oligomerization appears to be important for the correct targeting of the transporters to the membrane (6), but only limited experimental evidence for human (h)SERT (17), hNET, and hDAT (11, 19, 44, 45) indicates a functional role for this three-dimensional organization. Furthermore, a countertransport model for monoamine transporters in which the conformational state of one monomer affects the functionality of the other has been recently proposed (38). However, recent results in our laboratory on GAT1 (41), obtained by combining electrophysiology with fluorescent resonance energy transfer (FRET) microscopy, indicated that although structural oligomerization of rat GAT1 occurs, the single subunits operate individually.
Experiments aimed to determine the subunit stoichiometry of transporter complexes usually include cross-linking analysis (11, 29, 44) or coprecipitation assays (14, 17, 20) and, more recently, FRET analysis (32, 33, 36, 37, 41, 43). The use of biochemical approaches to study proteins overexpressed in heterologous systems may give ambiguous results because of unnatural aggregation of intracellular proteins. A concatamer strategy also may be useful in investigating oligomerization. This method was first applied to voltage-gated K+ channels (13). Coinjections or linked constructs of cDNA coding for different subunits have been successfully used together with electrophysiology to identify the stoichiometry and the quaternary structure of different electrogenic membrane proteins. Several aspects have been studied with this method, from structure to more subtle features related to the molecular mechanisms of gating and activation (8, 12, 16, 18, 23–26, 30, 46, 49). Many other membrane proteins, such as aquaporins (27), the sodium pump (5), ligand-gated ion channels (28), the lactose permease (34), and the renal type IIa Na+/Pi transporter (21), also have been studied using the concatamer approach.
In the present study we used the analysis of electrophysiological properties of concatenated and coinjected constructs together with FRET experiments to investigate the possible relevance of oligomerization in the functioning of two neutral amino acid transporters belonging to the NSS family. To this purpose, the two transporters cloned from M. sexta represent valid investigation tools. CAATCH1 and KAAT1, heterologously expressed in Xenopus laevis oocytes, give rise to transport-associated currents, which are clearly distinguishable between the two proteins. Differences in amplitude, kinetics, and voltage-dependence have been observed, as well as differences in substrate selectivity, which were confirmed by amino acid uptake assays (1, 3, 4, 7, 31, 42).
To also determine whether KAAT1 and CAATCH1 form functional oligomers, we constructed four concatameric proteins consisting of one KAAT1 protein covalently linked to another KAAT1 (K-K concatamer) or to CAATCH1 (K-C concatamer), and vice versa (C-C concatamer and C-K concatamer). Eight constructs in which the two transporters were linked to yellow (YFP) or cyan fluorescent protein (CFP), in either the NH2 or COOH terminus, also were prepared to investigate the oligomer formation and estimate the relative distance between the different subunits by FRET analysis.
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MATERIALS AND METHODS |
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For electrophysiological investigations, the two transporters were concatenated in homo- and heteroconcatameric constructs by a linker peptide (LST). Briefly the second cDNA coding for KAAT1 or CAATCH1, mutated to insert an HindIII site before the ATG (5'-CGT TTT TAT TTT TAA TTT TCT TTC AAA GCT TTC CAC CAT GAA TGA CGG CC-3' for KAAT1; 5'-TC GTT TTT ATT TTT AAT TTT CTT TCA AAG CTT TCC ACC ATG AAT GAC GGC C-3' for CAATCH1), was inserted in KAAT1-pAMV-PA or CAATCH1-pAMV-PA between two HindIII sites, one created by point mutation, to eliminate the stop codon of the transporters (5'-CGG GTG CTT ACA GGC GTA ATA TTA ATA AGC TTT ATT AAT TAA TTA ATA ATA C-3' for KAAT1; 5'-CGG GTG CTT ACA GGC GTA ATA TTA ATA AGC TTA TAA AAT ATT ATG TAA AAT ATG-3' for CAATCH1), and the other present in the polylinker, in frame with the ATG of inserted cDNA.
All constructs were verified by sequencing (MWG Biotech, Sequencing Service, Ebersberg, Germany). All enzymes were supplied by Promega Italia (Milan, Italy).
cRNA preparation and Xenopus laevis oocyte expression. The experimental procedure has been described in detail elsewhere (2). The cDNAs encoding the original cotransporters and the concatenated construct were linearized with NotI. cRNAs were in vitro synthesized in the presence of Cap Analog and 200 units of T7 RNA polymerase.
X. laevis frogs were anesthetized in 0.10% (wt/vol) MS222 (tricaine methansulfonate) solution in tap water; portions of ovary were removed through an incision on the abdomen, and the frogs were humanely killed after the collection. 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 stage V and VI oocytes were selected and injected with 12.5 ng of the appropriate cRNA in 50 nl of water, using a manual microinjection system (Drummond). In coinjection experiments, oocytes were injected with 6.25 ng of each cRNA for a 1:1 ratio and with 3.125 and 9.375 ng for a 3:1 ratio. The oocytes were incubated at 18°C for 3–4 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 were performed.
Electrophysiology and data analysis. A two-microelectrode voltage clamp was used to perform electrophysiological experiments (Geneclamp 500B; 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 –160 to +20 mV in 20-mV steps. Two 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 Instruments) (9).
Cell culture and transfection. tsA201 cells were grown in Dulbecco's modified Eagle's medium (Sigma-Aldrich, Milan, Italy) supplemented with 10% heat-inactivated fetal calf serum, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin and were kept in a 5% CO2 humidified atmosphere at 37°C. Cells were plated in 35-mm dishes (1.4 x 105 cells/dish) and transfected with 1 µg of total DNA per dish using Lipofectamine 2000 (Invitrogen, Milan, Italy). In single transfection experiments, 400 ng of plasmid were used. In cotransfection experiments, the different constructs were always used in a ratio of 1:1, at 400 ng each. An empty vector (pcDNA3.1; Invitrogen) was used to normalize the total amount of transfected plasmid. Cells were replated on round glass coverslips 24 h after the transfection and used 24 h after the replating procedure.
Fluorescence microscopy and image acquisition. tsA201 cells were observed 48 h after transfection. Fluorescence images were acquired with a Retiga 2000R cooled charge-coupled device (CCD) camera (QImaging, Burnaby, BC, Canada) mounted on a Zeiss Axiovert IM35 inverted microscope and through a Zeiss fluar x40 oil-immersion objective. Excitation was obtained by a 150-W xenon arc lamp coupled to a filter wheel (CAIRN Research, Faversham, UK) delivering the light to the microscope through a light guide. Excitation filters were 430/25 nm for ECFP and 500/20 nm for EYFP; a 50% neutral density filter was used on the 500-nm excitation optic corridor. A double dichroic filter (86002 BS) was placed on the excitation light path to reflect both excitation wavelengths and pass both emissions. Emitted fluorescence reached an optosplit image splitter (CAIRN Research) fitted with a 495DCLP beam splitter and two emission filters at 470/30 and 535/30 nm for ECFP and EYFP, respectively. With this configuration, donor, acceptor, and FRET images could be obtained by changing only the excitation filters. The image splitter allowed us, for each excitation wavelength, to capture the two images (through the 470- and 535-nm emission filters) in the same frame on the CCD chip. To minimize photobleaching effects, we interposed an electronically driven shutter between the excitation filters and the dichroic mirror so that the cells were only excited for the time necessary to acquire the images. Under these conditions, significant photobleaching of YFP occurred only after long-lasting exposures (>30 min). Images of the same set were acquired under identical conditions. All filters were obtained from Chroma Technology (Rockingham, VT).
Images analysis and FRET calculation.
Images were analyzed with Image Pro Plus software (Media Cybernetics, Silver Spring, MD). NFRET images were constructed using the three-filter set method, according to the following analytical procedure introduced by Xia and Liu (47). Relative background images were subtracted (obtained by acquiring images in an area of the sample without cells, with the respective excitation configuration and CCD integration time). Independent thresholding on the images was necessary to avoid image-processing artifacts. Donor and acceptor fluorescence bleed-through were calculated from cells transfected with KAAT1-ECFP and KAAT1-EYFP alone, according to Gordon et al. (10), and were subtracted from the FRET image to obtain the net FRET (nF) image; nF normalization was then performed by dividing the nF image by the square root of the donor x acceptor product (47);
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Solutions. In electrophysiological experiments, the external control solution had the following composition (in mM): 98 NaCl, 1 MgCl2, 1.8 CaCl2, and 5 HEPES free acid; in the other solutions, NaCl was replaced by KCl or tetramethylammonium (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, proline) at 500 µM were added to induce transport-associated currents. Solutions were superfused by gravity onto the oocyte by a pipette tip placed very close (1–2 mm) to the cell. In fluorescence experiments, the extracellular control solution contained (in mM) 135 NaCl, 4 KCl, 1 MgCl2, 2 CaCl2, 6 glucose, and 10 HEPES-NaOH at pH 7.35.
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RESULTS |
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Transport-associated currents in the presence of Na+. The behavior of KAAT1, CAATCH1, the homoconcatamers K-K (KAAT1_KAAT1) and C-C (CAATCH1_CAATCH1) and the heteroconcatamers C-K (CAATCH1_KAAT1) and K-C (KAAT_CAATCH1) was initially investigated by measuring the transport-associated currents at a holding potential of –60 mV when a Na+ solution containing 500 µM of one of the three amino acids (leucine, threonine, or proline) was applied. The results are shown in Fig. 1, where it is possible to compare the electrophysiological profiles of the wild-type proteins with those of the forced constructs. On the right side of the traces, a schematic representation of the corresponding constructs is illustrated.
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50% of the threonine-induced current. Conversely, in the bottom two traces of Fig. 1A, in CAATCH1 and in its homoconcatamer C-C, leucine reduced the holding current, interpreted as a block of the sodium leakage current (7), whereas the threonine-induced current was 80% of the maximal current elicited by proline. In the middle of Fig. 1A, the behavior of the heteroconcatamers K-C and C-K is illustrated. Their selectivity profiles are similar but do not correspond to any one of the original proteins: whereas leucine induced very small inward currents, the currents associated with the transport of the other two amino acids at this voltage were quite similar. Figure 1 also shows that the current amplitudes among the recordings are different: in addition to the individual variability among oocytes, the currents generated by the concatenated constructs are smaller than those from the wild-type proteins in oocytes from the same batch. This effect was already observed in electrophysiological experiments with concatenated transport proteins (21) and may be due to impaired translation of constructs of larger dimensions. Current-voltage relationships. The behavior at a single membrane potential, however, may be misleading, because the transport-associated currents elicited by different amino acids have characteristic voltage dependencies. Following these initial observations, the investigation was extended to other voltages, to the currents in the presence of K+, and also including oocytes coinjected with cRNA coding for the wild types in different proportions. Figures 2 and 3 show the current-voltage relationships derived from experiments in the presence of Na+ and K+, respectively, with the indicated amino acids for the wild-type KAAT1 and CAATCH1, for the concatenated construct KK, KC, CC, and CK, and with 1:1 coinjection; furthermore, the calculated sum of the currents recorded from the wild-type proteins is also shown. The data for the leucine current at –160 mV, from the other coinjection proportions, are shown in Fig. 4.
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Confirming the results at –60 mV, it is possible to observe that the homoconcatenated constructs K-K and C-C show the same behavior as the wild-type proteins, in both Na+ (Fig. 2, A, B, G, and H) and in K+ (Fig. 3, A, B, G, and H), maintaining the most important characteristics at all membrane potentials. In the presence of Na+, leucine generates inward currents 20–25% of the maximal current recorded in threonine for KAAT1 and K-K (Fig. 2, A and B), whereas for CAATCH1 as well as the concatamer C-C, leucine blocks the leakage current producing an apparent outward current, and the maximal transport current is associated with the transport of proline (Fig. 2, G and H).
In the presence of K+, in KAAT1 and the concatamer K-K (Fig. 3, A and B), the maximal current is associated with the leucine transport, whereas the proline-induced current is ten times less at –160 mV. In CAATCH1 and its homoconcatamer C-C, leucine blocks the potassium leakage current, and the current associated with the proline transport is about one-third of the maximal current obtained in the presence of threonine at –160 mV (Fig. 3, G and H).
In the middle of Figs. 2 and 3, where the heteroconcatamers (C and D), the coinjection data (E), and the calculated sum of KAAT1 and CAATCH1 independently injected (F) are shown, it is possible to notice that in every case the current-voltage curves are very similar at all potentials tested, but again they do not correspond to those obtained from the wild-type and homoconcatenated proteins.
In detail, comparing the current-voltage plots of the heterocomplexes with the KAAT1 and K-K currents in the presence of Na+, the currents associated with the transport of threonine and proline can be superimposed at all potentials, whereas the leucine current is reduced to 15% of the maximal current (Fig. 2, C–F). Greater changes are visible in K+, where the reduction of the leucine transport-associated current is more marked. The heteroconcatameric transporters K-C (Fig. 3C) and C-K (Fig. 3D) again showed the same behavior of the coinjection, which is also similar to the sum of the currents measured in oocytes separately expressing the two proteins, as indicated by the fact that in these current-voltage relationships, leucine caused a 50% inhibition of the KAAT1 wild-type current. Threonine becomes the most potent substrate in the oocytes expressing CAATCH1 and KAAT1 either as forced dimer or as independent coexpression as in those expressing CAATCH1 alone. Finally, proline induced an inward current at negative potentials in all subunit compositions.
The relative leucine currents at –160 mV (ILeu/IPro in the presence of Na+ and ILeu/IThr in the presence of K+) for wild-type, homo-, and heteroconcatamers and from different ratios of coinjection are shown in Fig. 4. The leucine-induced currents are generally in good agreement with the percentage of injected CAATCH1, suggesting an independent functioning of each subunit.
Fluorescence microscopy and FRET analysis. To validate the independent functionality of the subunits revealed by electrophysiology, we needed to determine the oligomerization of KAAT1 and CAATCH1 and also whether they may form heteroligomers. We therefore tagged both proteins with CFP and YFP at either the NH2 or COOH termini and tested all the constructs for FRET on an epifluorescence setup equipped with a CCD camera. Independent transfection of tsA201 cells with the different constructs gave rise to fluorescence signals mainly localized at the plasma membrane (not shown). The membrane distribution, however, may be visualized by looking at the CFP and YFP filter channels in Fig. 5, an example of the FRET experiment. As a further control, all the constructs also were tested electrophysiologically in patch-clamp experiments (data not shown): both KAAT1 and CAATCH1 with the fluorescent proteins linked at either the NH2 or the COOH termini behaved in a wild-type fashion, indicating that the presence of the CYP/YFP does not influence the ability of the two proteins to transport neutral amino acids. Having demonstrated the full functionality of the tagged transporters, we next assessed whether KAAT1 and CAATCH1 exist as oligomers.
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Cotransfection of KAAT1-YFP and CAATCH1-CFP (and also CAATCH1-YFP and KAAT1-CFP) revealed high NFRET values (not statistically different from the COOH-terminus tagged transporters forming homoligomers), therefore indicating the ability of the two proteins to form mixed oligomers in addition to their ability to exist as complexes composed of identical subunits.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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With these assumptions, and considering that the structure is probably a tetramer (a dimer of dimer) as suggested by the FRET data and by the crystal structure of LeuTAa (48), the leucine current is expected to be 25% of KAAT1 wild type when 50% of KAAT1 and 50% of CAATCH1 are expressed in the oocyte membrane together as coinjection or concatamers. The observed 50% loss of function (Figs. 2–4) for the 1:1 ratio is instead consistent with the existence of functionally independent transport pathways in each subunit.
The superimposability between the current-voltage curves obtained by coinjecting the two wild-type proteins in equal proportions, although subject to the assumption of equal expression efficiency of the two cRNAs, and by injecting the forced heteroligomers, clearly points to an independent behavior of the two components. The FRET analysis results tell us that homo- and heteroligomerization may occur between KAAT1 and CAATCH1. Furthermore, for all the cotransfections, higher NFRET values were obtained when the fluorescent proteins were positioned at the COOH terminus. NFRET gradually decreases with the displacement of one or both the fluorescent proteins to the NH2-terminal positions of the transporters. The value of NFRET, independently of the subunit composition (i.e., in homo- and heteroligomers), suggests the closeness of the COOH termini of the different subunits in the three-dimensional organization, in very good agreement with the recent structural model of LeuTAa (48).
The negative control obtained cotransfecting two plasma membrane proteins not believed to interact (KAAT1 and 5HT1A) shows, however, relatively higher NFRET values compared with the cotransfection of the fluorescent proteins alone (see Fig. 6). This result is in agreement with previous observations (10, 47) and may be explained as a possible protein-protein weak interaction between the transporter and the serotonin receptor or, most likely, to transient fluorophore interaction by diffusion, a side effect of the overexpression of the two proteins, in this case further enhanced by the structural constraint of the membrane localization.
On the whole, the functional and FRET results reported in this study indicate that the M. sexta neutral amino acid transporters KAAT1 and CAATCH1 form structural oligomers in which, however, each subunit maintains its own independent transport activity, similar to the behavior observed in other transporters of the NSS superfamily (41).
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GRANTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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ACKNOWLEDGMENTS |
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Present addresses: A. Soragna, Laboratory of Membrane Physiology, Institute of General Physiology and Biological Chemistry "G. Esposito," University of Milan, Via Trentacoste 2, 20134 Milan, Italy; V. Frangione, Dipartimento di Scienze Cliniche e Biologiche, University of Insubria, Via Dunant 5, 21100 Varese, 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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