Experimental Model and Subject Details
Cell lines
Full-grown Xenopus laevis oocytes were maintained at 18oC in post-injection NDE solution (96 mM NaCl, 2 mM KCl, 1 mM MgCl2, 5 mM HEPES, 2.5 mM pyruvate, 0.05 mg/mL gentamicin sulphate, and 1.8 mM CaCl2 at pH 7.6).
Human embryonic kidney 293 (HEK293) cells stably expressing the rat isoform of GAT1 (rGAT1) were used (rGAT1-HEK293 cells) in batch release assays. The generation and maintenance of stable cell lines expressing rGAT1 was conducted as previously described by other for other SLC6 transporters [74]. rGAT1-HEK293 cells were maintained in high glucose (4.5 g/L) and L-glutamine-containing DMEM, supplemented with 10% FBS and G418 (250 µg/mL) in a humidified atmosphere (37°C, 5% CO2) and in a sub-confluent state.
CHO cells were grown and maintained in DMEM containing L-glutamine (PAA laboratories, Austria) with gentamicin 50 mg/L, geneticin 500 µg/mL, 10% foetal calf serum on 100 mm-diameter cell culture dishes at 37°C in an atmosphere of 5% CO2 and 95% air [75].
Method Details
Heterologous Expression in X. laevis oocytes
The detailed experimental procedure has been described elsewhere [76]. Briefly, the cDNAs encoding rat GAT1 in the pAMV vector, and Madin-Darby canine kidney (MDCK) canine BGT-1 in the pSPORT1 vector were linearized using NotI, and the in vitro cRNA synthesis was done in the presence of Cap Analog 10 mM and T7 RNA polymerase 200 U.
Oocytes were collected from X. laevis female frogs, under anaesthesia, by performing laparotomy on the abdomen, to remove portions of the ovary. Detailed procedures are described elsewhere [25]. For the heterologous expression of the GABA transporters, stage IV oocytes were micro-injected with cRNA encoding the target protein i.e., rGAT1 and cBGT-1 (12.5 ng/ 50 nL) and incubated for the expression period (3-4 days) at 18oC before the experimental procedure.
The oocytes used in this work were either bought from EcoCyte Bioscience GmbH, Germany or donated by a collaborator with approved experimental protocol by the Committee of the “Organismo Preposto al Benessere Animale” of the University of Insubria, Varese, Italy, and nationally by Ministero della Salute (n. 449/2021-PR), Italy; and used in agreement with the Art.18 of decreto legislativo 4 marzo 2014, n. 26 and Art. 26 in directive 2010/63/eu of the European parliament and of the council of 22 September 2010.
Electrophysiology
The two-electrode voltage clamp (TEVC) Oocyte ClampOC-725B, Warner Instruments, Hamden, CT, USA was used for controlling voltages and recording currents. The clamped oocytes were maintained under continuous perfusion of the basic buffer solution ND98 (NaCl 98 mM, CaCl2 1.8 mM, MgCl2 1 mM, HEPES 5 mM at pH7.6) with a flow rate of 1.5 mL/min. For dose-response curves and competitive assay experiments, solution exchange was performed using an in-house developed gravity-driven digitally controlled perfusion control system. Signals were filtered at 0.1 kHz and sampled at 1 kHz. The voltage-jump experiments were performed by the voltage pulse control protocol consisting of 10 square pulses (0.8 s long) from -140 mV to +40 mV (at 20 mV increments). All recorded currents were digitalized using Axon CNS 1440B Digidata system controlled by pClamp 11.2.2 (Molecular Devices, USA).
For whole-cell patch clamp experiments using CHO cells expressing human GAT1 on the Patchliner™ (Nanion Technologies GmbH, Germany), the cell-harvesting was performed right after the setup of the instrument, to be transferred directly into the CellHotelTM. The cells were automatically transferred from the CellHotelTM to each of the eight wells of 4X chips for Patchliner™ that was set in the whole-cell mode with the holding membrane potential at -80 mV.
The internal solution (10 mM NaCl, 110 mM KF, 10 mM EGTA, 10 mM KCl, and 10 mM HEPES at pH 7.2) was provided inside the cells, followed by the external solution outside (Ca+2-rich, 130 mM NaCl, 4 mM KCl, 10 mM CaCl2, 1 mM MgCl2, 5 mM D-Glucose monohydrate, 10 mM HEPES at pH 7.4) for seal enhancement. For the recording on the Patchliner™, only cells showing a seal resistance >300 MΩ were taken into consideration for the analysis. After the seal was established, a double wash with external solution (standard, 140 mM NaCl, 4 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 5 mM D-Glucose monohydrate, 10 mM HEPES at pH 7.4) was performed to remove the extra Ca2+. The following fluidic protocol was followed for all betaine concentrations: 6 s perfusion of 60 µL of betaine-containing external solution (speed 10 µL/s) followed by 18 s perfusion of 180 µL of external solution (standard) to wash away the betaine (speed 10 µL/s).
Substrate detection in X. laevis oocytes using LC-MS/MS
To perform the substrate uptake experiment, the X. laevis oocytes expressing rGAT1 were incubated in a fresh Petri dish containing the substrate+ND98 for 25-30 minutes at 18oC. To remove any presence of the extracellular substrate post-incubation, the oocytes were carefully washed thrice with ice-cold ND96 for two minutes. At the end of the washing, the oocytes (in the group of 5) were transferred in a fresh Eppendorf containing CH3OH:CHCl3:H2O in proportion of 3:1:1 v/v with the solvent to oocytes ratio of 1:100. The oocytes were homogenized using water ultra-sonicator and then centrifuged at 13,000xg for eight minutes at 4oC. The supernatant from these samples was removed, and the pallet containing the cytosol content with oocyte debris were diluted in H2O (MilliQ, Merck, Italy) to extract the hydrophilic compounds. These samples were centrifuged at 13,000xg for eight minutes at 4oC and the aqueous top part containing cytosol content was extracted and used immediately for LC-MS/MS detection or stored at -80oC to use later (up to three months).
Betaine and GABA standard stock solutions (1 mM in 0.1% formic acid) were prepared in MS grade water. For quantitative mass spectrometry, a Finnigan LXQ linear ion trap mass spectrometer, equipped with an ESI ion source (Thermo Electron Corporation, CA, USA) was used. The analyses were performed in positive (spray voltage 4.5 kV, capillary temperature 270oC) and in the multiple-reaction monitoring (MRM) mode. The tuning parameters, the optimization of collision energy for each substance, and the choice of target compound fragments were conducted in continuous flow mode by using standard solutions at a concentration of 5 μM. The MRM acquisitions for GABA and betaine were accomplished by monitoring the 114/89 and 118/59 transitions, respectively. The HPLC analysis was performed using a Finnigan Surveyor MS plus HPLC system (Thermo Electron Corporation, CA, USA). For both GABA and betaine quantitation, separation was achieved using the C18 column (ACQUITY UPLC Peptide BEH C18 Column, 300 Å, 1.7 µm, 2.1 mm×150 mm). The mobile phase was composed of (A) water with 0.1% (v/v) formic acid and (B) acetonitrile (100%) plus 0.1% (v/v) formic acid with a flow rate 200 µL/min; gradient 0–7.0 min/2% (v/v) B, 7–10 min/2–50% (v/v) B; 10-15 min/50-2% (v/v) B.
To verify the sensitivity and linearity of the LC-MS/MS response to GABA and betaine, calibration curves showing the relative peak areas plotted against analytes concentrations (range 2–16 µM) were created. GABA and betaine contents were expressed as relative intensities (peak areas) among samples.
Radiolabelled GABA release assay
The batch-release assays have been conducted as described before [77, 78]. In brief, the transfected HEK293 cells overexpressing rGAT1 were grown overnight onto poly-D-lysine coated 96-well plates (4×104 cells/well). After removing DMEM, the cells were loaded with 0.01µM [3H]-GABA in Krebs-Ringer HEPES buffer (KHB; NaCl 120 mM, KCl 3 mM, CaCl2 2 mM, MgCl2 2 mM, glucose 20 mM, HEPES 10 mM at pH 7.3) for 20 min (5% CO2, 37oC). After the incubation, the cells were brought to room temperature and washed five times with KHB during 15min. Afterwards, cells were kept during 10 min either in KHB or KHB + monensin (mon) 10 μM and the resulting supernatant was discarded. The batch-release assay started by applying either KHB or KHB + mon 10 μM (100 μL; every 2min, four times). Subsequently, the substance of the interest (in KHB or KHB + mon 10μM) was added at a specific concentration (every 2 min, five times). The resulting supernatants were collected and transferred to the neighbour wells. Tiagabine (10 μM) and GABA (1-300 μM) were used as negative and positive controls, respectively. Three independent experiments were performed in duplicate for every compound and respective concentration of interest. At the end, liquid scintillation cocktail was added to the wells with remaining cells, with transferred supernatant and to the wells used for total uptake and activity measurements. The total radioactivity present in the supernatant and in the remaining cells was set as 100%, and the amount of [3H]-GABA present in the supernatant was expressed as percentage of the total.
Molecular docking and molecular dynamics simulations
In the absence of a crystal or cryo-EM structure of GAT1 in the outward-facing conformation, we selected the Alphafold homology model to analyse the stability of the docked and bound betaine [43, 44]. The docking experiments were performed using GOLDscore scoring function on GOLD molecular docking software (The Cambridge Crystallographic Data Centre, UK). For MD simulations, the bound-ions were added to the GAT1 model using the outward-open human SERT crystal structure (PDB ID: 5I71) [45] as a reference. The generated system was converted from full-atom into a coarse grain representation using the MARTINI force field [79-81], with a membrane composition of 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC): cholesterol containing membrane (POPC:CHOL 70:30 mol%) [82] solvated in water with 150mM NaCl. After 1μs of coarse-grain simulation, while restraining the protein structure, the membrane was equilibrated. The coarse-grained system was then converted to an all-atom representation [83] in which the transporter was replaced by the original GAT1 model to avoid factitious structural imprecisions induced by the double coordinate conversion of the protocol. Betaine was positioned according to the docking results. Possible atom overlaps between the reinserted protein and the relaxed membrane were loosened using the membed procedure [84] as previously described [85]. We used the amber ff99SB-ILDN force field[86] to describe GAT1, ions and the solvent, and Slipid [87] for POPC and cholesterol. Previously reported structural analysis of the orthologous SERT structure [45], suggests that the residue E467 is protonated. All simulations were carried out with GROMACS version 2021.4 [88]. Three replicas of the final assembled system were energy-minimised and equilibrated in four steps of 2.5 ns, each by stepwise releasing the position restraints (1,000, 100, 10, 1 kJ/mol/nm) that are active on the Cα atoms and the bound chloride ion [89, 90]. Per each trajectory, the production run was carried for 500ns after removing all position restraints. The temperature was maintained at 310 K using the v-rescale (τ = 0.5 ps) thermostat [91], while separately coupling protein, membrane, and solvent. The pressure was maintained at 1 bar using the Parrinello-Rahman barostat [92] in a semi-isotropic manner and applying a coupling constant of 20.1 ps. Long-range electrostatic interactions were described using the smooth particle mesh Ewald method [93] applying a cutoff of 0.9 nm. The van der Waals interactions were described using the Lennard Jones potentials applying a cutoff of 0.9 nm. Long-range corrections for energy and pressure were applied. Coordinates of all atoms were recorded every 5 ps. The complete set of parameters of the production run can be found in the supplementary materials (Code S1).
Quantification and statistical analysis
Electrophysiological analysis
For TEVC, data analysis was performed using Clampfit 11.2 (Molecular Devices, USA). The dose-response currents were calculated as the maximum current amplitude during the perfusion, and the curves were fitted by non-linear regression to the Logistic equation using Originpro 8 (OriginLab Corporation, USA)..The pre-steady state currents in rGAT1 were isolated by subtraction of the corresponding trace in the presence of SKF89976a 30µM, as this molecule blocks all of the transport-associated steady state and transient currents [29, 30]. The resultant traces were fitted with single exponentials to obtain decay time constant and voltage (τ-V) curves. The same traces were used to obtain the charge dislocation-voltage (Q-V) curves by fitting with Boltzmann equation to the sigmoidal curve, which also provided maximal displaceable charge Qmax [30, 40]. The further derivation of τ-V and Q-V curves yielded the unidirectional rate constants inrate (α) and outrate (β) (Eq. 1).
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)
Equation 1: The Boltzmann equation quantifies Qin, total displaced charge. Where Qmax is the maximal displaceable charge, V0.5 is the voltage at which half maximal charge is displaced (i.e., midpoint of the sigmoidal), σ represents the slope factor. The inrate and outrate constants provide the unidirectional constants α and β respectively.
The data collected with Patchliner™ were saved as ASCII files and successively analysed with the software OriginPro 2021 (OriginLab Corporation, USA). The traces were filtered (using a weighted Adjacent-Averaging filter with ten points of window) and for each cell the baseline was subtracted to the respective betaine traces. The transport current was read as average ± SD (standard deviation) of the current value between 4-6 seconds after betaine perfusion. It is to be noticed that the current in each well is generated by the sum of currents coming from four cells simultaneously since each well of a 4X chip contains four holes for cell attachment.