IntroductionThe betaine/γ-aminobutyric acid (GABA) transporter 1 (BGT1) was first discovered and cloned in Madin-Darby canine kidney (MDCK) cells and subsequently identified in the liver and brain1,2,3,4, where it participates in various physiological processes. In renal medullary cells, BGT1 transports and accumulates betaine at high concentrations to counteract extracellular hyperosmolarity1,5, thereby preventing apoptosis induced by osmotic stress from high NaCl and urea concentrations6,7. Homocysteine is a toxic metabolite, and its accumulation in the body increases the risk of cardiovascular diseases8,9,10. In the liver, betaine is transported into hepatocytes, where it donates a methyl group to homocysteine and converts it into methionine11,12,13, which maintains homocysteine levels within the normal range to prevent the toxic effects associated with its accumulation. In the brain, BGT1 has been reported to localize to astrocytes14, where it likely limits the diffusion of GABA to adjacent synapses, maintaining the fidelity of synaptic signal transmission15,16. Furthermore, BGT1 expression is elevated in the brain during status epilepticus17,18. Given that BGT1-selective inhibitors have demonstrated antiepileptic effects19,20, BGT1 has garnered increasing attention in the development of antiepileptic drugs21,22,23.Owing to its ability to transport GABA and sequence homology, BGT1 is classified within the GABA transporter (GAT) family, a subgroup of solute carrier family 6 (SLC6). Its activity is dependent on Na+ and Cl–5,24. Recently, the structures of several neurotransmitter/sodium symporter (NSS) proteins, such as GAT125,26,27, noradrenaline transporter (NET)28,29,30,31, dopamine transporter (DAT)32,33,34,35, and serotonin transporter (SERT)36,37,38, have been resolved. However, these proteins transport their substrates with only two sodium ions, unlike BGT1, which cotransports three sodium ions39. This unique sodium ion coupling results in a stronger uphill transport driving force and inhibition of reverse transport40, which is critical for the accumulation of high concentrations of betaine in renal medullary cells. Despite its significance, the exact binding site for the third Na+ ion remains unclear. Moreover, unlike GAT1–3, which exclusively take up GABA, BGT1 is capable of transporting both GABA and betaine41. However, the molecular basis of this dual substrate selectivity remains unknown.Here, we resolved the structures of BGT1 in the substrate-bound state and an inward-open state obtained without exogenous substrate addition during purification. These structures reveal the network of interactions between BGT1 and its substrates and ions, leading to the identification of a potential Na3-binding site. Additionally, we captured the conformational transition of BGT1 from the occluded state to the inward-open state. Our structural and biochemical characterization of BGT1 provides detailed insights into its substrate specificity and transport mechanisms.ResultsStructural determination and architecture of BGT1To elucidate the substrate binding and transport mechanisms of BGT1, we employed single-particle cryo-electron microscopy (cryo-EM) to determine its structure. In recent years, the structures of proteins in the SLC6 family have increasingly been resolved, often involving the use of thermostabilizing mutations or truncations to optimize their biochemical properties28,33. For this study, we expressed and purified full-length, wild-type (WT) BGT1 for structural determination. Specifically, we used n-dodecyl-β-D-maltopyranoside (DDM) to solubilize and purify BGT1, with digitonin buffer used during the size-exclusion chromatography (SEC) experiments (Supplementary Fig. S1a). The BGT1 sample was subsequently reconstituted into nanodiscs composed of brain polar lipids and MSP1D1E3 (Supplementary Fig. S1b, c). We determined the structures of BGT1 in complex with two substrates at concentrations of 10 mM GABA and 20 mM betaine, as well as an inward-open structure obtained in the absence of exogenous substrate, achieving resolutions of 2.7, 2.6, and 3.2 Å, respectively (Fig. 1a–c; Supplementary Fig. S2 and Table S1).Fig. 1: Cryo-EM structure of BGT1.Full size imagea Cryo-EM map of nanodisc-reconstituted BGT1betaine at a contour level of 7.5 σ in ChimeraX. The size of the BGT1 molecule is labeled. BGT1 and the nanodisc are represented by cyan and transparent gray, respectively. b Overall structure of BGT1betaine with labeled transmembrane helices and intracellular and extracellular loops. The betaine molecule is not shown in the model. c Structures of BGT1 in the inward-open state, GABA-bound state, and betaine-bound state are colored green, gold, and cyan, respectively.The overall structure of human BGT1 is approximately 77 Å long and 64 Å wide (Fig. 1a) and displays a classic LeuT-like fold28,33,42,43, containing 12 transmembrane helices (TMs) in which TMs 1–5 and TMs 6–10 exhibit an inverted pseudo-twofold symmetry (Fig. 1b). The helices TM1 and TM6 unwind in the middle of the transmembrane region, which is crucial for most of the interactions with the substrates and ions concentrated in this area43. The N- and C-termini of BGT1 are both located on the intracellular side, but owing to their disorder, we were unable to observe the N-terminus (residues 1–42) and part of the C-terminus (residues 585–614) in the cryo-EM map. However, in our structure, we observed extensive interactions between the C-terminus and the intracellular loop (IL) 1/IL5 (Supplementary Fig. S3a), which stabilized C-terminal residues (amino acids 561–584) and enabled us to resolve clear density for this region. The conserved disulfide bond in the GAT family was observed in BGT1, formed by Cys157 and Cys166 (Supplementary Fig. S3b), which stabilizes an ordered structure in part of extracellular loop (EL) 2. Two glycosylation sites, Asn171 and Asn183, are predicted on EL244. However, the segment of EL2 from L170 to P180 was not observed, nor were glycosylations detected, likely due to their flexibility. In the inward-open state, BGT1 (BGT1inward-open) creates a pathway from the intracellular membrane surface to the substrate-binding pocket. Despite the absence of exogenously added substrates during purification, residual density was observed within the substrate-binding pocket, which may originate from a small amount of endogenously bound GABA derived from HEK293F cells27. We also observed density at the conserved chloride ion-binding site in the BGT1inward-open structure, suggesting that the chloride ion contributes to the structural stability (Supplementary Fig. S3c). Additionally, we identified one binding site for cholesterol and one for cholesteryl hemisuccinate (CHS) in the BGT1inward-open structure: one situated between TM3 and TM4 and the other within a groove formed by TM9, TM10, and TM12.Substrate binding and selectivity of BGT1To elucidate the substrate-binding mode of BGT1, we determined the structures of BGT1 bound to GABA (BGT1GABA) and betaine (BGT1betaine) (Fig. 2a, b). In our maps, we found the density that could accommodate the substrate (Fig. 2c). The substrate is in the mid-region of BGT1, equidistant from the intracellular and extracellular surfaces of the membrane. The binding pocket is inaccessible from both the extracellular and intracellular sides (Fig. 2d), indicating that the BGT1GABA structure adopts an occluded conformation. The substrate-binding pocket is surrounded by TM1, TM3, TM6, and TM8, with residues G57 in TM1 (G57TM1), Y133TM3, S294TM6, A296TM6, Q299TM6, and S395TM8 maintaining a stable substrate-binding environment. Specifically, the backbone nitrogen of G57TM1 and the hydroxyl oxygen of Y133TM3 form hydrogen bonds with the carbonyl oxygen of GABA. The side-chain carbonyl oxygen of Q299TM6 forms a hydrogen bond with the amino nitrogen of GABA (Fig. 2e). Additionally, five water molecules identified near GABA in the structure participate in a complex hydrogen bond network with the amino end of the substrate and residues Q299TM6, F293TM6, I297TM6, S395TM8 and Q396TM8 (Fig. 2e; Supplementary Fig. S4a). These water molecules likely play a critical role in substrate binding in a mechanism similar to those observed in other transporters, such as DAT, GlyT1, and GAT126,33,43. Hydrophobic residues, including L129TM3, F293TM6 and A296TM6, together with polar residues, including S294TM6, S395TM8 and C399TM8, stabilize the binding pocket (Fig. 2e). [3H]GABA uptake assays revealed that the G57S and Q299A mutations led to a significant reduction in GABA transport activity in BGT1 and that the Y133A, A296I and S395A mutations caused the loss of transport activity, although these mutant proteins were delivered to the plasma membrane (Fig. 2f; Supplementary Fig. S5). Conversely, replacements such as L129A and F293A had little impact on transport activity (Fig. 2f). These results indicate that the binding of the substrate is predominantly mediated by hydrogen bonds, while hydrophobic residues create a suitable hydrophobic environment and provide sufficient space to accommodate the substrate. A comparison with the GABA-bound structure of GAT126 and GAT345 revealed that interactions at the carboxyl of GABA are conserved. In contrast, recognition of the GABA amino group differed substantially among the three subtypes. In BGT1, Q299TM6 forms a hydrogen bond with the amino nitrogen of GABA. This interaction appears to be specific to BGT1, as the equivalent position is occupied by leucine in both GAT1 and GAT3. In GAT1, the amino group adopts a different orientation and forms a hydrogen bond with Y60TM1, a residue unique to GAT1 that is replaced by glutamate in BGT1 and GAT3. In GAT3, the amino group is positioned close to the phenyl ring of F308TM6 and is stabilized through a cation–π interaction. Although the corresponding phenylalanine is conserved in BGT1 and GAT1, it does not interact with GABA in those structures. Together, these observations reveal that the recognition of the GABA carboxylate group is conserved, whereas the recognition of the amino group is subtype-specific (Fig. 2g).Fig. 2: Substrate binding of BGT1.Full size imagea, b Overall structures of BGT1 in complex with GABA (a) and betaine (b). Substrates, Na+ and Cl‒ are depicted as blue, purple and green spheres, respectively. c Chemical structure (top) and cryo-EM density (bottom) of GABA and betaine contoured at 4 σ in PyMOL. d Cross section of the electrostatic surface of BGT1; the substrates are shown as sticks and spheres. e Substrate binding pockets of BGT1GABA. Residues involved in GABA coordination are shown as sticks and labeled. Interactions are presented with black dashes. Na+, Cl‒ and water molecules are depicted as purple, green and red spheres, respectively. f The transport activities of WT BGT1 and its mutants related to substrate binding were quantified using [3H]-labeled GABA uptake assays. The transport activities of the WT and each mutant were adjusted based on their expression levels. The transport activity of each mutant was normalized to that of the WT. A 3-min incubation was performed to ensure that measurements were taken within the linear range. The data represent the means ± SEM (error bars); n = 3 biologically independent experiments. ****P