Cryo-EM structure of GABA transporter 1 reveals substrate recognition and transport mechanism

Abstract

The inhibitory neurotransmitter γ-aminobutyric acid (GABA) is cleared from the synaptic cleft by the sodium- and chloride-coupled GABA transporter GAT1. Inhibition of GAT1 prolongs the GABAergic signaling at the synapse and is a strategy to treat certain forms of epilepsy. In this study, we present the cryo-electron microscopy structure of Rattus norvegicus GABA transporter 1 (rGAT1) at a resolution of 3.1 Å. The structure elucidation was facilitated by epitope transfer of a fragment-antigen binding (Fab) interaction site from the Drosophila dopamine transporter (dDAT) to rGAT1. The structure reveals rGAT1 in a cytosol-facing conformation, with a linear density in the primary binding site that accommodates a molecule of GABA, a displaced ion density proximal to Na site 1 and a bound chloride ion. A unique insertion in TM10 aids the formation of a compact, closed extracellular gate. Besides yielding mechanistic insights into ion and substrate recognition, our study will enable the rational design of specific antiepileptics.

Interaction of GAT1 with sodium ions: from efficient recruitment to stabilisation of substrate and conformation

“…The importance of sodium for substrate transport has been characterised at the macroscopic and the kinetic level (Hilgemann and Lu, 1999;Iversen and Snyder, 1968;Mager et al, 1993) and showed that GABA uptake is energised by the downhill concentration gradient of the co-transported sodium (Kanner, 1978). Structural data of transporters from the SLC6 family have revealed the binding sites of substrate and co-transport ions and the main conformations of the transport cycle of GAT1 (Nayak et al, 2023;Zhu et al, 2023). We confirmed through direct observations that NA2 functions to stabilise GAT1 in the outward-open conformation and showed that Na2 achieves this by immobilising the scaffold and bundle domains in relation to each other.…”

“…The motion of the bundle domain relative to the scaffold domain allows for alternating access to the central substrate binding site S1 (Forrest et al, 2008). Very recently, several inward-facing GAT1 structures and a substrate, sodium and chloridebound inward-occluded structure were published (Motiwala et al, 2022;Nayak et al, 2023). These conformations together with an AlphaFold2 model of the outward-facing state have allowed to formulate the translocation cycle for GAT1 (Nayak et al, 2023;Zhu et al, 2023) which consists of outward-open, occluded and inward-open states.…”

“…Very recently, several inward-facing GAT1 structures and a substrate, sodium and chloride-bound inward-occluded structure were published (Motiwala et al, 2022; Nayak et al, 2023). These conformations together with an AlphaFold2 model of the outward-facing state have allowed to formulate the translocation cycle for GAT1 (Nayak et al, 2023; Zhu et al, 2023) which consists of outward-open, occluded and inward-open states. Interestingly, while the conformations of the outward-open and the occluded structures are very similar to the respective conformations observed in other SLC6 family members (Coleman et al, 2016; Singh et al, 2008; Yamashita et al, 2005; Yang and Gouaux, 2021), confirming shared structural changes across the SLC6 family, SLC6 transporter structures differ in the conformation of TM1a of the inward-facing state, whereby for GAT1 several conformations of TM1a were observed (Motiwala et al, 2022; Nayak et al, 2023; Zhu et al, 2023).…”

“…These conformations together with an AlphaFold2 model of the outward-facing state have allowed to formulate the translocation cycle for GAT1 (Nayak et al, 2023; Zhu et al, 2023) which consists of outward-open, occluded and inward-open states. Interestingly, while the conformations of the outward-open and the occluded structures are very similar to the respective conformations observed in other SLC6 family members (Coleman et al, 2016; Singh et al, 2008; Yamashita et al, 2005; Yang and Gouaux, 2021), confirming shared structural changes across the SLC6 family, SLC6 transporter structures differ in the conformation of TM1a of the inward-facing state, whereby for GAT1 several conformations of TM1a were observed (Motiwala et al, 2022; Nayak et al, 2023; Zhu et al, 2023). For LeuT we have shown that the large motions of TM1a as observed in the micelle solubilized crystal structure (doi: 10.1038/nature10737) are incompatible within the membrane environment (Sohail et al, 2016), suggesting that the conformation of TM1a is especially sensitive to the environment.…”

“…The concentrative uptake of GABA is enabled by the co-transport with two sodium ions and one chloride (Bhatt et al, 2023; Hilgemann and Lu, 1999; Keynan and Kanner, 1988). The chloride binding site and the two sodium binding sites (NA1 and NA2) are located in the centre of the transporter, next to the unwound regions on TM1 and TM6, which form the substrate binding site (S1) together with TM3 and TM8 (Nayak et al, 2023; Zhu et al, 2023; Zomot et al, 2007). Structural considerations on LeuT (Krishnamurthy and Gouaux, 2012; Yamashita et al, 2005), MD simulations (Szöllősi and Stockner, 2022) and biochemical studies (Tavoulari et al, 2016) indicated that the sodium bound to the NA2 (Na2) is involved in stabilisation of the outward-open state by connecting TM1 of the bundle with TM8 of the scaffold.…”