Structure and allosteric inhibition of excitatory amino acid transporter 1

Abstract

Human members of the solute carrier 1 (SLC1) family of transporters take up excitatory neurotransmitters in the brain and amino acids in peripheral organs. Dysregulation of their functions is associated to neurodegenerative disorders and cancer. Here we present the first crystal structures of a thermostabilized human SLC1 transporter, the excitatory amino acid transporter 1 (EAAT1), with and without allosteric and competitive inhibitors bound. The structures show novel architectural features of the human transporters, including intra- and extracellular domains with potential roles in transport function, as well as regulation by lipids and post-translational modifications. The coordination of the inhibitor in the structures and the change in the transporter dynamics measured by hydrogen-deuterium exchange mass spectrometry, reveal an allosteric mechanism of inhibition, whereby the transporter is locked in the outward-facing states of the transport cycle. Our results provide unprecedented insights into the molecular mechanisms of function and pharmacology of human SLC1 transporters.

Allosteric gate modulation confers K + coupling in glutamate transporters

“…Molecular simulations of K + binding to EAAT1 are based on the recently determined crystal structure of an engineered Na + ‐ and aspartate‐bound EAAT1 (Canul‐Tec et al , ). This protein contains several thermostabilizing mutations, some of which might cause slight structural deviations from the WT structure.…”

“…This protein contains several thermostabilizing mutations, some of which might cause slight structural deviations from the WT structure. However, the thermostabilized transporter exhibits robust Na + ‐ and K + ‐dependent glutamate uptake, indicating that the mechanisms of Na + and K + coupling are preserved (Canul‐Tec et al , ). Moreover, simulations of EAAT1 and Glt Ph provided consistent results about K + ‐binding sites, the effects of K + binding as well as on HP2 dynamic.…”

Extracellular ATP and glutamate drive pyruvate production and energy demand to regulate mitochondrial respiration in astrocytes

“…It may be taken up, through the main astroglial glutamate transporters GLT-1/EAAT2/ Slc1a2 and GLAST/EAAT1/Slc1a3 (Canul-Tec et al, 2017, Kanai et al, 2013, and/or it may activate glutamate receptors in astrocytes, mainly metabotropic receptors (Panatier & Robitaille;2016;Sun et al, 2013), or even NMDA receptors (Jimenez-Blasco, Santofimia-Castaño, Gonzalez, Almeida, & Bolaños, 2015). It may be taken up, through the main astroglial glutamate transporters GLT-1/EAAT2/ Slc1a2 and GLAST/EAAT1/Slc1a3 (Canul-Tec et al, 2017, Kanai et al, 2013, and/or it may activate glutamate receptors in astrocytes, mainly metabotropic receptors (Panatier & Robitaille;2016;Sun et al, 2013), or even NMDA receptors (Jimenez-Blasco, Santofimia-Castaño, Gonzalez, Almeida, & Bolaños, 2015).…”

AP1G1 is involved in cetuximab-mediated downregulation of ASCT2-EGFR complex and sensitization of human head and neck squamous cell carcinoma cells to ROS-induced apoptosis

“…ASCT2 is a major transporter of glutamine uptake in cancer cells [16–18], and glutamine uptake and metabolism (e.g., through glutathione biosynthesis and glutaminolysis) are critical for survival and proliferation of cancer cells that are metabolically addicted to glutamine [19–23]. Directly targeting ASCT2, however, is technically challenging because ASCT2 also has important physiological functions in normal tissues and the crystallographic structure of human ASCT2 remains to be fully elucidated [24,25]. …”