Substrate and inhibitor binding of human GABA transporter 3

  • Structure. 2025 Dec 4;33(12):2049-2057.e5. doi: 10.1016/j.str.2025.08.012.
Hao Xu  1 ,  Yimin Zhang  2 ,  Qinru Bai  3 ,  Lingli He  4 ,  Qihao Chen  3 ,  Yunlong Qiu  3 ,  Renjie Li  3 ,  Jie Yu  5 ,  Jun Zhao  6 ,  Yan Zhao  7
Affiliations
  • 1. Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui, China; Key Laboratory of Biomacromolecules (CAS), National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
  • 2. Interdisciplinary Research Center on Biology and Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 201210, China; University of Chinese Academy of Sciences, Beijing 100049, China.
  • 3. Key Laboratory of Biomacromolecules (CAS), National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; College of Life Sciences, University of Chinese Academy of Sciences, Beijing 100049, China.
  • 4. Chinese Institutes for Medical Research, Beijing, China.
  • 5. Interdisciplinary Research Center on Biology and Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 201210, China. Electronic address: [email protected].
  • 6. Peking University Institute of Advanced Agricultural Sciences, Shandong Laboratory of Advanced Agricultural Sciences at Weifang, Weifang, China. Electronic address: [email protected].
  • 7. Key Laboratory of Biomacromolecules (CAS), National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China. Electronic address: [email protected].
PMID: 40914153 DOI: 10.1016/j.str.2025.08.012
Abstract

GABA (g-aminobutyric acid) transporter 3 (GAT3) is primarily found in glial cells and is essential for regulating GABA homeostasis in the central nervous system by mediating GABA uptake. Consequently, GAT3 has emerged as a significant therapeutic target for the treatment of epilepsy. In this study, we present the cryoelectron microscopy (cryo-EM) structures of GAT3 bound to its substrate GABA, the selective inhibitor SNAP-5114, and in the substrate-free state. GAT3 binds to GABA in an inward-facing conformation, while SNAP-5114 occupies the GABA-binding pocket and is stabilized by extensive interactions with surrounding residues. Functional studies reveal that E66 plays a pivotal role in determining the substrate-binding mode and specificity of SNAP-5114 binding. Taken together, our study clarifies the GABA binding mechanism of GAT3 and reveals the molecular basis for the specific inhibition of SNAP-5114, offering valuable insights for developing GAT3 subtypes selective inhibitors, which hold potential as a treatment for epilepsy.

Keywords
GABA transporter; GAT3; SNAP-5114; cryo-EM structure.
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