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Excitatory amino acid transporters (EAATs) take up the neurotransmitter glutamate from the synaptic cleft and maintain glutamate concentrations below neurotoxic levels. Recently, the crystal structures of thermostable EAAT1 variants have been reported; however, little is understood regarding the functional mechanism of the transmembrane domain (TM) 4b-4c loop, which contains more than 50 amino acids in mammalian EAATs that are absent in prokaryotic homologues. To explore the spatial position and function of TM4 during the transport cycle, we introduced pairwise cysteine substitutions between the TM4b-4c loop and TM8 in a cysteine-less version of EAAT1, CL-EAAT1. We observed pronounced inhibition of transport by Cu(II)(1,10-phenanthroline) 3 (CuPh) for doubly substituted V238C/I469C and A243C/I469C variants, but not for corresponding singly substituted CL-EAAT1 or for more than 20 other double-cysteine variants. Dithiothreitol treatment partially restored the uptake activity of the CuPh-treated V238C/I469C and A243C/I469C doubly substituted variants, confirming that the effects of CuPh on these variants were due to the formation of intramolecular disulfide bonds. Glutamate, KCl, and d,l-threo-β-benzyloxy-aspartate weakened CuPh inhibition of the V238C/I469C variant, but only KCl weakened CuPh inhibition of the V243C/I469C variant, suggesting that the TM4b-4c loop and TM8 are separated from each other in the inward-facing conformations of EAAT1. Our results suggest that the TM4b-4c loop and TM8 are positioned in close proximity during the transport cycle and are less closely spaced in the inward-facing conformation.

Paired-Cysteine Scanning Reveals Conformationally Sensitive Proximity between the TM4b-4c Loop and TM8 of the Glutamate Transporter EAAT1