TM4 of the glutamate transporter GLT-1 experiences substrate-induced motion during the transport cycle

Excitatory amino acid transporter 2 (EAAT2), also known as glial glutamate transporter type 1 (GLT-1), plays an important role in maintaining the extracellular glutamate concentrations below neurotoxic levels. The highly conserved TM2 transmembrane domain of GLT-1 maintains a stable position during the transport cycle; however, the effect of the transport cycle on the topology of TM4 in not well established. To further reveal the function of TM4, two cysteine pairs between TM2 and TM4 were introduced using site-directed mutagenesis. A significant decrease of transport activity was observed in the I93C/V241C and I97C/V241C mutants upon application of the oxidative cross-linking reagent, copper (II) (1,10-phenanthroline)₃ (CuPh), which suggests that a conformational shift is essential for transporter activity. Furthermore, the decrease in activity by CuPh crosslinking was enhanced in external media with glutamate or potassium, which suggests that TM2 and TM4 assume closer proximity in the inward-facing conformation of the transporter. Our results suggest that the TM4 domain of GLT-1, and potentially other glutamate transporters, undergoes a complex conformational shift during substrate translocation, which involves an increase in the proximity of the TM2 and TM4 domains in the inward-facing conformation.