Substrate-Induced Motion between TM4 and TM7 of the Glutamate Transporter EAAT1 Revealed by Paired Cysteine Mutagenesis

In order to maintain efficient synaptic communication, glutamate transporters re-uptake glutamate from the synaptic cleft and prevent glutamate concentrations from reaching neurotoxic levels. Mammalian EAATs contain a stretch of over 50 amino acids in TM4b-4c loop that is absent in the bacterial protein. To investigate the spatial proximity and functional significance of residues in glutamate transporter, cysteine pairs were introduced at positions A243 of TM4b-4c loop and T396 or A414 of TM7, respectively. An inhibition of transport by Cu(II)(1,10-phenanthroline)3 and cadmium ions is observed in the double mutants A243C/T396C and A243C/S414C, but not in the corresponding single mutants. Treatment with dithiothreitol after CuPh restored much of the transport activity. Inhibition by Cu(II)(1,10-phenanthroline)3 and cadmium was only observed when the cysteine pairs were introduced in the same polypeptide. Therefore, we suggested that the formation of these disulfide bonds occurred intra-molecularly. Glutamate, potassium and DL-TBOA facilitated cross-linking in the A243C/T396C transporter and this suggested that TM4b-4c loop and β-bridge region in TM7 were drawn into close proximity to each other in the inward-facing and outward-facing conformation of EAAT1. However, glutamate and potassium prevented cross-linking in A243C/A414C, and this suggested that TM4b-4c loop and the tip of TM7 descended to a proximal region in the inward-facing conformation of the transporter. Thus, these data provide evidence that substrate-induced structural rearrangements occur between TM4b-4c loop and TM7 during the transport cycle.