TY - JOUR T1 - Cysteine Scanning Mutagenesis of TM4b-4c Loop of Glutamate Transporter EAAT1 Reveals Three Conformationally Sensitive Residues JF - Molecular Pharmacology JO - Mol Pharmacol DO - 10.1124/mol.117.111245 SP - mol.117.111245 AU - Wenlong Zhang AU - Xiuping Zhang AU - Shaogang Qu Y1 - 2018/01/01 UR - http://molpharm.aspetjournals.org/content/early/2018/04/13/mol.117.111245.abstract N2 - Glutamatergic synaptic transmission is terminated by members of the excitatory amino acid transporters (EAATs) that recycle glutamate from the synaptic cleft by transporting it into neuron and glial cells. To probe the structural role of the TM4b-4c loop of EAATs in glutamate transport, each of its 57 amino acid residues were mutated to cysteine. Thirteen of the single mutants have very low transport activity. Aqueous accessibility of the introduced cysteines from the remaining mutants was then explored with membrane-permeant and membrane-impermeant sulfhydryl reagents under different conditions. F190C, V238C and A243C were affected by MTSET while Q189C, F190C, V238C, A243C and L244C were sensitive to MTSEA. Q189C and L244C transport activity was diminished in the presence of potassium, which is expected to favor the outward-facing conformation of the transporter. Inversely, L244C was protected by glutamate. The modification of A243C by MTSEA was enhanced by either potassium and glutamate or DL-TBOA. From these results, we suggest residues F190C, V238C, A243C may be located near the extracellular surface and TM4b-4c loop forms multiple reentrant membrane loops at the cell surface. Alternatively, F190C, V238C, and A243C may function in the transport pathway which is exposed to MTSET. In addition, Q189C, A243C and L244C are conformationally sensitive and may play a role in the transport cycle. ER -