the accessibility in the external part of the tm5 of the glutamate transporter eaat1 is conformationally sensitive during the transport cycle的可访问性谷氨酸转运体的外部部分tm5 eaat1构象上敏感在运输周期.pdf

The Accessibility in the External Part of the TM5 of the Glutamate Transporter EAAT1 Is Conformationally Sensitive during the Transport Cycle 1 2 Xiuping Zhang , Shaogang Qu * 1 China-America Cancer Research Institute, Guangdong Medical College, Dongguan, Guangdong, China, 2 Department of Immunology, Southern Medical University, Guangzhou, Guangdong, China Abstract Background: Excitatory amino acid transporter 1 (EAAT1) is a glutamate transporter which is a key element in the termination of the synaptic actions of glutamate. It serves to keep the extracellular glutamate concentration below neurotoxic level. However the functional significance and the change of accessibility of residues in transmembrane domain (TM) 5 of the EAAT1 are not clear yet. Methodology/Principal Findings: We used cysteine mutagenesis with treatments with membrane-impermeable sulfhydryl reagent MTSET [(2-trimethylammonium) methanethiosulfonate] to investigate the change of accessibility of TM5. Cysteine mutants were introduced from position 291 to 300 of the cysteine-less version of EAAT1. We checked the activity and kinetic parameters of the mutants before and after treatments with MTSET, furthermore we analyzed the effect of the substrate and blocker on the inhibition of the cysteine mutants by MTSET. Inhibition of transport by MTSET was observed in the mutants L296C, I297C and G299C, while the activity of K300C got higher after exposure to MTSET. Vmax of L296C and G299C got lower while that of K300C got higher after treated by MTSET. The L296C, G299C, K300C single cysteine mutants showed a conformationally sensitive reactivity pattern. The sensitivity of L296C to MTSET was potentiated by glutamate and TBOA,but the sensitivity of G299C to MTSET was potentiated only by TBOA.