Multiple Consequences of Mutating Two Conserved β-Bridge Forming Residues in the Translocation Cycle of a Neuronal Glutamate Transporter

摘要: Glutamate transporters remove this neurotransmitter from the synapse in an electrogenic process. After sodium-coupled glutamate translocation, cycle is completed by obligatory outward translocation of potassium. In crystal structure archaeal homologue, two conserved residues form a β-bridge, which points away binding pocket. neuronal transporter EAAC1, equivalent are asparagine 366 and aspartate 368. Substitution mutants N366Q D368E, but not N366D D368N, show glutamate-induced inwardly rectifying steady-state currents, their apparent substrate affinity dramatically decreased. Such reflect net uptake observed with reciprocal double mutant N366D/D368N. Remarkably, exhibits slow substrate-induced voltage-dependent capacitative transient currents. These currents apparently reversible step, because interaction potassium largely impaired. Moreover, when analogous GLT-1 reconstituted into liposomes, exchange radioactive unlabeled acidic amino acids observed. Our results suggest that it 368 important during step. On other hand, side chains these themselves required for subsequent relocation