Multiple consequences of mutating two conserved beta-bridge forming residues in the translocation cycle of a neuronal glutamate transporter.
Glutamate transporters remove this neurotransmitter from the synapse in an electrogenic process. After sodium-coupled glutamate translocation, the cycle is completed by obligatory outward translocation of potassium. In the crystal structure of an archaeal homologue, two conserved residues form a beta-bridge, which points away from the binding pocket. In the neuronal glutamate transporter EAAC1, the equivalent residues are asparagine 366 and aspartate 368. Substitution mutants N366Q and D368E, but not N366D and D368N, show glutamate-induced inwardly rectifying steady-state currents, but their apparent substrate affinity is dramatically decreased. Such currents, which reflect electrogenic net uptake of substrate are not observed with the reciprocal double mutant N366D/D368N. Remarkably, the double mutant exhibits slow substrate-induced voltage-dependent capacitative transient currents. These currents apparently reflect the reversible sodium-coupled glutamate translocation step, because the interaction of the double mutant with potassium is largely impaired. Moreover, when the analogous double mutant in the glutamate transporter GLT-1 is reconstituted into liposomes, a slow exchange of radioactive and unlabeled acidic amino acids is observed. Our results suggest that it is the interaction of asparagine 366 and aspartate 368 that is important during the glutamate translocation step. On the other hand, the side chains of these residues themselves are required for the subsequent potassium relocation step.
Rosental
Noa
N
Department of Biochemistry, Hebrew University Hadassah Medical School, P. O. Box 12272, Jerusalem 91120, Israel.
Bendahan
Annie
A
Kanner
Baruch I
BI
D000818:Animals; D001692:Biological Transport; D050580:Excitatory Amino Acid Transporter 3 / Q000737:chemistry* / Q000502:physiology; D018698:Glutamic Acid / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D009154:Mutation; D011817:Rabbits; D013329:Structure-Activity Relationship
D050580:Excitatory Amino Acid Transporter 3; D018698:Glutamic Acid