Arginine 445 controls the coupling between glutamate and cations in the neuronal transporter EAAC-1.
The substrate-binding sites in membrane transporters are alternately accessible from either side of the membrane, but the molecular basis of how this alternate opening of internal and external gates is achieved is largely unknown. Here we present data indicating that, in the neuronal electrogenic sodium- and potassium-coupled glutamate transporter EAAC-1, the substrate-binding site and one of the gates, or a residue controlling the gating process, are in close physical proximity. Arginine 445, located only two residues away from a residue implicated in glutamate binding (Bendahan, A., Armon, A., Madani, N., Kavanaugh, M. P., and Kanner, B. I. (2000) J. Biol. Chem. 275, 37436-37442), has been mutated to serine (R445S). Upon expression in oocytes, measurements of l-[(3)H]-glutamate transport under voltage clamp reveal that the charge/flux ratio for l-glutamate at -60 mV is approximately 30-fold higher than that of the wild type. Also, with d-aspartate, R445S exhibits an approximately 15-fold increase in this ratio. In contrast to the wild type, the reversal potential of the substrate-dependent currents in R445S shifts to more negative potentials when either the external sodium or potassium concentration is decreased. These findings indicate that these two cations are the main current carriers in the R445S mutant. Introduction of a methionine or a glutamine, but not a lysine, at position 445 gives rise to a phenotype similar to R445S. Therefore, it seems that the elimination of a positive charge in the vicinity of the substrate-binding site converts the transporter into a glutamate-gated cation-conducting pathway.
Borre
Lars
L
Department of Biochemistry, Hadassah Medical School, Hebrew University, Jerusalem 91120, Israel.
Kanner
Baruch I
BI
D027322:Amino Acid Transport System X-AG / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001120:Arginine; D001665:Binding Sites; D002412:Cations; D050491:Glutamate Plasma Membrane Transport Proteins; D018698:Glutamic Acid / Q000378:metabolism*; D009154:Mutation; D011817:Rabbits; D013379:Substrate Specificity; D027981:Symporters / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D027322:Amino Acid Transport System X-AG; D002412:Cations; D050491:Glutamate Plasma Membrane Transport Proteins; D027981:Symporters; D018698:Glutamic Acid; D001120:Arginine