TY - JOUR
T1 - A conserved aspartate residue located at the extracellular end of the binding pocket controls cation interactions in brain glutamate transporters
AU - Rosental, Noa
AU - Gameiro, Armanda
AU - Grewer, Christof
AU - Kanner, Baruch I.
PY - 2011/12/2
Y1 - 2011/12/2
N2 - In the brain, transporters of the major excitatory neurotransmitter glutamate remove their substrate from the synaptic cleft to allow optimal glutamatergic neurotransmission. Their transport cycle consists of two sequential translocation steps, namely cotransport of glutamic acid with three Na + ions, followed by countertransport of K + . Recent studies, based on several crystal structures of the archeal homologue Glt Ph, indicate that glutamate translocation occurs by an elevator-like mechanism. The resolution of these structures was not sufficiently high to unambiguously identify the sites of Na + binding, but functional and computational studies suggest some candidate sites. In the Glt Ph structure, a conserved aspartate residue (Asp-390) is located adjacent to a conserved tyrosine residue, previously shown to be a molecular determinant of ion selectivity in the brain glutamate transporter GLT-1. In this study, we characterize mutants of Asp-440 of the neuronal transporter EAAC1, which is the counterpart of Asp-390 of Glt Ph. Except for substitution by glutamate, this residue is functionally irreplaceable. Using biochemical and electrophysiological approaches, we conclude that although D440E is intrinsically capable of net flux, this mutant behaves as an exchanger under physiological conditions, due to increased and decreased apparent affinities for Na + and K +, respectively. Our present and previous data are compatible with the idea that the conserved tyrosine and aspartate residues, located at the external end of the binding pocket, may serve as a transient or stable cation binding site in the glutamate transporters.
AB - In the brain, transporters of the major excitatory neurotransmitter glutamate remove their substrate from the synaptic cleft to allow optimal glutamatergic neurotransmission. Their transport cycle consists of two sequential translocation steps, namely cotransport of glutamic acid with three Na + ions, followed by countertransport of K + . Recent studies, based on several crystal structures of the archeal homologue Glt Ph, indicate that glutamate translocation occurs by an elevator-like mechanism. The resolution of these structures was not sufficiently high to unambiguously identify the sites of Na + binding, but functional and computational studies suggest some candidate sites. In the Glt Ph structure, a conserved aspartate residue (Asp-390) is located adjacent to a conserved tyrosine residue, previously shown to be a molecular determinant of ion selectivity in the brain glutamate transporter GLT-1. In this study, we characterize mutants of Asp-440 of the neuronal transporter EAAC1, which is the counterpart of Asp-390 of Glt Ph. Except for substitution by glutamate, this residue is functionally irreplaceable. Using biochemical and electrophysiological approaches, we conclude that although D440E is intrinsically capable of net flux, this mutant behaves as an exchanger under physiological conditions, due to increased and decreased apparent affinities for Na + and K +, respectively. Our present and previous data are compatible with the idea that the conserved tyrosine and aspartate residues, located at the external end of the binding pocket, may serve as a transient or stable cation binding site in the glutamate transporters.
UR - http://www.scopus.com/inward/record.url?scp=82355184476&partnerID=8YFLogxK
U2 - 10.1074/jbc.M111.291021
DO - 10.1074/jbc.M111.291021
M3 - ???researchoutput.researchoutputtypes.contributiontojournal.article???
C2 - 21984827
AN - SCOPUS:82355184476
SN - 0021-9258
VL - 286
SP - 41381
EP - 41390
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 48
ER -