A conserved Aspartate Residue Located at the Extracellular End of the Binding
Pocket Controls Cation Interactions in Brain Glutamate Transporters
Noa Rosental and Baruch Kanner
Abstract
Glutamate transporters located in the brain maintain low synaptic concentrations of the
neurotransmitter by coupling its flux to that of Na+ and H+, which are co-transported with
the neurotransmitter, and to that of K+, which is transported in the opposite direction.
Aspartate 440 in the neuronal glutamate transporter EAAC1 is highly conserved and
located at the extracellular end of the substrate binding pocket of the archeal homologue
GltPh. Here we probe the role of Asp-440 in glutamate transport, by studying substitution
mutants. With the exception of the D440E mutant, all other substitutions resulted in
inactive transport. Electrophysiological analysis of oocytes expressing EAAC1-WT and
D440E indicates that this mutant operates mainly in the exchange mode. Using
radioactive uptake in proteoliposomes under net flux conditions as an assay, we found
that D440E has a higher apparent affinity for Na+ than EAAC1-WT, but its apparent
affinity for K+ was lowered. These observations explain why under physiological
conditions D440E behaves as an obligate exchanger and show the critical importance of
the carboxylate side-chain at position 440 in controlling the cation interactions of
EAAC1.