Mechanism of cation binding to the glutamate transporter EAAC1 probed with mutation of the conserved amino acid residue Thr101

Zhen Tao, Noa Rosental, Baruch I. Kanner, Armanda Gameiro, Juddy Mwaura, Christof Grewer*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

44 Scopus citations

Abstract

The glutamate transporter excitatory amino acid carrier 1 (EAAC1) catalyzes the co-transport of three Na+ ions, one H+ ion, and one glutamate molecule into the cell, in exchange for one K+ ion. Na + binding to the glutamate-free form of the transporter generates a high affinity binding site for glutamate and is thus required for transport. Moreover, sodium binding to the transporters induces a basal anion conductance, which is further activated by glutamate. Here, we used the [Na+] dependence of this conductance as a read-out of Na+ binding to the substrate-free transporter to study the impact of a highly conserved amino acid residue, Thr101, in transmembrane domain 3. The apparent affinity of substrate-free EAAC1 for Na+ was dramatically decreased by the T101A but not by the T101S mutation. Interestingly, in further contrast to EAAC1 WT, in the T101A mutant this [Na+] dependence was biphasic. This behavior can be explained by assuming that the binding of two Na+ ions prior to glutamate binding is required to generate a high affinity substrate binding site. In contrast to the dramatic effect of the T101A mutation on Na+ binding, other properties of the transporter, such as its ability to transport glutamate, were impaired but not eliminated. Our results are consistent with the existence of a cation binding site deeply buried in the membrane and involving interactions with the side chain oxygens of Thr101 and Asp367. A theoretical valence screening approach confirms that the predicted site of cation interaction has the potential to be a novel, so far undetected sodium binding site.

Original languageEnglish
Pages (from-to)17725-17733
Number of pages9
JournalJournal of Biological Chemistry
Volume285
Issue number23
DOIs
StatePublished - 4 Jun 2010

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