Dataset Information

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

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, Thr(101), 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 Thr(101) and Asp(367). 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.

SUBMITTER: Tao Z

PROVIDER: S-EPMC2878536 | biostudies-literature | 2010 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

Tao Zhen Z Rosental Noa N Kanner Baruch I BI Gameiro Armanda A Mwaura Juddy J Grewer Christof C

The Journal of biological chemistry 20100408 23

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(+)] depe ...[more]

PMID: 20378543

Similar Datasets

Project description:The neuronal glutamate transporter EAAC1 contains several conserved acidic amino acids in its transmembrane domain, which are possibly important in catalyzing transport and/or binding of co/countertransported cations. Here, we have studied the effects of neutralization by site-directed mutagenesis of three of these amino acid side chains, glutamate 373, aspartate 439, and aspartate 454, on the functional properties of the transporter. Transport was analyzed by whole-cell current recording from EAAC1-expressing mammalian cells after applying jumps in voltage, substrate, or cation concentration. Neutralization mutations in positions 373 and 454, although eliminating steady-state glutamate transport, have little effect on the kinetics and thermodynamics of Na(+) and glutamate binding, suggesting that these two positions do not constitute the sites of Na(+) and glutamate association with EAAC1. In contrast, the D439N mutation resulted in an approximately 10-fold decrease of apparent affinity of the glutamate-bound transporter form for Na(+), and an approximately 2,000-fold reduction in the rate of Na(+) binding, whereas the kinetics and thermodynamics of Na(+) binding to the glutamate-free transporter were almost unchanged compared to EAAC1(WT). Furthermore, the D439N mutation converted l-glutamate, THA, and PDC, which are activating substrates for the wild-type anion conductance, but not l-aspartate, into transient inhibitors of the EAAC1(D439) anion conductance. Activation of the anion conductance by l-glutamate was biphasic, allowing us to directly analyze binding of two of the three cotransported Na(+) ions as a function of time and [Na(+)]. The data can be explained with a model in which the D439N mutation results in a dramatic slowing of Na(+) binding and a reduced affinity of the substrate-bound EAAC1 for Na(+). We propose that the bound substrate controls the rate and the extent of Na(+) interaction with the transporter, depending on the amino acid side chain in position 439.

Dataset Information

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

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure