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 ∼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 EAAC1WT. 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 EAAC1D439 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.
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1 April 2007
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March 26 2007
Cooperation of the Conserved Aspartate 439 and Bound Amino Acid Substrate Is Important for High-Affinity Na+ Binding to the Glutamate Transporter EAAC1
Zhen Tao,
Zhen Tao
University of Miami School of Medicine, Miami, FL 33136
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Christof Grewer
Christof Grewer
University of Miami School of Medicine, Miami, FL 33136
Search for other works by this author on:
Zhen Tao
University of Miami School of Medicine, Miami, FL 33136
Christof Grewer
University of Miami School of Medicine, Miami, FL 33136
Correspondence to Christof Grewer: [email protected]
Abbreviations used in this paper: EAAC1, excitatory amino acid carrier 1; EAAT, excitatory amino acid transporter; HEK, human embryonic kidney; PDC, l-trans-2,4-pyrrolidine dicarboxcylic acid; RL, reentrant loop; TBOA, dl-threo-β-benzyloxyaspartate; THA, d,l-threo-β-hydroxyaspartic acid; TM, transmembrane domain.
Received:
October 10 2006
Accepted:
March 07 2007
Online ISSN: 1540-7748
Print ISSN: 0022-1295
The Rockefeller University Press
2007
J Gen Physiol (2007) 129 (4): 331–344.
Article history
Received:
October 10 2006
Accepted:
March 07 2007
Citation
Zhen Tao, Christof Grewer; Cooperation of the Conserved Aspartate 439 and Bound Amino Acid Substrate Is Important for High-Affinity Na+ Binding to the Glutamate Transporter EAAC1 . J Gen Physiol 1 April 2007; 129 (4): 331–344. doi: https://doi.org/10.1085/jgp.200609678
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