Cooperation of the Conserved Aspartate 439 and Bound Amino Acid Substrate Is Important for High-Affinity Na~+ Binding to the Glutamate Transporter EAAC1

摘要

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 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.