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.