Is ASP-454 Necessary for Na+ and K+ Binding in the Glutamate Transporter EAAC1?

Abstract

The glutamate transporter Excitatory Amino Acid Carrier 1 (EAAC1) catalyzes rapid removal of glutamate, the major excitatory neurotransmitter in the brain, from the extracellular space, preventing its accumulation to neurotoxic levels. Uphill substrate transport is coupled to co-transport of 3 Na+, 1 H+, and counter-transport of 1 K+ ion. The cation binding sites are yet to be fully identified in this transporter. Here, we investigate the role of the highly conserved D454 residue which is thought to contribute its side chain to coordinating these cations. D454 was mutated to the non-ionizable asparagine, as well as alanine. K+-induced relocation was impaired in EAAC1-D454N, as the transporter no longer catalyzed forward transport. Our findings indicated that K+ could still bind to EAAC1-D454N, but with reduced apparent affinity. Na+/glutamate exchange was functional in EAAC1-D454N, as demonstrated by the presence of transient currents following rapid glutamate application and voltage jumps. In the D454A mutant, glutamate application inhibited inward leak anion currents, and transient currents in response to voltage jumps were abolished. This is consistent with Na+ binding being impaired in this transporter. K+ binding was also eliminated by the D454A mutation. Although D454 is proposed to participate in Na+ binding, its protonation state is yet to be established. pKa calculations for the bacterial homologue Gltph indicate that D405 (analogous to D454) is protonated at physiological pH with a highly perturbed pKa in the range of 7.6 to >14. Valences calculated using the Poison-Boltzmann equation for EAAC1 with D454 protonated (0.45) were close in value to the experimental values obtained for EAAC1-D54N (0.71) and EAAC1-WT (0.64). Together, our results suggest that D454 is protonated under physiological conditions, and participates in Na+ as well as K+ binding.