Protonation State of a Conserved Acidic Amino Acid Involved in Na⁺ Binding to the Glutamate Transporter EAAC1

Abstract

Substrate transport by glutamate transporters is coupled to the co-transport of 3 Na + ions and counter-transport of 1 K + ion. The highly conserved Asp454, which may be negatively charged, is of interest as its side chain may coordinate cations and/or contribute to charge compensation. Mutation to the nonionizable Asn resulted in a transporter that no longer catalyzed forward transport. However, Na + /glutamate exchange was still functional, as demonstrated by the presence of transient currents following rapid substrate application and voltage jumps. While the kinetics of Na + / glutamate exchange were slowed, the apparent valence (z) of the charge moved in EAAC1 D454N (0.71) was similar to that of EAAC1 WT (0.64). Valences calculated using the Poisson−Boltzmann equation were close to the experimental values for EAAC1 D454N (0.55), and with D454 protonated (0.45). In addition, pK a calculations performed for the bacterial homologue GltPh revealed a highly perturbed pK a (7.6 to >14) for D405 residue (analogous to D454), consistent with this site being protonated at physiological pH. In contrast to the D454N mutation, substitution to alanine resulted in a transporter that still bound glutamate, but could not translocate it. The results are consistent with molecular dynamics simulations, showing that the alanine but not the asparagine mutation resulted in defective Na + coordination. Our results raise the possibility that the protonated state of D454 supports transporter function.

SLC1 glutamate transporters

“…Consistent with the charge compensation mechanism described in the previous paragraph, inward charge movement was observed in response to rapid glutamate concentration jumps, with an apparent valence of about +0.45, consistent with Poisson-Boltzmann calculations of the valence of +0.2 to +0.4, depending on the protonation state of the highly-conserved D454 residue [72,133]. Interestingly, transient currents in response to glutamate concentration jumps decay with two exponential components [128,219], both of which are also present when the transporter is restricted to access states associated with translocation, in the Na + /glutamate-exchange mode.…”

“…1C, in which substrate/cation binding/release steps are coordinated with conformational changes to allow for the proposed alternating access functionality. Interestingly, voltage dependence appears to be distributed over many different binding/translocation steps [72,133], including Na + dissociation on the intracellular side [231], resulting in a relatively shallow voltage dependence of steady state glutamate transport, considering the large number of positive charges co-transported with glutamate (3 Na + , 1 H + ). If these charges were transported across the membrane in a single step, transport would be strongly inhibited upon depolarization.…”

Electrogenic Steps Associated with Substrate Binding to the Neuronal Glutamate Transporter EAAC1

“…6, D and E, position of A334E indicated by arrows, membrane potential color-coded from 0 mV (blue) to Ϫ100 mV (red)). Whereas the valence associated with this transition is positive for the wildtype transporter (18), as determined from experiments as well as computation (z ϭ ϩ0.15; Fig. 6F), the computed valence for the mutant transporter is large and negative (z ϭ Ϫ0.71 (Fig.…”

“…Therefore, series resistance was not compensated. For voltage jump experiments, series resistance compensation of 40 -50% was applied in order to accelerate the capacitive charging of the membrane in response to the changes of the membrane potential (18).…”

“…2, A and B) based on structural models of the open loop, glutamate-free and closed loop, glutamate-bound configurations ( Fig. 2C; based on EAAC1 homology models generated using Glt Ph as a template (18,27)). Solving the Poisson-Boltzmann equation allows the computation of the electrostatic energy of the two states (open loop and closed loop/glutamate-bound), which yields the valence of the transition, when computed as a function of the membrane potential ( Fig.…”