title: 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.
authors.affiliation: Department of Chemistry, Binghamton University, Binghamton, NY 13902, USA.
authors.forename: Juddy
authors.initials: J
authors.lastname: Mwaura
authors.forename: Zhen
authors.initials: Z
authors.lastname: Tao
authors.forename: Herbert
authors.initials: H
authors.lastname: James
authors.forename: Thomas
authors.initials: T
authors.lastname: Albers
authors.forename: Alexander
authors.initials: A
authors.lastname: Schwartz
authors.forename: Christof
authors.initials: C
authors.lastname: Grewer
mesh_terms: D024342:Amino Acids, Acidic / Q000378:metabolism*; D001692:Biological Transport; D050580:Excitatory Amino Acid Transporter 3 / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D018408:Patch-Clamp Techniques; D012964:Sodium / Q000378:metabolism*
chemical_list: D024342:Amino Acids, Acidic; D050580:Excitatory Amino Acid Transporter 3; D018698:Glutamic Acid; D012964:Sodium