Transport direction determines the kinetics of substrate transport by the glutamate transporter EAAC1.
Glutamate transport by the excitatory amino acid carrier EAAC1 is known to be reversible. Thus, glutamate can either be taken up into cells, or it can be released from cells through reverse transport, depending on the electrochemical gradient of the co- and countertransported ions. However, it is unknown how fast and by which reverse transport mechanism glutamate can be released from cells. Here, we determined the steady- and pre-steady-state kinetics of reverse glutamate transport with submillisecond time resolution. First, our results suggest that glutamate and Na(+) dissociate from their cytoplasmic binding sites sequentially, with glutamate dissociating first, followed by the three cotransported Na(+) ions. Second, the kinetics of glutamate transport depend strongly on transport direction, with reverse transport being faster but less voltage-dependent than forward transport. Third, electrogenicity is distributed over several reverse transport steps, including intracellular Na(+) binding, reverse translocation, and reverse relocation of the K(+)-bound EAAC1. We propose a kinetic model, which is based on a "first-in-first-out" mechanism, suggesting that glutamate association, with its extracellular binding site as well as dissociation from its intracellular binding site, precedes association and dissociation of at least one Na(+) ion. Our model can be used to predict rates of glutamate release from neurons under physiological and pathophysiological conditions.
Zhang
Zhou
Z
University of Miami School of Medicine, 1600 NW 10th Avenue, Miami, FL 33136, USA.
Tao
Zhen
Z
Gameiro
Armanda
A
Barcelona
Stephanie
S
Braams
Simona
S
Rauen
Thomas
T
Grewer
Christof
C
D001692:Biological Transport; D002460:Cell Line; D050580:Excitatory Amino Acid Transporter 3 / Q000378:metabolism*; D018698:Glutamic Acid / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008962:Models, Theoretical
D050580:Excitatory Amino Acid Transporter 3; C493400:SLC1A1 protein, human; D018698:Glutamic Acid