Glutamate Translocation of the Neuronal Glutamate Transporter EAAC1 Occurs within Milliseconds

• Published in: Proceedings of the National Academy of Sciences - PNAS 2000-08, Vol.97 (17), p.9706-9711
• Main Authors: Grewer, Christof, Watzke, Natalie, Wiessner, Michael, Rauen, Thomas
• Format: Article
• Language: English
• Subjects: Amino Acid Transport System X-AG; Amino acid transport system XAG; Animals; Anions; Anions - metabolism; Biological Sciences; Biological Transport - drug effects; Carrier Proteins - genetics; Carrier Proteins - metabolism; Cell Line; Cell Membrane - drug effects; Cell Membrane - metabolism; Electric Conductivity; Electric current; Electric potential; Excitatory Amino Acid Transporter 3; Glutamate Plasma Membrane Transport Proteins; Glutamic Acid - metabolism; Glutamic Acid - pharmacology; HEK293 cells; Humans; Ions; Kinetics; Lasers; Molecular biology; Neurology; Neurons - chemistry; Photolysis; Photolysis - radiation effects; Potassium - metabolism; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Response time; Retina; Sodium - metabolism; Steady state current; Symporters; Time constants; Transfection
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.160170397

The activity of glutamate transporters is essential for the temporal and spatial regulation of the neurotransmitter concentration in the synaptic cleft, and thus, is crucial for proper excitatory signaling. Initial steps in the process of glutamate transport take place within a time scale of microseconds to milliseconds. Here we compare the steady-state and pre-steady-state kinetics of the neuronal heterologously expressed glutamate transporter EAAC1, cloned from the mammalian retina. Rapid transporter dynamics, as measured by using whole-cell current recordings, were resolved by applying the laser-pulse photolysis technique of caged glutamate with a time resolution of 100 μ s. EAAC1-mediated pre-steady-state currents are composed of two components: A transport current generated by substrate-coupled charge translocation across the membrane and an anion current that is not stoichiometrically coupled to glutamate transport. The two currents were temporally resolved and studied independently. Our results indicate a rapid glutamate-binding step occurring on a submillisecond time scale that precedes subsequent slower electrogenic glutamate translocation across the membrane within a few milliseconds. The voltage-dependent steady-state turnover time constant of the transporter is about 1/10 as fast, indicating that glutamate translocation is not rate limiting. A third process, the transition to an anion-conducting state, is delayed with respect to the onset of glutamate transport. These rapid transporter reaction steps are summarized in a sequential shuttle model that quantitatively accounts for the results obtained here and are discussed regarding their functional importance for glutamatergic neurotransmission in the central nervous system.