Glutamate translocation of the neuronal glutamate transporter EAAC1 occurswithin milliseconds

The activity of glutamate transporters is essential for the temporal and sp atial regulation of the neurotransmitter concentration in the synaptic clef t, and thus, is crucial for proper excitatory signaling, Initial steps in t he process of glutamate transport take place within a time scale of microse conds to milliseconds. Here we compare the steady-state and pre-steady-stat e kinetics of the neuronal heterologously expressed glutamate transporter E AAC1, cloned from the mammalian retina. Rapid transporter dynamics, as meas ured by using whole-cell current recordings, were resolved by applying the laser-pulse photolysis technique of caged glutamate with a time resolution of 100 mu s. EAAC1-mediated pre-steady-state currents are composed of two c omponents: A transport current generated by substrate-coupled charge transl ocation across the membrane and an anion current that is not stoichiometric ally coupled to glutamate transport. The two currents were temporally resol ved and studied independently. Our results indicate a rapid glutamate-bindi ng step occurring on a submillisecond time scale that precedes subsequent s lower 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 translocat ion is not rate limiting. A third process, the transition to an anion-condu cting state, is delayed with respect to the onset of glutamate transport. T hese rapid transporter reaction steps are summarized in a sequential shuttl e model that quantitatively accounts for the results obtained here and are discussed regarding their functional importance for glutamatergic neurotran smission in the central nervous system.