Isolation of glutamate transport-coupled charge flux and estimation of glutamate uptake at the climbing fiber-Purkinje cell synapse

Excitatory amino acid transporters (EAATs) located on neurons and glia are responsible for limiting extracellular glutamate concentrations, but specific contributions made by neuronal and glial EAATs have not been determined. At climbing fiber to Purkinje cell (PC) synapses in cerebellum, a fraction of released glutamate is rapidly bound and inactivated by neuronal EAATs located on postsynaptic PCs. Because transport involves a stoichiometric movement of ions and is electrogenic, postsynaptic currents mediated by EAATs should permit precise calculation of the amount of postsynaptic glutamate uptake. However, this is possible only if a stoichiometric EAAT current can be isolated from all other contaminating signals. We used synaptic stimulation and photolysis of caged glutamate to characterize the current in PCs that is resistant to high concentrations of glutamate receptor antagonists. Some of this response is inhibited by the high-affinity EAAT antagonist TBOA (dl-threo-beta-benzyloxyaspartic acid), whereas the remaining current shows properties inconsistent with glutamate transport. By subtracting this residual non-EAAT current from the response recorded in glutamate receptor antagonists, we have obtained an estimate of postsynaptic uptake near physiological temperature. Analysis of such synaptic EAAT currents suggests that, on average, postsynaptic EAATs take up approximately 1,300,000 glutamate molecules in response to a single climbing fiber action potential.