Excitatory amino acid transporters (EAATs) are secondary active, electrogenic transporters which translocate L-glutamate (glu) against its concentration gradient using the co-transport of 3 Na+, 1 H+, and the counter-transport of 1 K+ ion. In addition, these carriers possess a thermodynamically uncoupled anion channel that fluxes Cl- but is promiscuous with several permeant anionic species. The roles of EAATs are to shape the spatio-temporal profile of released glu in both the synaptic cleft and extra-synaptic regions as well as maintaining a low ambient extracellular concentration of glu. This transport activity regulates activation of glu receptors and thus regulates excitatory neurotransmission.
Using a combination of techniques, we were successful in identifying inward oriented transporter conformations which allow transitions to open channels states. This observation was enabled by our development of a novel method to isolate EAAT1 in the inward facing conformation. While constrained to these conformations, currents with the same macroscopic amplitudes as conducting states mediated by the outward facing, Na+ bound states were observed. The persistence of currents is indicative of a channel gating mechanism that is insensitive to transporter orientation and that the anion channel is open during the majority of the transport cycle. Additional conducting states allows for a larger contribution of the anion channel function of EAATs to shape cellular function then previously assumed.
Next we investigated the gating mechanism of the anion channel. We assayed for the ability of Na+ to gate the anion channel in both glial (EAAT1 and EAAT2) and neuronal (EAAT3 and EAAT4) isoforms. We discovered that the glial isoforms are not gated by Na+ but are leak channels with an open probability and single channel conductance that is insensitive to Na+ concentrations. In contrast, neuronal EAAT isoforms EAAT3 and EAAT4 both display Na+ dependent channel activity. This is the first example of a significant functional difference between glial and neuronal transporter isoforms of the solute carrier 1 (SLC1) family. The research presented here allows for a greater understanding of low open probability channel states and the possible contributions of the EAAT anion channel to the functioning of the nervous system
