Glutamate Transporter-Associated Anion Channels

Abstract

Excitatory amino acid transporters (EAATs) do not only mediate secondary-active glutamate transport, but also anion-selective currents. EAAT anion currents are small in the absence and increase upon application of L-glutamate due to substrate-dependent gating of EAAT anion channels. Anion channel gating can be described by a kinetic scheme that is based on the glutamate transport cycle and in which anion channel opening is associated with certain states. All voltage- and substrate-dependent conformational changes of EAAT4 anion channels are linked to transitions within the transport cycle, and there are no indications for additional substrate- or voltage-dependent anion channel opening and closing transitions. To account for the substrate dependence of macroscopic currents different transporter states might either exhibit distinct unitary conductances or distinct anion channel open probabilities. Macroscopic current recordings and noise analysis revealed a single channel conductance of 1 pS at symmetrical NO3- for EAAT4 anion channels in the absence as well as in the presence of glutamate (Kovermann et al. (2010) J Biol Chem 285:23676-23686). Stochastic simulations and noise analysis of simulated currents demonstrated that noise analysis is indeed able to distinguish between variable unitary current amplitudes and open probabilities (Machtens et al. Channels (in press)). Our findings indicate that open states branch from the transport cycle, and that - to ensure detailed balance - the uptake cycle does not progress as long the channel is open, resulting in a switch between transport and anion conduction mode. This model correctly describes alterations of anion currents upon variation of voltage and substrate concentrations. It furthermore predicts that conditions that modify the EAAT anion channel open probability modify apparent rate constants in the uptake cycle. To test this prediction we currently employ voltage-clamp fluorometry on EAATs carrying mutations that modify opening/closing transitions of EAAT anion channels.