Sulfhydryl Modification Of Cysteine Substitutions In The Second Hairpin Loop (HP2) Alters The Ion Permeation Properties Of The Glutamate Transporter, EAAT1

Abstract

In the mammalian CNS, excitatory amino acid transporters (EAATs) limit glutamatergic signalling and regulate extracellular glutamate concentrations. EAATs have been shown to function not only as secondary active transporters, but also as anion-selective channels that regulate excitability. Under some conditions, EAATs have also been shown to mediate cation-selective conductances. The C-terminal portion of the protein contains two membrane spanning domains (TM7, TM8) and two hairpin loops (HP1, HP2), which are critical for substrate translocation, however, the anion permeation pathway has been linked to additional domains within the N-terminus of the protein (TM2). Application of bulky MTS-reagents to several cysteine substitution mutants in HP2 abolishes glutamate translocation, but preserves the anion conductance, suggesting that the anion permeation and substrate transport pathways are physically separated. Interestingly, MTS-modification of some of these mutants results in substantially greater macroscopic current amplitude. We have used two-electrode voltage clamp in Xenopus oocytes and whole-cell patch-clamp in mammalian cells to determine the basis for the change in ion permeation properties for a series of mutants that exhibit this phenotype. We examined the substrate-gated anion currents associated with one mutant, M451C EAAT1, in Xenopus oocytes and observed a profound time- and voltage-dependent deactivation of the macroscopic current at depolarizing potentials, suggesting that MTS-modification increases channel open probability. After modification, extracellular substitution with the impermeant anion gluconate, resulted in an outward current at positive potentials, suggesting that the current includes a major cation component. We are currently exploring the relationships between the substrate translocation pathway with this novel cation current and the enhanced anion current induced by modification, with the aim of better understanding the molecular determinants involved in EAAT-associated channel gating and permeation.