A Conserved Aspartate Determines Pore Properties of Anion Channels Associated with Excitatory Amino Acid Transporter 4 (EAAT4)

David Ewers,Jan-Philipp Machtens,Peter Kovermann,Christoph Fahlke

doi:10.1074/jbc.m110.126557

Abstract

Excitatory amino acid transporter (EAAT) glutamate transporters function not only as secondary active glutamate transporters but also as anion channels. Recently, a conserved aspartic acid (Asp(112)) within the intracellular loop near to the end of transmembrane domain 2 was proposed as a major determinant of substrate-dependent gating of the anion channel associated with the glial glutamate transporter EAAT1. We studied the corresponding mutation (D117A) in another EAAT isoform, EAAT4, using heterologous expression in mammalian cells, whole cell patch clamp, and noise analysis. In EAAT4, D117A modifies unitary conductances, relative anion permeabilities, as well as gating of associated anion channels. EAAT4 anion channel gating is characterized by two voltage-dependent gating processes with inverse voltage dependence. In wild type EAAT4, external l-glutamate modifies the voltage dependence as well as the minimum open probabilities of both gates, resulting in concentration-dependent changes of the number of open channels. Not only transport substrates but also anions affect wild type EAAT4 channel gating. External anions increase the open probability and slow down relaxation constants of one gating process that is activated by depolarization. D117A abolishes the anion and glutamate dependence of EAAT4 anion currents and shifts the voltage dependence of EAAT4 anion channel activation by more than 200 mV to more positive potentials. D117A is the first reported mutation that changes the unitary conductance of an EAAT anion channel. The finding that mutating a pore-forming residue modifies gating illustrates the close linkage between pore conformation and voltage- and substrate-dependent gating in EAAT4 anion channels.

Highlights

Whereas key processes underlying glutamate transport have been identified in recent years (8 –15), molecular determinants of the Excitatory amino acid transporter (EAAT) anion conductance still need to be clarified
We took a similar approach on EAAT anion channels and focused on a point mutation that was first reported by Vandenberg and co-workers [19]
Internal dialysis with Naϩ-based internal solutions and the use of NO3Ϫ as main internal and external anion resulted in EAAT4 anion currents that are significantly larger than uptake currents and endogenous currents and permitted measurements of EAAT4 anion currents in isolation [5]

Summary

EXPERIMENTAL PROCEDURES

Heterologous Expression of WT and Mutant EAAT4 in Mammalian Cells—pcDNA3.1(Ϫ) rEAAT4 was kindly provided by Dr J. More than 80% of the series resistance was compensated by an analog procedure, and cells with current amplitudes of Ͼ12 nA were excluded from analysis. Relative open probabilities were calculated from instantaneous tail current amplitudes at ϩ135 or Ϫ135 mV after 200-ms steps of variable voltages. Anion permeability sequences for WT EAAT4 and D117A EAAT4 were obtained from measurements with the pipette solution containing 110 mM NaCl, 2 mM MgCl2, 5 mM EGTA, 10 mM HEPES, pH 7.4, in the presence of saturating external L-glutamate (100 ␮M). Reversal potentials were determined from resulting current-voltage relationships, and permeability ratios were calculated from Equation 1. Permeability ratios for divalent anions were calculated from Equation 2

Erev ϭ

RESULTS

DISCUSSION