Abstract
EAAT glutamate transporters do not only function as secondary-active glutamate transporters but also as anion channels. EAAT anion channel activity depends on transport substrates. For most isoforms, it is negligible without external Na(+) and increased by external glutamate. We here investigated gating of EAAT4 anion channels with various cations and amino acid substrates using patch clamp experiments on a mammalian cell line. We demonstrate that Li(+) can substitute for Na(+) in supporting substrate-activated anion currents, albeit with changed voltage dependence. Anion currents were recorded in glutamate, aspartate, and cysteine, and distinct time and voltage dependences were observed. For each substrate, gating was different in external Na(+) or Li(+). All features of voltage-dependent and substrate-specific anion channel gating can be described by a simplified nine-state model of the transport cycle in which only amino acid substrate-bound states assume high anion channel open probabilities. The kinetic scheme suggests that the substrate dependence of channel gating is exclusively caused by differences in substrate association and translocation. Moreover, the voltage dependence of anion channel gating arises predominantly from electrogenic cation binding and membrane translocation of the transporter. We conclude that all voltage- and substrate-dependent conformational changes of the EAAT4 anion channel are linked to transitions within the transport cycle.
Highlights
23780 JOURNAL OF BIOLOGICAL CHEMISTRY anion currents represent the predominant transporter-mediated current component [7,8,9,10]
To further probe the existence of such conformational changes, we studied the substrate dependence of EAAT4 anion channels by comparing channel gating in Naϩ and Liϩ as well as in glutamate, aspartate, and cysteine
We analyzed EAAT4 anion currents under subsequent perfusion of cells with various amino acid substrates and cations and tested whether changes in anion channel gating could be explained in terms of variable interactions of the different substrates with the transporter
Summary
23780 JOURNAL OF BIOLOGICAL CHEMISTRY anion currents represent the predominant transporter-mediated current component [7,8,9,10]. Parameters were estimated by fitting the model simultaneously to experimentally determined relative open probabilities, time courses of current relaxations, concentration-response curves with Naϩ or Liϩ as well as with glutamate, aspartate, or cysteine. EAAT4 anion currents in the presence of other amino acid substrates, L-aspartate or L-cysteine, in combination with external Naϩ or Liϩ.
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