Modulation of anion channel gating by C-terminal domains in excitatory amino acid transporters

Authors

• SG Amara
• D Torres-Salazar

Abstract

Excitatory Amino Acid Transporters (EAATs) clear synaptically-released glutamate and thus limit the duration and extent of excitatory signaling in the CNS. EAATs possess two functions: they couple glutamate transport to the inward movement of sodium ions and they also regulate cellular excitability through an intrinsic substrate-gated anion channel activity. Although the critical elements of the transport mechanism are known, the relationship between substrate translocation and channel gating remains unclear. We constructed a computational model of EAAT4 including the cytoplasmic C-terminus that suggests potential interactions between charged residues in the C-terminus and in a conserved region of transmembrane domain 3 (TM3). Using electrophysiological recordings of EAAT4 constructs expressed in Xenopus oocytes, we found that truncation of the C-terminus alters channel gating, drives the channel into an open state and disrupts glutamate transport, supporting a role for C-terminal residues in the structural coupling between substrate transport and anion channel opening. In addition, mutations in either TM3 or the C-terminus alter anion permeability ratio suggesting that both domains influence permeation. We next designed cell permeable-peptides that correspond to both regions and applied different concentrations of either peptide to oocytes expressing the full-length WT EAAT4. Application of either peptide to EAAT4-exressing oocytes yielded results identical to the phenotype observed with the full C-terminal truncation: glutamate-independent currents consistent with an open channel state and a reduced transport activity. These studies indicate that the C-terminus and its interaction with TM3 have a major influence on the complex structural dynamics that regulate anion channel gating and permeation.

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