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J Biol Chem. 2015 Sep 18;290(38):22977-90. doi: 10.1074/jbc.M115.660860. Epub 2015 Jul 22.

A Mutation in Transmembrane Domain 7 (TM7) of Excitatory Amino Acid Transporters Disrupts the Substrate-dependent Gating of the Intrinsic Anion Conductance and Drives the Channel into a Constitutively Open State.

Author information

1
From the National Institute of Mental Health, Bethesda, Maryland 20892 and delany.torressalazar@nih.gov.
2
the Department of Neurobiology, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15213.
3
From the National Institute of Mental Health, Bethesda, Maryland 20892 and.
4
From the National Institute of Mental Health, Bethesda, Maryland 20892 and the Department of Neurobiology, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15213 susan.amara@nih.gov.

Abstract

In the mammalian central nervous system, excitatory amino acid transporters (EAATs) are responsible for the clearance of glutamate after synaptic release. This energetically demanding activity is crucial for precise neuronal communication and for maintaining extracellular glutamate concentrations below neurotoxic levels. In addition to their ability to recapture glutamate from the extracellular space, EAATs exhibit a sodium- and glutamate-gated anion conductance. Here we show that substitution of a conserved positively charged residue (Arg-388, hEAAT1) in transmembrane domain 7 with a negatively charged amino acid eliminates the ability of glutamate to further activate the anion conductance. When expressed in oocytes, R388D or R388E mutants show large anion currents that display no further increase in amplitude after application of saturating concentrations of Na(+) and glutamate. They also show a substantially reduced transport activity. The mutant transporters appear to exist preferentially in a sodium- and glutamate-independent constitutive open channel state that rarely transitions to complete the transport cycle. In addition, the accessibility of cytoplasmic residues to membrane-permeant modifying reagents supports the idea that this substrate-independent open state correlates with an intermediate outward facing conformation of the transporter. Our data provide additional insights into the mechanism by which substrates gate the anion conductance in EAATs and suggest that in EAAT1, Arg-388 is a critical element for the structural coupling between the substrate translocation and the gating mechanisms of the EAAT-associated anion channel.

KEYWORDS:

chloride channel; glutamate; glutamate transporter; neurotransmitter; neurotransmitter transport; synaptic transmission; voltage-dependent anion channel (VDAC)

PMID:
26203187
PMCID:
PMC4645584
DOI:
10.1074/jbc.M115.660860
[Indexed for MEDLINE]
Free PMC Article

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