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@ARTICLE{Kolen:884257,
author = {Kolen, Bettina and Kortzak, Daniel and Franzen, Arne and
Fahlke, Christoph},
title = {{A}n amino-terminal point mutation increases {EAAT}2 anion
currents without affecting glutamate transport rates},
journal = {The journal of biological chemistry},
volume = {295},
number = {44},
issn = {1083-351X},
address = {Bethesda, Md.},
publisher = {Soc.72889},
reportid = {FZJ-2020-03152},
pages = {14936-14947},
year = {2020},
abstract = {Excitatory amino acid transporters (EAATs) are prototypical
dual function proteins that function as coupled
glutamate/Na+/H+/K+ transporters and as anion-selective
channels. Both transport functions are intimately
intertwined at the structural level: Secondary active
glutamate transport is based on elevator-like movements of
the mobile transport domain across the membrane, and the
lateral movement of this domain results in anion channel
opening. This particular anion channel gating mechanism
predicts the existence of mutant transporters with changed
anion channel properties, but without alteration in
glutamate transport. We here report that the L46P mutation
in the human EAAT2 transporter fulfills this prediction. L46
is a pore-forming residue of the EAAT2 anion channels at the
cytoplasmic entrance into the ion conduction pathway. In
whole-cell patch clamp recordings, we observed larger
macroscopic anion current amplitudes for L46P than for WT
EAAT2. Rapid l-glutamate application under forward transport
conditions demonstrated that L46P does not reduce the
transport rate of individual transporters. In contrast,
changes in selectivity made gluconate permeant in L46P
EAAT2, and nonstationary noise analysis revealed slightly
increased unitary current amplitudes in mutant EAAT2 anion
channels. We used unitary current amplitudes and individual
transport rates to quantify absolute open probabilities of
EAAT2 anion channels from ratios of anion currents by
glutamate uptake currents. This analysis revealed up to
7-fold increased absolute open probability of L46P EAAT2
anion channels. Our results reveal an important determinant
of the diameter of EAAT2 anion pore and demonstrate the
existence of anion channel gating processes outside the EAAT
uptake cycle.},
cin = {IBI-1},
ddc = {540},
cid = {I:(DE-Juel1)IBI-1-20200312},
pnm = {552 - Engineering Cell Function (POF3-552)},
pid = {G:(DE-HGF)POF3-552},
typ = {PUB:(DE-HGF)16},
pubmed = {32820048},
UT = {WOS:000588414100012},
doi = {10.1074/jbc.RA120.013704},
url = {https://juser.fz-juelich.de/record/884257},
}