• Home
  • Search Results
  • Investigating substrate-induced motion between the scaffold and transport domains in the glutamate transporter EAAT1.

Investigating substrate-induced motion between the scaffold and transport domains in the glutamate transporter EAAT1.

Molecular pharmacology (2014-10-01)
Xiuliang Rong, Elia Zomot, Xiuping Zhang, Shaogang Qu
ABSTRACT

Excitatory amino acid transporter 1 plays an important role in keeping the synaptic glutamate concentration below neurotoxic levels by translocating this neurotransmitter into the cell. Both reentrant hairpin loops, HP1 and -2, have been shown to take part in binding the substrate and the more deeply buried sodium ion, and might therefore be a part of the intra- or extracellular gate of the transporter. However, the shape of the motion of either loop relative to transmembrane domain (TM) 4 during the transport cycle has not yet been fully resolved. Using copper(II) (1,10-phenanthroline)3 (CuPh) for cross-linking cysteine pairs, we found strong inhibition of transport when A243C (TM4) was combined with S366C (HP1), I453C (HP2), or T456C (HP2). These findings were reinforced by the impact of cadmium on transport activity, and both approaches consistently showed that proximity was exclusively intramonomeric. Under conditions that promote the inward-facing state, inhibition by CuPh in A243C/S366C was reduced, while the opposite was seen when the outward-facing one was stabilized, suggesting that the two positions are farther apart in the former conformation than in the latter. Surprisingly, maximal cross-linking of A243C with I453C or T456C was not observed under conditions that promote the inward-facing state. Altogether, our data suggest that the transporter may undergo complex relative movement between these positions on TM4 and HP1/HP2 during the transport cycle.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
L-Glutamic acid, ReagentPlus®, ≥99% (HPLC)
Sigma-Aldrich
L-Aspartic acid, reagent grade, ≥98% (HPLC)
Sigma-Aldrich
L-Glutamic acid, from non-animal source, meets EP testing specifications, suitable for cell culture, 98.5-100.5%
Sigma-Aldrich
L-Glutamic acid, BioUltra, ≥99.5% (NT)
Sigma-Aldrich
L-Aspartic acid, from non-animal source, meets EP, USP testing specifications, suitable for cell culture, 98.5-101.0%
Sigma-Aldrich
L-Aspartic acid, BioXtra, ≥99% (HPLC)
Sigma-Aldrich
Cadmium, powder, −100 mesh, 99.5% trace metals basis
Sigma-Aldrich
DL-Aspartic acid, ≥99% (TLC)
Sigma-Aldrich
L-Aspartic acid potassium salt, ≥98% (HPLC)
Supelco
L-Glutamic acid, Pharmaceutical Secondary Standard; Certified Reference Material
Sigma-Aldrich
D-Glutamic acid, ≥99% (TLC)
SAFC
L-Aspartic acid
Glutamic acid, European Pharmacopoeia (EP) Reference Standard
Sigma-Aldrich
Cadmium, granular, 30-80 mesh, ≥99%
Sigma-Aldrich
L-Aspartic acid, BioUltra, ≥99.5% (T)
Sigma-Aldrich
Cadmium, granular, ≥99%, 5-20 mesh
Sigma-Aldrich
L-Glutamic acid hydrochloride, ≥99% (HPLC)
Sigma-Aldrich
L-Glutamic acid, FCC
Supelco
L-Aspartic acid, Pharmaceutical Secondary Standard; Certified Reference Material
Aspartic acid, European Pharmacopoeia (EP) Reference Standard
Supelco
L-Glutamic acid, certified reference material, TraceCERT®
Sigma-Aldrich
L-Aspartic acid, ≥98%, FG
Supelco
L-Aspartic acid, certified reference material, TraceCERT®
Cadmium, foil, 100x100mm, thickness 1.0mm, as rolled, 99.99%
Cadmium, foil, 4mm disks, thickness 0.25mm, as rolled, 99.99+%
Cadmium, wire reel, 0.1m, diameter 0.70mm, as drawn, 99.99+%
Cadmium, wire reel, 0.1m, diameter 1.2mm, as drawn, 99.99+%
Cadmium, wire reel, 0.1m, diameter 2.0mm, hard, 99.99+%
Cadmium, wire reel, 0.1m, diameter 1.0mm, hard, 99.999%
Cadmium, wire reel, 0.2m, diameter 0.25mm, hard, 99.99+%