Fig. 2: Glutamate binding site of the hEAAT2.
From: Structural basis of ligand binding modes of human EAAT2
a Overall structure of the hEAAT2Glu homotrimer with glutamate (colored spheres) viewed from the membrane plane. b Slice through the molecular surface of a single protomer of hEAAT2Glu showing glutamate (sticks) and its EM density (blue mesh). c Detailed binding site for glutamate (yellow) with its key interacting residues (sticks). d 2D plot representing the interactions of glutamate with its surrounding residues by LigPlot+. e Comparison of the glutamate-related current densities between the tested mutants and wild-type hEAAT2. Currents were recorded at 1000 μM glutamate and presented after Na+ dependent leak current was subtracted. Sample sizes (n) tested for wild-type hEAAT2, D475ATM8, or R478ATM8 are 5, 4, 5 cells. One-way ANOVA was followed by the Bonferroni’s post hoc test, ****P < 0.0001. f WAY-213613 dose-response relationships for the D475ATM8, R478ATM8, or S441GHP2 mediated currents. WAY-213613 was varied at the following concentrations: 0.3 μM, 1 μM, 3 μM, 10 μM, 30 μM, and 100 μM for D475ATM8 and R478ATM8; 0.03 μM, 0.1 μM, 0.3 μM, 1 μM, 3 μM, 10 μM, and 30 μM for S441GHP2. Currents were normalized to the maximal current recorded after application of 30 μM or 100 μM WAY-213613. Sample sizes (n) tested from low to high concentrations are listed as follows: n = 4, 5, 5, 5, 5, and 9 cells for D475ATM8; n = 5, 5, 5, 4, 5, and 12 cells for R478ATM8; n = 4, 4, 4, 5, 5, 4, and 7 cells for S441GHP2. The lines represent the best fits to a Michaelis-Menten-like equation. The data of wild-type hEAAT2 from Fig. 1b was compared with those of the tested mutants. g Structural comparison of the transport domains between hEAAT2Glu (pale green) and hEAAT3Apo (PDB ID: 6X3F, gray). h Glutamate dose-response relationship for the S441GHP2 mediated currents at the varied concentrations of 0.1 μM, 0.3 μM, 1 μM, 3 μM, 10 μM, and 30 μM. The lines represent the best fit to a Michaelis–Menten-like equation with an average apparent Km of 0.40 ± 0.03 μM. Currents were normalized to the maximal current recorded after the application of 30 μM glutamate. Sample sizes (n) tested from low to high concentrations are 4, 5, 5, 5, 12, and 5 cells. In the figures, e, f and h, all experiments were executed at 0 mV and the data were presented as mean values ± SD.
