This inhibition was absent from WT GluA2 in equivalent conditions (Figure 6D). The lack of inhibition at high glutamate concentration was not due to chelation of zinc into zinc-glutamate complexes because we still observed robust inhibition when the patches were washed with 1 μM zinc, 500 μM L-glutamate, and 9,500 μM D-glutamate, which barely activates the receptor but should chelate zinc equally well. The glutamate dependence of trapping was similar to that of the A665C mutant, with a maximum extent of trapping at 348 μM glutamate (Figure 6E). Thus, the zinc-binding site created by the HHH mutant, which was suggested by

the crosslinked crystal structure, selleck chemicals also traps a partially bound state and does so with more specificity than the A665C mutant. Structural modeling of the HHH mutant built using our LBD tetramer structure, where only residues 436–440 and 455–457 (those flanking the HHH substitutions) were repositioned using energy minimization, shows that a zinc Selleckchem Forskolin ion can be cradled by the three histidines (Figure 6F). The repositioned residues all lie in loop regions, and the rmsd measured at the Cα atoms of these residues is only 0.75 Å. These observations constitute strong evidence that the crystallized CA conformation occurs in the full-length receptor when some, but

not all, of the ligand-binding sites are occupied by glutamate. Structural modeling was pursued to examine possible consequences of OA-to-CA

conformational transitions that occur in conjunction with LBD closure in subunits B and D on ion channel pore opening. First, the closure of subunits B and D in the crystal structure of the crosslinked LBD tetramer was modeled by superimposing the structure of a closed, glutamate-bound LBD (PDB ID 1FTJ; chain A) (Armstrong and Gouaux, 2000) at helices D and J in lobe 1. Next, the TMD from the full-length GluA2 crystal structure was allowed to relax energetically to accommodate the LBDs (Figure S7). In this model, the inner transmembrane helices (M3) are predicted to widen at the Linifanib (ABT-869) ion channel gate between subunits B and D by ∼11 Å, as measured between Cα atoms of T625. It should be noted that NMA was attempted with both the full-length GluA2 structure and the isolated TMD, but the ion channel gate could not be opened in either case, likely due to the tight network of residues around the gate. Over 80 crystal structures of the isolated GluA2 LBD have been reported to date (Pøhlsgaard et al., 2011). These structures, in concert with biochemical and biophysical experiments, and molecular simulation studies, have characterized the processes of ligand binding and domain closure, which are directly linked to receptor activation (Armstrong and Gouaux, 2000 and Dong and Zhou, 2011). Less is known, however, about the possible intersubunit conformational rearrangements in iGluR tetramers that could underlie ion channel gating.