Figure 8.

Additional deletions of GluN1-ATD subdomains support an epitope located near the hinge of the top and bottom lobes and reveal a degree of antibody heterogeneity. A–C, Binding of patients' antibodies is blocked by deletion of an α-helix spanning residues 144–156, which is near N368/G369. Deletion of the top lobe (top del) of the ATD, however, has variable effects. It can preserve antibody staining at levels close to that of wild-type (A), destroy antibody staining (B), or increase antibody staining (C). D, Deletion of residues 144–156 uniformly destroys antibody binding (n = 4 patients, ****p < 0.0001 vs wild-type, Student's t test), while deletion of the top lobe has different effects on different patients (n = 15 patients, p > 0.05 vs wild-type, Student's t test). E, Individual staining intensities of ATD top lobe deletion versus wild-type. F, Model of ATD with proposed antibody binding site and effect of G369 mutations. Open-cleft conformation of the ATD both allows antibody binding and leads to channel opening, and the nearby α-helix spanning residues 144–156 also seems to play a role. Antibody binding to this open conformation then stabilizes the conformation and prolongs open time. The proximity of the top lobe of the ATD makes it likely that a small shift in epitope location could result in the variable staining patterns of the ATD top lobe deletion mutants. G, Substitution of larger residues at position 369 block antibody binding and promote a closed-cleft conformation that keeps the channel closed.