Figure 5.

Mapping TARP γ-2 and TARP γ-8 Residues Involved in AMPAR Interaction

(A) Alignment of rat type 1a (γ-2, γ-3) and type 1b (γ-4, γ-8) TARP extracellular loops, Ex1 and Ex2. Conserved residues are shaded brown, and residues highly conserved throughout the Cacng family (γ-1 to γ-8) are boxed in gray. The four cysteines are highlighted in yellow. Curly brackets above the γ-2 (green) and below γ-8 alignment (blue) indicate regions in the center of Ex1 interacting with the AMPAR extracellular domains (NTD and LBD).

(B) TARP array encompassing the Ex1 and Ex2 segments of γ-2 (green box) and γ-8 (blue box) probed with the NTD. (Upper panel) Nonspecific signal, resulting from direct anti-GluA2 antibody binding to the membrane (AB control). The Ex1 and Ex2 regions for both TARPs are denoted (dashed line). (Lower panel) the same membrane was exposed to the rat GluA2 NTD followed by probing with anti-GluA2 AB. The membrane exposure time is as indicated.

See Table S2 for peptide sequences.

(C) γ-2 and γ-8 arrays probed with GluA2 LBD and GluK2 LBD. The negative controls with anti-FLAG AB did not show any signal and thus are not shown. Membranes were then incubated with FLAG-tagged GluA2 LBD or GluK2 LBD and probed with anti-FLAG AB. (Upper panel) GluA2 LBD interaction with γ-2 (same peptides as in Figure 5B, green box). (Central panel) GluA2 LBD interaction with γ-8 (same peptides as in B, blue box). (Lower panel) the γ-2 membrane previously probed with GluA2 LBD (top panel) was regenerated and probed with the FLAG-tagged GluK2 LBD, which produced no clear binding. Regeneration of this membrane resulted in a clear binding pattern when reprobed with the GluA2 LBD that matched the one shown in (C, top).

(D) Schematic representation of TARP structure with the GluA2 NTD and LBD interacting parts of the Ex1 and Ex2 loops highlighted in orange and the highly conserved GLWR motif indicated.