Volume 96, Issue 5, Pages 989-1001 (December 2017) Getting a Handle on Neuropharmacology by Targeting Receptor-Associated Proteins  Michael P. Maher, Jose A. Matta, Shenyan Gu, Mark Seierstad, David S. Bredt  Neuron  Volume 96, Issue 5, Pages 989-1001 (December 2017) DOI: 10.1016/j.neuron.2017.10.001 Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 1 Strategies for the Discovery of Ion Channel-Associated Proteins (A) Biochemical purification of receptors can identify ion channel-associated proteins (e.g., calcium channel auxiliary subunits). (B) Deconvoluting the mechanism of action of drugs can uncover ion channel components (e.g., sulfonylureas and ATP-sensitive potassium channel [KATP]). (C) Forward genetic screens can identify ion channel auxiliary subunits (e.g., SOL-1 and GLR1). (D) Genomic cloning strategies, either using RNAi or cDNA overexpression (shown here), can isolate receptor-associated proteins (e.g., NACHO for α7 nAChR). Neuron 2017 96, 989-1001DOI: (10.1016/j.neuron.2017.10.001) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 2 Features of AMPAR Modulators Selective for TARP γ-8 (A) Structures of negative modulators selective for TARP γ-8. (B) Outside-out recordings of glutamate-evoked currents in patches from cells expressing human GluA1 fused to human TARP γ-8 or γ-2 in the presence (red) and absence (black) of a saturating concentration of JNJ-55511118 (from Maher et al., 2016). (C) Potency of inhibition for the compounds shown in (A) for key combinations of GluA and TARP subunits and for neurons from the hippocampus and cerebellum. Potency is expressed as pIC50 = −log(IC50[M]). Data were derived from Maher et al. (2016) and Gardinier et al. (2016). Error bars represent SEM. (D) Autoradiogram of a mouse coronal brain slice labeled with [3H]JNJ-56022486 (from Maher et al., 2016). (E) Expression of the CACNG8 gene by in situ hybridization in a coronal section of mouse brain, downloaded from the Allen Mouse Brain Atlas (Lein et al., 2007) (copyright 2015 Allen Institute for Brain Science, http://mouse.brain-map.org/experiment/show/72108823). (F) Sequence alignments for human type 1 TARP transmembrane regions TM3 and TM4. The approximate borders of the transmembrane domains were determined from the cryo-EM structures. Highlights and symbols indicate residues that affect potency (#, cyan) and selectivity (Δ, yellow) of the negative modulators. Also shown are additional residues (○, green) that were identified as potentially lining the binding pocket for JNJ-55511118. Neuron 2017 96, 989-1001DOI: (10.1016/j.neuron.2017.10.001) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 3 Pharmacological Binding Sites in the GluA2 Tetramer Complexed with Four TARPs (A) Structure of rat GluA2 co-expressed with γ-2. This structure was derived from PDB: 5KK2 (Zhao et al., 2016), modified by changing the residues in the transmembrane domain to their corresponding TARP γ-8 forms, and adding the missing side chains. This structure lacks the intracellular domains of the proteins as well as several residues of the TARPs in the extracellular space. Alternating GluA subunits are shown in green and red. Alternating TARP subunits are shown in blue and orange. Known pharmacological binding sites for the agonist, positive allosteric modulator (PAM), and non-competitive antagonist (1-(4-Aminophenyl)-3-methylcarbamyl-4-methyl-3,4-dihydro-7,8-methylenedioxy-5H-2,3-benzodiazepine [GYKI]) are marked (circles). (B) Expanded view of the TARP-GluA interface at the outer edge of the membrane. Residues known to affect selectivity and/or potency are highlighted in yellow. The stick figure shows the structure of JNJ-55511118 in the putative binding pocket. (C) Same as (B), rotated 90° to view from the pore region with the GluA subunits removed. The side chains of three tyrosines that line the putative binding pocket are shown (sticks). Neuron 2017 96, 989-1001DOI: (10.1016/j.neuron.2017.10.001) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 4 Assembly and Trafficking of nAChRs Unassembled nAChRs are susceptible to ER associated degradation (ERAD.) This process involves translocation from the ER, ubiquitination, and proteasomal degradation. NACHO, RIC-3, and nicotine act as chaperones in the ER to promote assembly of nAChRs, which are then trafficked to the cell surface. Lynx proteins associate with nAChRs to influence trafficking from the ER and modulate gating of nAChRs at the cell surface. Neuron 2017 96, 989-1001DOI: (10.1016/j.neuron.2017.10.001) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 5 nAChR Stoichiometry and Ligand Binding Homomeric nAChRs contain 5 binding sites for ACh (blue triangles) at the interphase between each α7 subunit. Heteromeric nAChRs contain 2 primary binding sites for ACh (blue triangles) at the interphase between α and β subunits (orthodox binding sites) and a third unorthodox binding site for ACh (green triangle) between the fifth accessory subunit (shaded) and an α subunit. The β3 and α5 nAChR subunits can only incorporate into the fifth accessory subunit and contain unorthodox binding sites. Potential allosteric modulators at β3 and α5 nAChR subunits (red circle) may present opportunities to selectively target nAChRs containing these subunits. Neuron 2017 96, 989-1001DOI: (10.1016/j.neuron.2017.10.001) Copyright © 2017 Elsevier Inc. Terms and Conditions