Indurthi DC , Lewis TM , Ahring PK , Balle T , Chebib M , Absalom NL .

	Fig 1. Saz-A and TC-2559 act selectively at the α4-β2 interface.A Schematic showing α4-β2 (black arrow) and α-α (grey arrow) binding sites for ACh at the 3α4:2β2, 2α4:3β2 and 3α4:2β2HQT receptors. B. Current traces of Saz-A (1μM) and C. TC-2559 (10μM) for 3α4:2β2, 2α4:3β2 and 3α4:2β2HQT receptor compared to maximum ACh currents on the same oocyte. Peak currents elicited by D Saz-A and E TC-2559 were normalized to the saturating concentration of ACh and fitted to non-linear curve fits. The injection ratios of α4:β2 that correspond to the 3α4:2β2 (blue) and the 2α4:3β2 (red) receptors are 4:1 and 1:4 respectively. Saz-A and TC-2559 do not activate the 3α4:2β2HQT (green) receptor that has three α4-α4-like interfaces. Dots represent the mean ± s.e.m.
	Fig 2. Saz-A and TC-2559 co-application with NS9283: A Schematic of the 3α4:2β2 receptor with the binding sites of Saz-A and TC-2559 at the α4-β2 interface, and NS9283 at the α4-α4 interface. Representative current trace of B. Saz-A and C. TC-2559, co-applied with NS9283 (10μM) (Green) compared to max ACh (1mM) current on 3α4:2β2 receptors D Saz-A and E TC-2559 concentration-response curves in the absence (blue) and presence (green) of 10 μM NS9283 normalized to 1 mM ACh at 3α4:2β2 receptors. Dots represent the mean ± s.e.m.
	Fig 3. Estimation of open probability.A Schematic depicting the hypothesized areas for NS206 binding [34]. B Representative current trace of the response of the 3α4:2β2 receptor to 1 mM ACh (blue) and 1 mM ACh co-applied with 10 μM NS206 to estimate open channel probability. C The level of NS206 (10μM) positive modulation of ACh currents for the WT. D Saz-A and E TC-2559 concentration-response curves in the absence (blue) and presence (green) of 10 μM NS9283 normalized to 1 mM ACh co-applied with 10 μM NS206 (Est Po,max) at 3α4:2β2 receptors. Dots represent the mean ± s.e.m.
	Fig 4. Model-1.A. Linear Model of receptor activation incorporating binding (AR), open (AO) and closed-desensitized (AD) states. The equilibrium constants are defined in terms of the microscopic rate constants: the ligand equilibrium dissociation constant is K = k−1 / k+1; the gating equilibrium constant is E = β / α; and the desensitization equilibrium constant D = d+1/d−1. B. Three schemes from 1–3, represent the proposed model to test whether NS9283 changes the gating (E), desensitization (D) or binding (K) equilibrium constants to elicit its effect. C. Saz-A (blue) and TC-2559 (maroon) by themselves and in the presence of NS9283 (Saz-A—Green; TC-2559—brown) were plotted (mean ± s.e.m.) (data from Fig 2E and 2F). The dotted curves represent predicted open-probability responses from the model for the control (Saz-A—Blue dotted; TC-2559 –Maroon dotted) and when gating constant (E) was multiplied by the constant CE (Saz-A—Green dotted; TC-2559—Brown dotted).
	Fig 5. Model-2.A. Linear Model of receptor activation incorporating an intermediate flip state (AF) between ligand bound (AR) and open (AO) states. Dotted box suggests that the gating step from Fig 4 is extended to include flipping step in this model. B. Proposed schemes represent when NS9283 changes the pre-activation (F) or gating (E) constants. The equilibrium constants were estimated using Scheme 2. All equilibrium constants are defined as previously, and F is equal to the forward rate/reverse rate. C TC-2559 and D Saz-A data plotted (mean ± s.e.m.) is same as Fig 4, while the dotted curves represent predicted open-probability curves from Models 2 where either the pre-activation constant (F green) or the gating constant (E red) were multiplied by the constant CF or CE respectively.
	Fig 6. Proposed model.A schematic of proposed receptor model for the activation of WT 3α4:2β2 nAChR. The model includes unbound (R), one agonist bound (AR), two agonist bound (A2R) and three agonist bound (A3R) receptor states. Intermediate ‘flip’ state (RF), Open state (RO) and desensitized state (RD) are shown. Blue frame includes receptor activation model for agonists (eg. Saz-A and TC-2559) that selective bind at the α4-β2 interface and red frame shows receptor activation for ligands selective for α4-α4 interface (NS9283). Dashed lines and grey states indicate transitions and states not discretely tested in our experiments but are considered likely to exist.

, Correction: Ligand Binding at the α4-α4 Agonist-Binding Site of the α4β2 nAChR Triggers Receptor Activation through a Pre-Activated Conformational State. 2016, Pubmed

, Correction: Ligand Binding at the α4-α4 Agonist-Binding Site of the α4β2 nAChR Triggers Receptor Activation through a Pre-Activated Conformational State. 2016, Pubmed
Absalom, Covalent trapping of methyllycaconitine at the α4-α4 interface of the α4β2 nicotinic acetylcholine receptor: antagonist binding site and mode of receptor inhibition revealed. 2013, Pubmed , Xenbase
Ahring, Engineered α4β2 nicotinic acetylcholine receptors as models for measuring agonist binding and effect at the orthosteric low-affinity α4-α4 interface. 2015, Pubmed , Xenbase
Akasu, Neuropeptides facilitate the desensitization of nicotinic acetylcholine-receptor in frog skeletal muscle endplate. 1984, Pubmed
Andersen, Functional relationships between agonist binding sites and coupling regions of homomeric Cys-loop receptors. 2011, Pubmed
Bain, A randomized, double-blind, placebo-controlled phase 2 study of α4β2 agonist ABT-894 in adults with ADHD. 2013, Pubmed
Benallegue, The additional ACh binding site at the α4(+)/α4(-) interface of the (α4β2)2α4 nicotinic ACh receptor contributes to desensitization. 2013, Pubmed , Xenbase
Carbone, Pentameric concatenated (alpha4)(2)(beta2)(3) and (alpha4)(3)(beta2)(2) nicotinic acetylcholine receptors: subunit arrangement determines functional expression. 2009, Pubmed , Xenbase
Colquhoun, Perspectives on: conformational coupling in ion channels: allosteric coupling in ligand-gated ion channels. 2012, Pubmed
Colquhoun, Binding, gating, affinity and efficacy: the interpretation of structure-activity relationships for agonists and of the effects of mutating receptors. 1998, Pubmed
Corradi, Unraveling mechanisms underlying partial agonism in 5-HT3A receptors. 2014, Pubmed
DEL CASTILLO, Interaction at end-plate receptors between different choline derivatives. 1957, Pubmed
Eaton, γ-aminobutyric acid type A α4, β2, and δ subunits assemble to produce more than one functionally distinct receptor type. 2014, Pubmed , Xenbase
Engelman, Presynaptic ionotropic receptors and control of transmitter release. 2004, Pubmed
Gielen, Benzodiazepines modulate GABAA receptors by regulating the preactivation step after GABA binding. 2012, Pubmed , Xenbase
Grosman, Mapping the conformational wave of acetylcholine receptor channel gating. 2000, Pubmed
Grupe, Unravelling the mechanism of action of NS9283, a positive allosteric modulator of (α4)3(β2)2 nicotinic ACh receptors. 2013, Pubmed
Harpsøe, Unraveling the high- and low-sensitivity agonist responses of nicotinic acetylcholine receptors. 2011, Pubmed
Jones, The impact of receptor desensitization on fast synaptic transmission. 1996, Pubmed
Jorenby, Efficacy of varenicline, an alpha4beta2 nicotinic acetylcholine receptor partial agonist, vs placebo or sustained-release bupropion for smoking cessation: a randomized controlled trial. 2006, Pubmed
Kuryatov, Nicotine acts as a pharmacological chaperone to up-regulate human alpha4beta2 acetylcholine receptors. 2005, Pubmed
Lape, On the nature of partial agonism in the nicotinic receptor superfamily. 2008, Pubmed
Li, The neuronal nicotinic alpha4beta2 receptor has a high maximal probability of being open. 2010, Pubmed
Mazzaferro, Additional acetylcholine (ACh) binding site at alpha4/alpha4 interface of (alpha4beta2)2alpha4 nicotinic receptor influences agonist sensitivity. 2011, Pubmed , Xenbase
Mazzaferro, Non-equivalent ligand selectivity of agonist sites in (α4β2)2α4 nicotinic acetylcholine receptors: a key determinant of agonist efficacy. 2014, Pubmed , Xenbase
Mike, Choline and acetylcholine have similar kinetic properties of activation and desensitization on the alpha7 nicotinic receptors in rat hippocampal neurons. 2000, Pubmed
Moroni, alpha4beta2 nicotinic receptors with high and low acetylcholine sensitivity: pharmacology, stoichiometry, and sensitivity to long-term exposure to nicotine. 2006, Pubmed , Xenbase
Moroni, Non-agonist-binding subunit interfaces confer distinct functional signatures to the alternate stoichiometries of the alpha4beta2 nicotinic receptor: an alpha4-alpha4 interface is required for Zn2+ potentiation. 2008, Pubmed , Xenbase
Mukhtasimova, Detection and trapping of intermediate states priming nicotinic receptor channel opening. 2009, Pubmed
Nelson, Alternate stoichiometries of alpha4beta2 nicotinic acetylcholine receptors. 2003, Pubmed , Xenbase
Olsen, Two distinct allosteric binding sites at α4β2 nicotinic acetylcholine receptors revealed by NS206 and NS9283 give unique insights to binding activity-associated linkage at Cys-loop receptors. 2013, Pubmed , Xenbase
Olsen, Structural and functional studies of the modulator NS9283 reveal agonist-like mechanism of action at α4β2 nicotinic acetylcholine receptors. 2014, Pubmed , Xenbase
Papke, Tricks of perspective: insights and limitations to the study of macroscopic currents for the analysis of nAChR activation and desensitization. 2010, Pubmed , Xenbase
Paterson, Neuronal nicotinic receptors in the human brain. 2000, Pubmed , Xenbase
Purohit, Unliganded gating of acetylcholine receptor channels. 2009, Pubmed
Rezvani, Cognitive effects of nicotine. 2001, Pubmed
Rüsch, Gating allosterism at a single class of etomidate sites on alpha1beta2gamma2L GABA A receptors accounts for both direct activation and agonist modulation. 2004, Pubmed , Xenbase
Shahsavar, Acetylcholine-Binding Protein Engineered to Mimic the α4-α4 Binding Pocket in α4β2 Nicotinic Acetylcholine Receptors Reveals Interface Specific Interactions Important for Binding and Activity. 2015, Pubmed , Xenbase
Tapia, Ca2+ permeability of the (alpha4)3(beta2)2 stoichiometry greatly exceeds that of (alpha4)2(beta2)3 human acetylcholine receptors. 2007, Pubmed , Xenbase
Timmermann, An allosteric modulator of the alpha7 nicotinic acetylcholine receptor possessing cognition-enhancing properties in vivo. 2007, Pubmed , Xenbase
Timmermann, Augmentation of cognitive function by NS9283, a stoichiometry-dependent positive allosteric modulator of α2- and α4-containing nicotinic acetylcholine receptors. 2012, Pubmed , Xenbase
Wang, An Accessory Agonist Binding Site Promotes Activation of α4β2* Nicotinic Acetylcholine Receptors. 2015, Pubmed , Xenbase
Wang, A Novel α2/α4 Subtype-selective Positive Allosteric Modulator of Nicotinic Acetylcholine Receptors Acting from the C-tail of an α Subunit. 2015, Pubmed
Williams, Investigation of the molecular mechanism of the α7 nicotinic acetylcholine receptor positive allosteric modulator PNU-120596 provides evidence for two distinct desensitized states. 2011, Pubmed , Xenbase
Williams, Positive allosteric modulators as an approach to nicotinic acetylcholine receptor-targeted therapeutics: advantages and limitations. 2011, Pubmed
Zwart, Sazetidine-A is a potent and selective agonist at native and recombinant alpha 4 beta 2 nicotinic acetylcholine receptors. 2008, Pubmed , Xenbase