Indurthi DC et al. (2016), Ligand Binding at the 4-4 Agonist-Binding Site ...

XB-ART-53871

PLoS One 2016 Aug 16;118:e0161154. doi: 10.1371/journal.pone.0161154.

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Ligand Binding at the 4-4 Agonist-Binding Site of the 42 nAChR Triggers Receptor Activation through a Pre-Activated Conformational State.

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

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The α4β2 nicotinic acetylcholine receptor (nAChR) is the most abundant subtype in the brain and exists in two functional stoichiometries: (α4)3(β2)2 and (α4)2(β2)3. A distinct feature of the (α4)3(β2)2 receptor is the biphasic activation response to the endogenous agonist acetylcholine, where it is activated with high potency and low efficacy when two α4-β2 binding sites are occupied and with low potency/high efficacy when a third α4-α4 binding site is occupied. Further, exogenous ligands can bind to the third α4-α4 binding site and potentiate the activation of the receptor by ACh that is bound at the two α4-β2 sites. We propose that perturbations of the recently described pre-activation step when a third binding site is occupied are a key driver of these distinct activation properties. To investigate this, we used a combination of simple linear kinetic models and voltage clamp electrophysiology to determine whether transitions into the pre-activated state were increased when three binding sites were occupied. We separated the binding at the two different sites with ligands selective for the α4-β2 site (Sazetidine-A and TC-2559) and the α4-α4 site (NS9283) and identified that when a third binding site was occupied, changes in the concentration-response curves were best explained by an increase in transitions into a pre-activated state. We propose that perturbations of transitions into a pre-activated state are essential to explain the activation properties of the (α4)3(β2)2 receptor by acetylcholine and other ligands. Considering the widespread clinical use of benzodiazepines, this discovery of a conserved mechanism that benzodiazepines and ACh potentiate receptor activation via a third binding site can be exploited to develop therapeutics with similar properties at other cys-loop receptors.

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	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.

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