Proposed model.

<p>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.</p

Parameters derived from fitting of data to the Hill Equation with a Hill co-efficient of one.

<p>Parameters derived from fitting of data to the Hill Equation with a Hill co-efficient of one.</p

Estimation of open probability.

<p><b><i>A</i></b> Schematic depicting the hypothesized areas for NS206 binding [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161154#pone.0161154.ref034" target="_blank">34</a>]. <b><i>B</i></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. <b><i>C</i></b> The level of NS206 (10μM) positive modulation of ACh currents for the WT. <b><i>D</i></b> Saz-A and <b><i>E</i></b> 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 <i>P</i>_{<i>o</i>,<i>max</i>}) at 3α4:2β2 receptors. Dots represent the mean ± s.e.m.</p

Ligand Binding at the α4-α4 Agonist-Binding Site of the α4β2 nAChR Triggers Receptor Activation through a Pre-Activated Conformational State - Fig 2

<p>Saz-A and TC-2559 co-application with NS9283: <b>A</b> 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><i>B</i>.</b> Saz-A and <b><i>C</i>.</b> TC-2559, co-applied with NS9283 (10μM) (Green) compared to max ACh (1mM) current on 3α4:2β2 receptors <b><i>D</i></b> Saz-A and <b><i>E</i></b> 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.</p

Kinetic equilibrium constants derived from three-step models proposed.

<p>Kinetic equilibrium constants derived from three-step models proposed.</p

Saz-A and TC-2559 act selectively at the α4-β2 interface.

<p><b>A</b> Schematic showing α4-β2 (black arrow) and α-α (grey arrow) binding sites for ACh at the 3α4:2β2, 2α4:3β2 and 3α4:2β2^HQT receptors. <b>B.</b> Current traces of Saz-A (1μM) and <b>C.</b> TC-2559 (10μM) for 3α4:2β2, 2α4:3β2 and 3α4:2β2^HQT receptor compared to maximum ACh currents on the same oocyte. Peak currents elicited by <b>D</b> Saz-A and <b>E</b> 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 3α4:2β2^HQT (green) receptor that has three α4-α4-like interfaces. Dots represent the mean ± s.e.m.</p