Abstract
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.
Highlights
Nicotinic acetylcholine receptors are members of the cys-loop receptor superfamily of ligand-gated ion channels (LGICs) and form a pentameric subunit arrangement around a PLOS ONE | DOI:10.1371/journal.pone.0161154 August 23, 2016nicotinic acetylcholine receptor (nAChR) Are Activated via a Pre-Activated State
By developing two linear kinetic models that can differentiate the transitions between receptor states, we show that the increased efficacy of activation when a third agonist (NS9283) is bound is due to a shift in the equilibrium from the ligand-bound closed state to a pre-activated or “flip” state
We used site-selective ligands coupled with two distinct linear kinetic models to interrogate how the activation mechanism is altered when the third α4-α4 agonist-binding site is occupied in addition to the two α4-β2 sites
Summary
Nicotinic acetylcholine receptors (nAChRs) are members of the cys-loop receptor superfamily of ligand-gated ion channels (LGICs) and form a pentameric subunit arrangement around a PLOS ONE | DOI:10.1371/journal.pone.0161154 August 23, 2016. Agonist binds at the extracellular domain to initiate a series of conformational changes leading to opening of the channel pore some 50 Å away at the transmembrane domain (TM2) This allosteric receptor activation can be described by kinetic models that define discrete, multiple transition states that are too short to be captured by spectroscopic or crystallographic techniques [22,23]. Acquiring single-channel recordings from α4β2 nAChRs has been difficult, but an alternative method using simpler linear models and whole-cell electrophysiological recordings has recently been described to explain how benzodiazepines potentiate GABA activation of GABAARs through binding at a third site homologous to the GABA-binding site [30] Similar to benzodiazepines, both ACh and NS9283, a ligand that augments cognitive function in rodents, bind at the third α4-α4 binding site of 3α4:2β2 nAChRs to potentiate receptor response, and may increase receptor activation via a similar mechanism. We propose that ligands that bind at a third homologous binding site will potentiate receptor activation by the same mechanism
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