Allosteric Control of Super-Agonism in a Ligand-Gated Ion Channel

Ligand-gated ion channels open in response to the binding of an agonist. The agonist binding site is typically located tens of Angstroms away from the channel gate, which lies within the membrane and thus the gating mechanism is considered a classic example of allostery within proteins. The multi-subunit nature of these proteins also means that modulatory effects on the gating process can also be mediated by several other distinct regions – so called allosteric modulatory sites. One of the most well-studied channels in this regard, is the nicotinic acetylcholine receptor. Super-agonists are compounds that can produce a greater maximal response than the endogenous ligand (acetylcholine in this case). They are able to stabilize the open state of the ion channel and this can have important consequences for neuronal signaling. Some super-agonist effects can be mediated through the orthosteric binding site, but others can be mediated by alternative binding sites. The latter are often much harder to identify and can depend very precisely on the subunit composition of the receptor. In this work we sought to identify the mechanism by which TC-2559, a known super-agonist that acts as such only at one particular combination of neuronal nicotinic acetylcholine receptors – the high sensitivity receptor which is comprised of 2 alpha subunits and 3 beta subunits (as opposed to the low sensitivity receptor which has 3 alpha and 2 beta subunits). By using advanced computational methods, supported by two-voltage electrode clamp experiments, we were able to show that TC-2559 not only binds to the orthosteric but also binds to the unique b2-b2 interface of the HS receptor. The binding of TC-2559 to this interface exerts unique interactions that other agonists are not able to make, but more importantly it induces changes in the interface that support that concept of an allosteric gain in overall efficacy. Our results highlight how allosteric control exists to modulate receptor function.