Topology of ligand binding sites on the nicotinic acetylcholine receptor

Abstract

The nicotinic acetylcholine receptor (AChR) presents two very well differentiated domains for ligand binding that account for different cholinergic properties. In the hydrophilic extracellular region of both α subunits there exist the binding sites for agonists such as the neurotransmitter acetylcholine (ACh) and for competitive antagonists such as d-tubocurarine. Agonists trigger the channel opening upon binding while competitive antagonists compete for the former ones and inhibit its pharmacological action. Identification of all residues involved in recognition and binding of agonist and competitive antagonists is a primary objective in order to understand which structural components are related to the physiological function of the AChR. The picture for the localisation of the agonist/competitive antagonist binding sites is now clearer in the light of newer and better experimental evidence. These sites are mainly located on both α subunits in a pocket approximately 30–35 Å above the surface membrane. Since both α subunits are sequentially identical, the observed high and low affinity for agonists on the receptor is conditioned by the interaction of the α subunit with the δ or the γ chain, respectively. This relationship is opposite for curare-related drugs. This molecular interaction takes place probably at the interface formed by the different subunits. The principal component for the agonist/competitive antagonist binding sites involves several aromatic residues, in addition to the cysteine pair at 192–193, in three loops-forming binding domains (loops A–C). Other residues such as the negatively charged aspartates and glutamates (loop D), Thr or Tyr (loop E), and Trp (loop F) from non-α subunits were also found to form the complementary component of the agonist/competitive antagonist binding sites. Neurotoxins such as α-, κ-bungarotoxin and several α-conotoxins seem to partially overlap with the agonist/competitive antagonist binding sites at multiple point of contacts. The α subunits also carry the binding site for certain acetylcholinesterase inhibitors such as eserine and for the neurotransmitter 5-hydroxytryptamine which activate the receptor without interacting with the classical agonist binding sites. The link between specific subunits by means of the binding of ACh molecules might play a pivotal role in the relative shift among receptor subunits. This conformational change would allow for the opening of the intrinsic receptor cation channel transducting the external chemical signal elicited by the agonist into membrane depolarisation. The ion flux activity can be inhibited by non-competitive inhibitors (NCIs). For this kind of drugs, a population of low-affinity binding sites has been found at the lipid–protein interface of the AChR. In addition, several high-affinity binding sites have been found to be located at different rings on the M2 transmembrane domain, namely luminal binding sites. In this regard, the serine ring is the locus for exogenous NCIs such as chlorpromazine, triphenylmethylphosphonium, the local anaesthetic QX-222, phencyclidine, and trifluoromethyliodophenyldiazirine. Trifluoromethyliodophenyldiazirine also binds to the valine ring, which is the postulated site for cembranoids. Additionally, the local anaesthetic meproadifen binding site seems to be located at the outer or extracellular ring. Interestingly, the M2 domain is also the locus for endogenous NCIs such as the neuropeptide substance P and the neurotransmitter 5-hydroxytryptamine. In contrast with this fact, experimental evidence supports the hypothesis for the existence of other NCI high-affinity binding sites located not at the channel lumen but at non-luminal binding domains. Among them, we can quote the binding site for exogenous NCIs such as quinacrine which is located at the lipid–protein interface of the αM1 domain, and ethidium which is believed to interact with the wall of the vestibule very far away from both the channel lumen and the lipid membrane surface. Additionally, endogenous NCIs such as progesterone, testosterone and glucocorticoids have been postulated to act through the lipid–protein interface or/and through the extracellular domain of the AChR. The most simple mechanism to describe the action of luminal NCIs is based on the assumption that these compounds enter the open channel, bind to different rings within the M2 transmembrane AChR domain, and block cation flux by sterically `plugging' the receptor pore. The existence of non-luminal NCI binding sites is not consistent with the open-channel-blocking mechanism. Instead, the presence of these kind of sites open the possibility for the regulation of cation permeation by an allosteric process. In turn, the allosteric mechanism can be viewed as the structural modification of the AChR channel by the binding of one NCI molecule to its specific high-affinity binding site. Within this perspective, although agonists at high concentrations resemble the pharmacological action of NCIs, the inhibitory binding site for agonists is postulated to be located at or close to the quinacrine locus. In turn, the quinacrine binding site is structurally related to the non-annular lipid domain of the AChR.