Luminal and non-luminal non-competitive inhibitor binding sites on the nicotinic acetylcholine receptor

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Abstract
The nicotinic acetylcholine receptor presents two very well differentiated domains for ligand binding that account for different cholinergic properties. In the hydrophilic extracellular region of the alpha subunit exist the binding sites for agonists such as the neurotransmitter acetylcholine, which upon binding trigger the channel opening, and for competitive antagonists such as d-tubocurarine, which compete for the former inhibiting its pharmacological action. For non-competitive inhibitors, a population of low-affinity binding sites have been found at the lipid-protein interface of the nicotinic acetylcholine receptor. In addition, at the M2 transmembrane domain, several high-affinity binding sites have been found for non-competitive inhibitors such as chlorpromazine, triphenylmethylphosphonium, the local anaesthetic QX-222 and the hydrophobic probe trifluoromethyl-iodophenyldiazirine. They are known as luminal binding sites. Although the local anaesthetic meproadifen seems to be located between the hydrophobic domains M2-M3, this locus is considered to form part of the channel mouth, thus this site can also be called a luminal binding site. In contraposition, experimental evidences support the hypothesis of the existence of other high-affinity binding sites for non-competitive inhibitors located not at the channel lumen, but at non-luminal binding domains. Among them, we can quote the binding site for quinacrine, which is located at the lipid-protein interface of the alpha M1 domain, and the binding site for ethidium, which is believed to interact with the wall of the vestibule very far away from both the lumen channel and the lipid membrane surface. The aim of this review is to discuss these recent findings relative to both structurally and functionally relevant aspects of non-competitive inhibitors of the nicotinic acetylcholine receptor. We will put special emphasis on the description of the localization of molecules with non-competitive antagonist properties that bind with high-affinity to luminal and non-luminal domains. The information described herein was principally obtained by means of methods such as photolabelling and site-directed mutagenesis in combination with patch-clamp. Our laboratory has contributed with data obtained by using biophysical approaches such as paramagnetic electron spin resonance and quantitative fluorescence spectroscopy.