LUMINAL AND NON-LUMINAL NONCOMPETITIVE INHIBITOR BINDING-SITES ON THENICOTINIC ACETYLCHOLINE-RECEPTOR (REVIEW)

The nicotinic acetylcholine receptor presents two very well differenti ated domains for ligand binding that account for different cholinergic properties. In the hydrophilic extracellular region of the alpha subu nit 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 inh ibitors, 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 bin ding sites have been found for non-competitive inhibitors such as chlo rpromazine, triphenylmethylphosphonium, the local anaesthetic QX-222 a nd the hydrophobic probe trifluoromethyl-iodophenyldiazirine. They are known as luminal binding sites. Although the local anaesthetic meproa difen 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, experime ntal evidences support the hypothesis of the existence of other high-a ffinity binding sites for non-competitive inhibitors located not at th e channel lumen, but at non-luminal binding domains. Among them, we ca n quote the binding site for quinacrine, which is located at the lipid -protein interface of the alpha M1 domain, and the binding site for et hidium, which is believed to interact with the wall of the vestibule v ery far away from both the lumen channel and the lipid membrane surfac e. The aim of this review is to discuss these recent findings relative to both structurally and functionally relevant aspects of non-competi tive inhibitors of the nicotinic acetylcholine receptor. We will put s pecial emphasis on the description of the localization of molecules wi th non-competitive antagonist properties that bind with high-affinity to luminal and non-luminal domains. The information described herein w as principally obtained by means of methods such as photolabelling and site-directed mutagenesis extracellular in combination with patch-cla mp. Our laboratory has contributed hydrophilic domain with data obtain ed by using biophysical approaches such as paramagnetic electron spin resonance and quantitative fluorescence spectroscopy.