Photo-induced covalent attachment of agonists as a tool to study allosteric mechanisms of nicotinic acetylcholine receptors.

Muscular and neuronal nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels and contain either two or five binding sites for acetylcholine (ACh). Binding of ACh molecules on the nAChR will trigger the fast opening of the channel and subsequent slow desensitization process. Neuronal alpha7 nicotinic receptors are made up of five identical subunits and possess five binding sites for ACh; this raises the question of how many sites must be occupied before channel opening. However, the effect of each ligand binding on gating is difficult to assess because of the reversible aspect of ligand binding at each site. One solution is to photochemically tether agonists to their binding sites. Such methodology has been applied elegantly and successfully on the homotetrameric cyclic-nucleotide-gated channels to evaluate the functional effects of each ligand binding on gating (Ruiz and Karpen, 1997). We therefore decided to develop a similar approach on Torpedo and neuronal alpha7 nAChRs with the photoactivatable agonist AC5 to investigate the effect of binding site occupancy on allosteric transitions of the receptor. In the dark, AC5 (see structure below) evokes robust currents on oocytes expressing Torpedo nAChR, displaying maximal amplitude comparable to ACh, with EC50 = 1.2 microM (Mourot et al., 2005). When the voltage-clamp oocyte was exposed to UV light in the presence of 30 microM AC5 for 50 s, there was a prolonged activation of the Torpedo nAChR, not reversible by washing, but inhibited by the noncompetitive blockers tetracaine and proadifen (see structure below). Both UV light and AC5 are required for this effect. However, further studies are required to determine whether the gradual decrease of the inward current reflects a slow desensitization process. AC5 is thus a potent photoactivatable agonist of the nAChR, which is able, upon UV irradiation, to incorporate covalently into the ACh-binding sites and to prolong activation of the nAChR. By extending this methodology to patch-clamp experiments, we will be able to incorporate one or several AC5s covalently into the muscular and neuronal nAChR at the single-channel level. Such study will help us understand the observed cooperative effect of gating and will contribute decisively to the controversial concerted vs sequential models for nAChR allosteric transitions.