Neuronal nicotinic alpha 7 receptor expressed in Xenopus oocytes presents five putative binding sites for methyllycaconitine.

Abstract

1. The recently isolated compound methyllyeaconitine (MLA) is a plant toxin which is a competitive inhibitor of nicotinic acetylcholine receptors (nAChRs). We found that homomeric a7 receptors display a very high sensitivity to MLA with an IC50 in the picomolar range. 2. The competitive nature of the a7 MLA blockade was reinforced by the observation that this compound has no action on wild-type serotoninergic receptors (5-HT3), whereas it is a powerful antagonist of chimaeric receptors a7-5-HT3. 1992;Drasdo, Caulfield, Bertrand, Bertrand & Wonnacott, 1992; Wonnacott, Albuquerque & Bertrand, 1993;Puchacz, Buisson, Bertrand & Lukas, 1994;Gopalakrishnan et al. 1995). The nicotinic acetylcholine receptors form a rather wide family whose members display quite distinct features either from the point of view of their patterns of expression or from their physiological and pharmacological properties (for reviews see Role, 1992;Clarke, 1993;Sargent, 1993;Galzi & Changeux, 1994;Lindstrom, 1995). In vertebrates, a-BuTX is a competitive antagonist of muscle nAChR, alflly8 or alfllye, and of homomeric neuronal nAChRs, a7, a8, a9, whereas all the other neuronal receptors formed by the assembly of a2, a3, c4 subunits with the non-a subunits (fl2 and /14) are completely insensitive to a-BuTX (for reviews see Role,

Nicotinic Acetylcholine Receptors

“…Work on the macroscopic kinetics of the onset and offset of antagonist action on recombinant α7/5HT3 chimaeras suggests methyllycaconitine affinity may be an order of magnitude higher than that for pure α7 receptors (Palma et al, 1996). Additionally, this study was consistent with the idea that homomeric receptors have indeed got five binding sites for the alkaloid (i.e.…”

“…up to one per subunit), if it was assumed that binding of one antagonist molecule is enough to block the response. Heteromeric receptors are also blocked by methyllycaconitine, but at much higher concentrations (tens of nM); recombinant α4β2 receptors recover from methyllycaconitine block with a time course consistent with the presence of two antagonist binding sites (Palma et al, 1996).…”

Desensitization of neuronal nicotinic receptors

“…Although conventional models (Fig. 2) have been used to explain desensitization of ␣7 receptors (Bertrand et al, 1992), a novel multistate model in which desensitization depends on the fractional agonist occupation (five states ϭ five ligand binding sites; Palma et al, 1996) has been proposed to explain the unusual behavior of ␣BTX-sensitive receptors in hypothalamic cells . The model predicts that high concentrations of ligand will cause complete desensitization (more fractional occupation of nonconducting states); conversely, low concentrations (less occupancy) will allow prolonged channel opening.…”

“…Moreover, because of the high affinity of desensitized states for agonists, the mutant ␣7 receptor is now sensitive to both ACh and nicotine in the concentration range that would cause desensitization of wildtype nAChRs (Revah et al, 1991). Interestingly, the desensitized conducting state of the mutant channel can be activated by some competitive antagonists, implying that antagonists, like agonists, can bind to desensitized states with high affinity (Bertrand et al, 1992;Palma et al, 1996;see Sabey et al, 1999 for ␣4␤2 receptors). Note that such binding to desensitized states of nAChRs has potential implications for up-regulation of receptor function during chronic exposure to antagonists Peng et al, 1994).…”

The long internal loop of the α3 subunit targets nAChRs to subdomains within individual synapses on neurons in vivo

“…Myc-tagged chimeric and wild-type subunit cDNAs were subcloned into PMT-3 expression vector in frame for injection into oocytes. Xenopus oocytes were prepared according to the standard procedure, nuclear injected with 10 nl of buffer containing 0.2 mg/ml cDNA, and maintained for two to three days before electrophysiological investigations 42 . Dual electrode voltage-clamp recordings were done using a Geneclamp amplifier (Axon Instruments) and data analyzed as described 42 .…”