Nicotinic acetylcholine-activated receptor-channels (nAChR) are found throughout the central and peripheral nervous systems. The primary structures of these pentameric proteins are homologous to GABA and glycine receptors, and patch-clamp studies have revealed many common features of channel gating in the presence of agonist. One feature common to nAChR, GABA, glycine and glutamate receptors is desensitization, whereby the channels lose their responsiveness during prolonged (seconds) or repetitive exposure to agonist. While the physiological role of desensitization is unclear, it has been proposed to participate in cell-level learning or to protect the endplate from prolonged depolarization. Because phenothiazine neuroleptics at micromolar concentrations potentiate desensitization, this effect might contribute to the side-effects of these drugs. As a result of previous studies of desensitization, many different molecular mechanisms have been proposed to account for nAChR desensitization kinetics. In this proposal, single-channel recordings from rat muscle nAChR will be used to achieve the long-term objective; to distinguish among classes of kinetic schemes and provide detailed mechanistic information about the actions of cholinergic agonists and neuroleptic drugs on desensitization. The experimental approach is to record agonist-evoked whole-cell currents from myoballs or single-channel currents from outside-out excised membrane patches. Analysis takes advantage of the fact that each proposed class of mechanism for desensitization makes specific predictions about types or sequences of molecular transitions that, if the mechanism were true, could be readily found in single-channel recordings. This conditional approach to analysis of single-channel data thus does not require modeling of detailed mechanisms because only unique transitions are used to test each class of model. In addition, models of the type usually proposed do not take into account recently described multiple open states or spontaneous openings of the nAChR (and other ligand-activated channels). Experiments described in this proposal will test whether either or both of the identifiable open states can act as a gateway to desensitization. Other experiments will elucidate the kinetics of desensitization for a single channel; a description at this level will remove much of the ambiguity in the interpretation of desensitization of macroscopic whole-cell currents or ion fluxes. Several experiments will examine desensitization kinetics in the presence of other cholinergic agonists or neuroleptics. The information that is obtained from these studies will increase the understanding of the underlying mechanism of desensitization, and provide a means for interpreting modulation by ligands.
