36] Purification and reconstitution of nicotinic acetylcholine receptor

Abstract

Publisher Summary This chapter describes the preparation of acetylcholine receptor (AChR)-rich native membranes, solubilized and purified AChR, and AChR reconstituted in liposomes. Triton X-100 efficiently solubilizes the AChR and maintains its snake curarimimetic toxin-binding activity and, with some alteration, its binding of acetylcholine (ACh) and other small ligands. Ligand-gated channel activity of the AChR cannot be recovered once it has been solubilized in Triton X-100, even though the solubilized AChR can be reincorporated into liposomes. Elapid snake venom contains basic polypeptide toxins, which bind to the ACh binding sites with high specificity and affinity. The ligand-gated channel activity of the AChR can be more reproducibly assayed after solubilization and reconstitution into liposomes. For reconstitution and flux assays, ligand-gated channel activity is more effectively reconstituted in a mixture of phosphatidylethanolamine (PE), phosphatidylserine (PS), and cholesterol than in asolectin.