Evidence that the M2 Membrane-Spanning Region Lines the Ion Channel Pore of the Nicotinic Receptor

• Published in: Science (American Association for the Advancement of Science) Vol. 242; no. 4885; pp. 1578 - 1581
• Main Authors: Leonard, Reid J., Labarca, Cesar G., Charnet, Pierre, Davidson, Norman, Lester, Henry A.
• Format: Journal Article
• Language: English
• Published: Washington, DC The American Association for the Advancement of Science 16.12.1988; American Association for the Advancement of Science
• Subjects: Acetylcholine; Acetylcholine receptors; Animals; Biological and medical sciences; Cell Membrane - physiology; Cholinergic receptors; Cloning, Molecular; Electric Conductivity; Electric current; Electric potential; Female; Fundamental and applied biological sciences. Psychology; Genetic mutation; Genetics; Ion channels; Ion Channels - physiology; Kinetics; Mammalian female genital system; Medical research; Membrane Potentials; Membrane proteins; Messenger RNA; Molecules; Morphology. Physiology; Mutagenesis; Mutation; Oocytes; Oocytes - physiology; Receptors; Receptors, Nicotinic - genetics; Receptors, Nicotinic - physiology; RNA, Messenger - genetics; Time constants; Transcription, Genetic; Vertebrates: reproduction; Xenopus; Transmembrane current; Gamete; Vertebrata; Cholinergic receptor; Plasma membrane; Amphibia; Electrophysiology; Toad; Salientia; Acetylcholine; Nicotinic receptor; Oocyte
• ISSN: 0036-8075; 1095-9203
• DOI: 10.1126/science.2462281

Site-directed mutagenesis and expression in Xenopus oocytes were used to study acetylcholine receptors in which serine residues (i) were replaced by alanines ($\alpha $, $\delta $ subunits) or (ii) replaced a phenylalanine ($\beta $ subunit) at a postulated polar site within the M2 transmembrane helix. As the number of serines decreased, there were decreases in the residence time and consequently the equilibrium binding affinity of QX-222, a quaternary ammonium anesthetic derivative thought to bind within the open channel. Receptors with three serine-to-alanine mutations also displayed a selective decrease in outward single-channel currents. Both the direction of this rectification and the voltage dependence of QX-222 blockade suggest that the residues mutated are within the aqueous pore of the receptor and near its cytoplasmic (inner) surface.