Stoichiometry of Recombinant Heteromeric Glycine Receptors Revealed by a Pore-Lining Region Point Mutation

• Published in: Receptors & channels Vol. 9; no. 6; pp. 353 - 361
• Main Authors: Burzomato, Valeria, Groot-Kormelink, Paul J., Sivilotti, Lucia G., Beato, Marco
• Format: Journal Article
• Language: English
• Published: England Taylor & Francis 2003
• Subjects: Cell Line; Central Nervous System Stimulants - pharmacology; DNA, Complementary - metabolism; Dose-Response Relationship, Drug; Electrophysiology; Glycine - chemistry; Glycine - pharmacology; Glycine Receptor; Heterologous Expression; Humans; Mutagenesis, Site-Directed; Mutation; Picrotoxin; Picrotoxin - pharmacology; Plasmids - metabolism; Point Mutation; Protein Structure, Tertiary; Receptors, Glycine - chemistry; Receptors, Glycine - genetics; Receptors, Nicotinic - chemistry; Recombinant Proteins - chemistry; Single-Channel Recording; Site-Directed Mutagenesis; Subunit Stoichiometry; Transfection; Trigeminal Caudal Nucleus - metabolism
• ISSN: 1060-6823; 1607-856X; 1543-5334
• DOI: 10.3109/714041016

Heteromeric glycine receptors mediate synaptic inhibition in the caudal areas of the adult mammalian central nervous system (CNS). These channels resemble other receptors in the nicotinic superfamily in that they are pentamers, but may differ in that they contain α and β subunits in a 3:2 rather than a 2:3 ratio. Evidence in favor of a 3α:2β stoichiometry of heteromeric glycine receptors comes from biochemical data and from the expression of chimeric subunits. We investigated this question using a potentially more direct approach and mutated the highly conserved hydrophobic residues in the middle (position 9′) of the pore-lining domain. This mutation increases agonist potency in all channels in the nicotinic superfamily and its effects are in first approximation proportional to the number of mutant subunit incorporated into the receptor. We expressed in HEK 293 cells wild-type glycine α1β receptors or receptors bearing the 9′ mutation on either the α or the β subunit, using an α:β plasmid ratio of 1:40 in the transfection. This resulted in negligible levels of contamination by homomeric α1 receptors, as proven by low picrotoxin potency and by the extreme rarity of high conductances in single channel recording. Our data show that the effects of the 9′ mutation on the receptor sensitivity to glycine were more marked when the α subunit bore the mutation. The magnitude of the leftward shift in the agonist dose-response curve for the two mutant combinations was in agreement with a subunit stoichiometry of 3α:2β.