title: Subunit-selective contribution to channel gating of the M4 domain of the nicotinic receptor.
abstract: The muscle nicotinic receptor (AChR) is a pentamer of four different subunits, each of which contains four transmembrane domains (M1-M4). We recently showed that channel opening and closing rates of the AChR depend on a hydrogen bond involving a threonine at position 14' of the M4 domain in the alpha-subunit. To determine whether residues in equivalent positions in non-alpha-subunits contribute to channel gating, we mutated deltaT14', betaT14', and epsilonS14' and evaluated changes in the kinetics of acetylcholine-activated currents. The mutation epsilonS14'A profoundly slows the rate of channel closing, an effect opposite to that produced by mutation of alphaT14'. Unlike mutations of alphaT14', epsilonS14'A does not affect the rate of channel opening. Mutations in deltaT14' and betaT14' do not affect channel opening or closing kinetics, showing that conserved residues are not functionally equivalent in all subunits. Whereas alphaT14'A and epsilonS14'A subunits contribute additively to the closing rate, they contribute nonadditively to the opening rate. Substitution of residues preserving the hydrogen bonding ability at position 14' produce nearly normal gating kinetics. Thus, we identify subunit-specific contributions to channel gating of equivalent residues in M4 and elucidate the underlying mechanistic and structural bases.
authors.affiliation: Instituto de Investigaciones Bioquímicas, UNS-CONICET, Bahía Blanca, Argentina. inbouzat@criba.edu.ar
authors.forename: Cecilia
authors.initials: C
authors.lastname: Bouzat
authors.forename: Fernanda
authors.initials: F
authors.lastname: Gumilar
authors.forename: María
authors.initials: M
authors.lastname: del Carmen Esandi
authors.forename: Steven M
authors.initials: SM
authors.lastname: Sine
mesh_terms: D000818:Animals; D002460:Cell Line; D002794:Choline / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004594:Electrophysiology; D006801:Humans; D006860:Hydrogen Bonding; D007700:Kinetics; D051379:Mice; D008956:Models, Chemical; D009132:Muscles / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D018408:Patch-Clamp Techniques; D017434:Protein Structure, Tertiary; D011978:Receptors, Nicotinic / Q000737:chemistry*; D013816:Thermodynamics; D013997:Time Factors
chemical_list: D018076:DNA, Complementary; D011978:Receptors, Nicotinic; D002794:Choline