title: Role of pairwise interactions between M1 and M2 domains of the nicotinic receptor in channel gating.
abstract: The adult form of the nicotinic acetylcholine receptor (AChR) consists of five subunits (alpha(2)betaepsilondelta), each having four transmembrane domains (M1-M4). The atomic model of the nicotinic acetylcholine receptor shows that the pore-lining M2 domains make no extensive contacts with the rest of the transmembrane domains. However, there are several sites where close appositions between segments occur. It has been suggested that the pair alphaM1-F15' and alphaM2-L11' is one of the potential interactions between segments. To determine experimentally if these residues are interacting and to explore if this interhelical interaction is essential for channel gating, we combined mutagenesis with single-channel kinetic analysis. Mutations in alphaM1-F15' lead to profound changes in the opening rate and slighter changes in the closing rate. Channel gating is impaired as the volume of the residue increases. Rate-equilibrium linear free-energy relationship analysis reveals an approximately 70% open-state-like environment for alphaM1-F15' at the transition state of the gating reaction, suggesting that it moves early during the gating process. Replacing the residue at alphaM1-15' by that at alphaM2-11' and vice versa profoundly alters gating, but the combination of the two mutations restores gating to near normal, indicating that alphaM1-F15' and alphaM2-L11' are interchangeable. Double-mutant cycle analysis shows that these residues are energetically coupled. Thus, the interaction between M1 and M2 plays a key role in channel gating.
authors.affiliation: Instituto de Investigaciones Bioquímicas, Universidad Nacional del Sur-CONICET, Bahía Blanca, Argentina.
authors.forename: Jeremías
authors.initials: J
authors.lastname: Corradi
authors.forename: Guillermo
authors.initials: G
authors.lastname: Spitzmaul
authors.forename: María José
authors.initials: MJ
authors.lastname: De Rosa
authors.forename: Marcelo
authors.initials: M
authors.lastname: Costabel
authors.forename: Cecilia
authors.initials: C
authors.lastname: Bouzat
mesh_terms: D000109:Acetylcholine / Q000378:metabolism; D000818:Animals; D018076:DNA, Complementary / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D018408:Patch-Clamp Techniques; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011978:Receptors, Nicotinic / Q000737:chemistry*; D013816:Thermodynamics
chemical_list: D018076:DNA, Complementary; D011978:Receptors, Nicotinic; D000109:Acetylcholine