title: Nicotinic receptor fourth transmembrane domain: hydrogen bonding by conserved threonine contributes to channel gating kinetics.
abstract: The fourth transmembrane domain (M4) of the nicotinic acetylcholine receptor (AChR) contributes to the kinetics of activation, yet its close association with the lipid bilayer makes it the outermost of the transmembrane domains. To investigate mechanistic and structural contributions of M4 to AChR activation, we systematically mutated alphaT422, a conserved residue that has been labeled by hydrophobic probes, and evaluated changes in rate constants underlying ACh binding and channel gating steps. Aromatic and nonpolar mutations of alphaT422 selectively affect the channel gating step, slowing the rate of opening two- to sevenfold, and speeding the rate of closing four- to ninefold. Additionally, kinetic modeling shows a second doubly liganded open state for aromatic and nonpolar mutations. In contrast, serine and asparagine mutations of alphaT422 largely preserve the kinetics of the wild-type AChR. Thus, rapid and efficient gating of the AChR channel depends on a hydrogen bond involving the side chain at position 422 of the M4 transmembrane domain.
authors.affiliation: Instituto de Investigaciones Bioquímicas, Universidad Nacional del Sur-Consejo Nacional de Investigaciones Científicas y Técnicas, 8000 Bahía Blanca, Argentina. inbouzat@criba.edu.ar
authors.forename: C
authors.initials: C
authors.lastname: Bouzat
authors.forename: F
authors.initials: F
authors.lastname: Barrantes
authors.forename: S
authors.initials: S
authors.lastname: Sine
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D004735:Energy Transfer; D006801:Humans; D006860:Hydrogen Bonding; D015640:Ion Channel Gating / Q000502:physiology*; D007668:Kidney / Q000166:cytology; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D018408:Patch-Clamp Techniques; D017434:Protein Structure, Tertiary; D011978:Receptors, Nicotinic / Q000737:chemistry* / Q000378:metabolism*; D013912:Threonine / Q000737:chemistry*
chemical_list: D011978:Receptors, Nicotinic; D013912:Threonine