title: Probing the role of backbone hydrogen bonding in a critical beta sheet of the extracellular domain of a cys-loop receptor.
abstract: Probing the sheet: The network of hydrogen bonds formed in the outer beta sheet of the nicotinic acetylcholine receptor (nAChR; see figure) is fairly robust and tolerates single amide-to-ester mutations throughout. However, eliminating two proximal hydrogen bonds completely destroys receptor function; this adds further support to gating models that ascribe important roles to these beta strands of the nAChR extracellular domain.Long-range communication is essential for the function of members of the Cys-loop family of neurotransmitter-gated ion channels. The involvement of the peptide backbone in binding-induced conformational changes that lead to channel gating in these membrane proteins is an interesting, but unresolved issue. To probe the role of the peptide backbone, we incorporated a series of alpha-hydroxy acid analogues into the beta-sheet-rich extracellular domain of the muscle subtype of the nicotinic acetylcholine receptor, the prototypical Cys-loop receptor. Specifically, mutations were made in beta strands 7 and 10 of the alpha subunit. A number of single backbone mutations in this region were well tolerated. However, simultaneous introduction of two proximal backbone mutations led to surface-expressed, nonfunctional receptors. Together, these data suggest that while the receptor is remarkably robust in its ability to tolerate single amide-to-ester mutations throughout these beta strands, more substantial perturbations to this region have a profound effect on the protein. These results support a model in which backbone movements in the outer beta sheet are important for receptor function.
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, 1200 E. California Boulevard, Pasadena, CA 91106, USA.
authors.forename: Kristin R
authors.initials: KR
authors.lastname: Gleitsman
authors.forename: Henry A
authors.initials: HA
authors.lastname: Lester
authors.forename: Dennis A
authors.initials: DA
authors.lastname: Dougherty
mesh_terms: D006860:Hydrogen Bonding; D015640:Ion Channel Gating / Q000502:physiology; D009154:Mutation; D018377:Neurotransmitter Agents / Q000737:chemistry; D017433:Protein Structure, Secondary; D011978:Receptors, Nicotinic / Q000737:chemistry* / Q000378:metabolism
chemical_list: D018377:Neurotransmitter Agents; D011978:Receptors, Nicotinic