Abstract P-7: Cryoelectron Microscopy Study of Water-Soluble Extracellular Domain of α7 Nicotinic Acetylcholine Receptor

Andrey V. Tsarev, Roman A. Kamyshinsky, Vasiliy I. Mikirtumov, Dmitriy S. Kulbatskii, Eugene O. Yablokov, Zakhar O. Shenkarev, Olga S. Sokolova, Ekaterina N. Lyukmanova


International Journal of Biomedicine.2019;9  Suppl_1:S19-S19.
Originally published June 29, 2019  


Background: Nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel, which is widely distributed both in the central and peripheral nervous system, and in some of the non-neuronal tissues, including epithelium and immune cells. The pathophysiology of a number of diseases is associated with dysfunctions of this receptor, including neurodegenerative and mental disorders like Alzheimer disease (AD) and schizophrenia. The nicotinic receptor of α7 type (α7-nAChR) plays important role in the memory and learning processes and is inhibited by soluble aggregates of β-amyloid peptide (Aβ). Aβ1-42 is the most toxic form of the amyloid peptide.
Methods: The water-soluble analogue of the ligand-binding extracellular domain of α7-nAChR was produced in Pichia pastoris and purified from a culture medium by Ni-NTA and size-exclusion chromatography. The equilibrium dissociation constant (Kd) of the complex of the recombinant domain with α-bungarotoxin was measured on the optical SPR biosensor Biacore 3000. Structures of the α7 domain alone and in the complex with oligomeric Aβ1-42 peptide were studied by cryoelectron microscopy (cryo-EM).
Results: The α7 ligand-binding domain has an increased stability in solution, and demonstrates ligand-binding characteristics similar to those of the native receptor (Kd of the domain/α-bungarotoxin complex 28±2nM). Statistical analysis of the cryo-EM images of the individual domain particles revealed the presence of a pentameric structure, confirming intact subunit assembly. Unfortunately, the domain demonstrated the preferable orientation on grids with the top view. Nevertheless, the 3D structure of the domain with a height ~ 7 nm, external diameter of ~ 9 nm, and the pore diameter of ~ 2 nm was reconstructed at 8.5 Å resolution. 2D classification of the cryo-EM images of the domain particles in the complex with Aβ1-42 revealed the conformational changes appeared due to interaction with the amyloid peptide.
Conclusion: Obtained results open new perspectives for structural studies of the nAChR ligand-binding domains in complex with the ligands which escape crystallization.

Download Abstract

cryo-electron microscopy • α7-nAChR • beta-amyloid peptide