Nicotinic acetylcholine receptor solubilization using lipid analog detergents: A key aspect toward receptor crystallization and functional characterization.
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Autor
Padilla Morales, Luis F.
Advisor
Lasalde-Dominicci, José A.Quesada, Orestes
Tipo
DissertationDegree Level
Ph.D.Fecha
2018-12Metadatos
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The nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel (LGIC) composed of five subunits (2α, 1β, 1γ, and 1δ) in a pentameric arrangement. The nAChR is a crucial component of the cholinergic pathway and manages the synaptic transmission between the brain and muscle. During the past five decades, the nAChR from T. californica has been extensively studied and used as a representative model for crystallization trials. There have been several accomplishments in the nAChR crystallization efforts, particularly using Cryo-EM and molecular engineering coupled with hanging drop crystallography. However, a high-resolution crystal structure of the native nAChR has not yet been developed, in part due to the poor understanding of the lipid-protein-detergent interactions occurring within the receptor. Lipidic matrixes as crystallization environments for membrane proteins have been used to improve the yield of membrane protein’s crystal structures. Nevertheless, little is known about the effect of the isolation methodologies (i.e., detergents, buffers, and endogenous lipid composition) towards the LCP. Our laboratory designed a lipid-based characterization approach and examined the stability, functionality, and mobility of the T. californica nAChR. Size exclusion chromatography was used to assay soluble stability, two electron voltage clamp (TEVC) to study ion channel functionality, and fluorescence recovery after photobleaching (FRAP) to assess nAChR mobility on the LCP. The evaluation of these criteria is essential to determine the appropriate conditions to conduct nAChR crystallization. Our studies showed it is possible to extract T. californica nAChR by employing lipid analog detergents and provided valuable insights for the preparation of functionally active nAChR-detergent complexes. We demonstrated that phospholipid-analog detergents with 16 carbon chains sustain nAChR function and stability.