CaltechTHESIS
  A Caltech Library Service

Topographical Studies of the Nicotinic Acetylcholine Receptor

Citation

Middlemas, David S. (1987) Topographical Studies of the Nicotinic Acetylcholine Receptor. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/se3p-8191. https://resolver.caltech.edu/CaltechTHESIS:04172019-103221069

Abstract

All four subunits of the nicotinic acetylcholine receptor in membrane vesicles isolated from Torpedo californica have been labeled with the photoactivated hydrophobic probe, [3H]adamantanediazirine, which selectively labels regions of integral membrane proteins in contact with the hydrocarbon core of the lipid bilayer. As all of the homologous subunits are exposed to the lipid bilayer, it is probable that they each interact with the surrounding membrane in a similar fashion.

All four subunits of the acetylcholine receptor in membrane vesicles isolated from Torpedo californica have been labeled with [3H]cholesteryl diazoacetate. As this probe incorporates into lipid bilayers analogously to cholesterol, this result indicates that acetylcholine receptor interacts with cholesterol. This investigation also demonstrates that this probe is a useful reagent for studying the interaction of cholesterol with membrane proteins.

Since the photogenerated carbene is situated near the lipid-water interface, this probe has potential as a topographic tool for mapping membrane protein structure. The labeling studies with both [3H]adamantanediazirine and [3H]cholesteryl diazoacetate support the concept that the acetylcholine receptor is a pseudosymmetric complex of homologous subunits, all of which interact with and span the membrane.

The syntheses of the fluorine-containing agonists for the Torpedo californica nicotinic acetylcholine receptor, fluoroacetylcholine bromide and p-fluorophenyltrimethylammonium iodide, are described. It is demonstrated that both are agonists using a cation flux assay with acetylcholine receptor enriched membrane vesicles. The potential for their use in ligand binding studies using 19F NMR spectroscopy is discussed.

The affinity cleavage reagent, p-thiocyanophenyltrimethylammonium iodide, specifically cleaves a peptide bond of the nicotinic acetylcholine receptor in membrane vesicles isolated from Torpedo californica. It is demonstrated that this reagent is an agonist using a cation flux assay. The cleavage is blocked by stoichiometric quantities of α-bungarotoxin. The yield of the cleavage reaction is reduced with addition of the agonist, phenyltrimethylammonium iodide. This affinity cleavage reaction provides evidence for a low-affinity binding site for agonists on the 60K subunit.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:Chemistry
Degree Grantor:California Institute of Technology
Division:Chemistry and Chemical Engineering
Major Option:Chemistry
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Raftery, Michael A.
Thesis Committee:
  • Richards, John H. (chair)
  • Raftery, Michael Augustine
  • Grubbs, Robert H.
  • Dervan, Peter B.
Defense Date:27 October 1986
Record Number:CaltechTHESIS:04172019-103221069
Persistent URL:https://resolver.caltech.edu/CaltechTHESIS:04172019-103221069
DOI:10.7907/se3p-8191
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:11479
Collection:CaltechTHESIS
Deposited By: Mel Ray
Deposited On:17 Apr 2019 18:20
Last Modified:16 Apr 2021 23:10

Thesis Files

[img]
Preview
PDF - Final Version
See Usage Policy.

28MB

Repository Staff Only: item control page