Determinants of agonist affinity on neuronal nicotinic acetylcholine receptors

Date of Award




Degree Name

Doctor of Philosophy (Ph.D.)


Molecular and Cellular Pharmacology

First Committee Member

Charles W. Luetje, Committee Chair


I used equilibrium binding analyses to characterize the agonist binding properties of six different rat neuronal nicotinic receptor subunit combinations expressed in Xenopus oocytes. Use of [3H]-epibatidine allowed detailed characterization of multiple neuronal nicotinic receptor subunit combinations ( a 2 b 2, a 2 b 4, a 3 b 2, a 3 b 4, a 4 b 2, a 4 b 4). Affinities for six additional agonists (acetylcholine, anabasine, cytisine, 1,1-dimethyl-4-phenylpiperazinium, lobeline and nicotine) were determined in competition assays. While small differences in affinity could be ascribed to different a subunits, the major factor in determining agonist affinity was the nature of the b subunit.To locate specific amino acid residues on b subunits responsible for differences in agonist affinity, I expressed a series of chimeras of the b 2 and b 4 subunits in combination with the a 2 subunit in Xenopus oocytes. Saturation and competition analyses were performed on a set of five chimeric b subunits: b 4-204- b 2, b 4-133- b 2, b 2-54- b 4, b 2-63- b 4, and b 2-80- b 4. Saturation and competition analyses demonstrated that the region between residues 54--63 contained the primary determinants of epibatidine, acetylcholine, nicotine and DMPP affinity. The affinity of lobeline contained determinants in multiple areas of the b subunits. The binding affinities of mutations of the b 2 subunit in the region 54--63 were analyzed. The primary determinant of ACh and nicotine affinity was found at position 59. The residues at positions 55, 56, and 63 were also significant determinants. A novel mutation at position 59 demonstrated that the charge on the side chain of this residue was not responsible for determining the affinities of ACh and nicotine. Mutation analyses failed to resolve determinants in this region for DMPP or [3H]-epibatidine affinity.The affinities of seven derivatives of epibatidine were determined for six wild type neuronal nAChRs ( a 2 b 2, a 3 b 2, a 4 b 2, a 2 b 4, a 3 b 4, and a 4 b 4) by competition analyses. The derivatives were generated by substituting the chlorine at the 6th position on the pyridyl group of epibatidine with one of the following groups: -Br, -F, -H, -NH 3, -OH, -OSO2CF3, or -N(CH 3)2. Changes at this position of epibatidine resulted in ligands that had a wide range of affinities. The largest difference in affinities between b 2 and b 4 containing receptors was observed for the -H derivative for the a 3 b 4 receptor (3500-fold). There were other large differences, which were primarily the result of the derivatives having a lower affinity for the b 4 containing receptors. Functional characterization of these derivatives is planned for future experiments.


Biology, Molecular; Biology, Neuroscience

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