Publication Date

2008-01-01

Availability

Open access

Degree Type

Thesis

Degree Name

Master of Science (MS)

Department

Molecular and Cellular Pharmacology (Medicine)

Date of Defense

2008-07-01

First Committee Member

Dr. Vladlen Slepak - Mentor

Second Committee Member

Dr. Kerry Burnstein - Mentor

Third Committee Member

Dr. Charles Luetje - Mentor

Abstract

Neuronal nicotinic acetylcholine receptors can have their function modulated by zinc. Depending on concentration and subunit composition, zinc either inhibits or potentiates receptor function. The zinc ion potentiates the alpha4beta4 receptor at non-agonist binding interfaces or "pseudo sites." Zinc potentiation is reduced if certain residues are mutated or spatially interfered with. The residue contributing most to this potentiation reduction effect is histidine 162 on the alpha4 subunit. The anthelmintic compound levamisole potentiates acetylcholine response of certain neuronal nicotinic receptors. Levamisole and its functional analogues morantel, oxantel, and pyrantel all were found to potentiate alpha4beta4 receptors at low (µM) concentrations and inhibit them at high (mM) concentrations. Oxantel showed the greatest degree of potentiation, about a third of the maximal zinc potentiation measured. Oxantel was screened using the substituted cysteine accessibility method (SCAM) against the residue histidine 162 as well as nearby alpha4 residues histidine 61 and glutamate 59 and the beta4 residue aspartate 195. Screening was carried out by mutating said residues into cysteine, followed by covalent linkage with a disulfide bridge of that residue with a methanethiosulfonate compound. SCAM experiments allowed testing of the effects of a single residue and the area immediately adjacent to it. Receptors that lost zinc potentiation capacity from site-directed mutagenesis at the his 162 residue and subsequent methanethiosulfonate reaction still showed regular potentiation following oxantel treatment. Although these compounds exhibit similar biphasic potentiation dose-response curves as zinc, their mechanism for potentiation is not through the same mechanism.

Keywords

Substituted Cysteine Accessibility Method; Ligand Gated Ion Channels

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