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Dissertation
Interactions of [psi]-conotoxin PrIIIE with the muscle-type nicotinic acetylcholine receptor
Doctor of Philosophy (Ph.D.), Drexel University
Apr 2012
DOI:
https://doi.org/10.17918/00000976
Abstract
Conotoxins are peptide toxins present in the venom of Conus marine snails. There are over 500 species of Conus giving rise to over 50,000 different peptide toxins. These toxins are highly selective for various ligand-gated and voltage-gated ion channel subtypes. Conotoxins have proven to be useful tools to probe the functional domains of ion channels. In addition, they can be used as therapeutic agents to target specific ion-channels. Conotoxin PrIIIE is a 22-amino acid peptide from the venom of Conus parius Reeve containing three disulfide bonds. It is a member of the [psi]-conotoxin family which targets the nicotinic acetylcholine receptor (nAChR). In this thesis, we demonstrate that PrIIIE is a non-competitive inhibitor (NCI) of the mouse muscle-type nicotinic acetylcholine receptor using an electrophysiological assay of receptors expressed in Xenopus oocytes. In addition, we developed a recombinant expression system to produce a biologically-active PrIIIE in Escherichia coli. We fused the coding sequence of PrIIIE to that of the small ubiquitin-like modifier (SUMO) and expressed the fusion protein in an E. coli strain with an oxidizing cytoplasm. We purified the fusion protein by immobilized metal affinity chromatography and further purified PrIIIE from cleaved SUMO using cation exchange chromatography. The recombinant peptide is functional, as demonstrated by two-electrode voltage clamp experiments. We used site-directed mutagenesis to create PrIIIE mutants and our recombinant expression system to produce them, and then tested the activity of the mutants using electrophysiological assays. Mutation of glutamate 14 to glutamine (E14Q) increases the apparent affinity of PrIIIE by ~3.5 fold. Lysine 19 and tyrosine 20, in loop 3, are not individually important for PrIIIE to inhibit the nAChR. Mutation of both arginines in loop 3 to serines (R17SR18S) resulted in a loss of activity for PrIIIE. We determined that only one positively charged arginine, either R17 or R18, but not both, is required for PrIIIE to inhibit the nAChR. We have identified key residues important for the inhibition of the mouse nAChR by PrIIIE. Future work includes identifying other residues important for PrIIIE to inhibit the nAChR and elucidating the mechanism of action for PrIIIE as a NCI.
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Details
- Title
- Interactions of [psi]-conotoxin PrIIIE with the muscle-type nicotinic acetylcholine receptor
- Creators
- Lisa Hernandez-Cuebas
- Contributors
- Michael M. White (Advisor)
- Awarding Institution
- Drexel University
- Degree Awarded
- Doctor of Philosophy (Ph.D.)
- Publisher
- Drexel University; Philadelphia, Pennsylvania
- Number of pages
- xi, 143 pages
- Resource Type
- Dissertation
- Language
- English
- Academic Unit
- Biochemistry and Molecular Biology; College of Medicine; Drexel University
- Other Identifier
- 991014970320304721