Peptide toxin bioengineering-advancement of fluorescent probe design for targeting human K+ channels

Bergeron, Zachary
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[Honolulu] : [University of Hawaii at Manoa], [December 2013]
Transmembrane receptor proteins such as Potassium (K+) Channels are paramount in cellular excitability, signal transduction pathways and various pathophysiological conditions. Fundamental understanding of channel proteins, such as the Large Conductance Ca2+-activated Potassium (K+) Channel (BK), may have implications in elucidating and treating a myriad of disease states such as cancer, and those that effect electrical conduction. Classically, patch clamp analysis has been used to characterize transmembrane receptors pharmacologically, while antibodies have been utilized in varying fashions (Immunohistochemistry, Western Blots etc.) to demonstrate anatomical localization of protein expression. Fluorescent bioengineered peptide-toxins represent a paradigm shift from the current probe technology used to investigate channel expression, and pending confirmation of in vivo target specificity, could potentially be reformulated for the treatment of advanced disease states such as the primary brain tumors associated with glioblastoma multiforme (GBM). The primary objectives of the investigation are: (i) To use Solid Phase Peptide Synthesis (SPPS) to efficiently bioengineer two unique isoforms of the BK-specific scorpion venom peptide, Iberiotoxin, with a key non-native amino acid substitution to allow direct bioconjugation of a fluorophore, while retaining biological activity and pharmacological specificity. (ii) To utilize well established patch clamp techniques to determine the kinetic parameters of IbTx[D19K] and IbTx[D19K]-LC-FAM. A comparison of the Fluorometric Imaging Plate Reader (FLIPR) Membrane Potential (FMP) assay can be used to quantify dose response activity of IbTx[D19K] and IbTx[D19K]-FITC by measuring concentration induced changes in membrane potential (Ω: Ohms). A comprehensive analysis of the two techniques will establish the superior approach amenable to the current investigation. (iii) The fluorescent probe IbTx[D19K]-FITC can be used to successfully study the expression and trafficking of Ca2+ activated K+ channel (BK) in a human cell line. Successful completion of these objectives will illustrate that the development of a comprehensive evaluation platform would enhance our ability to study ion-channel physiology in real-time. This platform could serve as a bioengineering template for the assembly of a comprehensive molecular toolbox of peptide probes, facilitating the investigation of a wide range of clinically significant ion channels. Domestic sources of peptides represent a reservoir of ready-made molecules that can be harvested for peptides amenable to the bioengineering process. In this study, local Hawaiian Conus species have been used to demonstrate effective approaches to peptide prospecting for expanding probe target distribution. These techniques, when implemented correctly, will expedite the development of potential therapies based on new found understanding of receptor physiology, and minimize adverse drug reactions during the advancement of investigational new drugs.
Ph.D. University of Hawaii at Manoa 2013.
Includes bibliographical references.
peptide, toxin, scorpion, K+, ion channel, BK, cancer
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