Studying interactions between peptide toxins and voltage-gated K+ channels at the molecular level

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dc.contributor.advisorPanyi, György
dc.contributor.authorNaseem, Muhammad Umair
dc.contributor.departmentMolekuláris Orvostudomány Doktori Iskolahu
dc.contributor.submitterdepDebreceni Egyetem::Általános Orvostudományi Kar::Biofizikai és Sejtbiológiai Intézet
dc.date.accessioned2023-06-05T11:49:20Z
dc.date.available2023-06-05T11:49:20Z
dc.date.created2023
dc.date.defended2023-06-20
dc.description.abstractThe voltage-gated Kv1.3 potassium ion channels express in immune cells and are implicated in a range of autoimmune diseases and neuroinflammatory disorders. Selective blocking of Kv1.3 using peptides isolated from scorpion venom holds a great potential in developing immunomodulatory therapies. We discovered and characterized a novel short peptide in the venom of C. margaritatus (Cm28). Cm28 obeys a unique primary structure, consists of 27 amino acid residues and has <40% similarity with other known α-KTxs from scorpions. Cm28 reversibly inhibited Kv1.2 and Kv1.3 channels with Kd values of 0.96 and 1.3 nM, respectively. The biophysical characterization of the block revealed that Cm28 is not a gating modifier, but rather a pore blocker. Cm28 is ~400-fold selective for Kv1.2 and Kv1.3 over Kv1.1 and did not inhibit a variety of other K+, Na+ and H+ channels at 150 nM concentration. Cm28 strongly downregulated the expression of two key early activation markers IL2R and CD40 ligand in stimulated human effector memory T cells. Cm28, due to its unique structure, may serve as a template for the generation of novel peptides targeting Kv1.3. The high affinity and selectivity of peptide toxins for Kv1.3 make them suitable for the development of visualization tools to study the expression and the pharmacokinetics of peptide toxins. We developed a fluorescent analogue of HsTX1[R14A] having an N-terminus Cy5 tag. We showed that Cy5-HsTX1[R14A] retained high affinity and selectivity for Kv1.3 (Kd ~0.9 nM), even against channels formed by Kv1.3-Kv1.5 tandem dimers. Furthermore, flow cytometry demonstrated that Cy5-HsTX1[R14A] can identify Kv1.3-expressing CHO cells. To generate ample amounts of Kv1.3 inhibitor toxins for pharmacology and therapeutic development processes we optimized Pichia pastoris expression system to produce ~36 mg/L of His-tagged margatoxin with >98% purity. This yield, which is 3-fold higher than has been previously reported, was achieved by optimizing the codon, the selection process, and the culturing conditions. Moreover, we showed that the His-tagged MgTx inhibited Kv1.2 and Kv1.3 channels with similar potency to the untagged MgTx, and significantly inhibited the IL2R and CD40 ligand in activated human effector memory T cells, thus, elimination of the tag removal reduces the cost of the production. These results suggest that Pichia expression system is a powerful method to produce the disulfide-rich peptide MgTx, the overexpression of similar peptides could be enhanced noticeably through optimization strategies, making it more cost-effective. In summary, the data presented in the PhD dissertation resulted in a novel ion channel inhibitor peptide (Cm28), a new visualization tool for Kv1.3 (Cy5-HsTX1[R14A]) and an optimized method to produce MgTx in the Pichia expression system.
dc.format.extent109
dc.identifier.urihttps://hdl.handle.net/2437/354604
dc.language.isoen
dc.subjectVoltage-gated potassium channel
dc.subjectKv1.3
dc.subjectAutoimmune diseases
dc.subjectScorpion toxins
dc.subjectMargatoxin
dc.subjectHsTX1 toxin
dc.subjectFluorescent-labeled toxin
dc.subjectPatch-clamp electrophysiology
dc.subjectFlow cytometry
dc.subjectRecombinant protein production
dc.subjectYeast expression system
dc.subjectHPLC
dc.subject.disciplineElméleti orvostudományokhu
dc.subject.sciencefieldOrvostudományokhu
dc.titleStudying interactions between peptide toxins and voltage-gated K+ channels at the molecular level
dc.title.translatedStudying interactions between peptide toxins and voltage-gated K+ channels at the molecular level
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