title: An engineered scorpion toxin analogue with improved Kv1.3 selectivity displays reduced conformational flexibility.
abstract: The voltage-gated Kv1.3 K(+) channel plays a key role in the activation of T lymphocytes. Kv1.3 blockers selectively suppress immune responses mediated by effector memory T cells, which indicates the great potential of selective Kv1.3 inhibitors in the therapy of certain autoimmune diseases. Anuroctoxin (AnTx), a 35-amino-acid scorpion toxin is a high affinity blocker of Kv1.3, but also blocks Kv1.2 with similar potency. We designed and produced three AnTx variants: ([F32T]-AnTx, [N17A]-AnTx, [N17A/F32T]-AnTx) using solid-phase synthesis with the goal of improving the selectivity of the toxin for Kv1.3 over Kv1.2 while keeping the high affinity for Kv1.3. We used the patch-clamp technique to determine the blocking potency of the synthetic toxins on hKv1.3, mKv1.1, hKv1.2 and hKCa3.1 channels. Of the three variants [N17A/F32T]-AnTx maintained the high affinity of the natural peptide for Kv1.3 but became more than 16000-fold selective over Kv1.2. NMR data and molecular dynamics simulations suggest that the more rigid structure with restricted conformational space of the double substituted toxin compared to the flexible wild-type one is an important determinant of toxin selectivity. Our results provide the foundation for the possibility of the production and future therapeutic application of additional, even more selective toxins targeting various ion channels.
authors.affiliation: Department of Biophysics and Cell Biology, University of Debrecen, Egyetem tér 1, Debrecen, H-4012, Hungary.
authors.forename: Adam
authors.initials: A
authors.lastname: Bartok
authors.affiliation: Department of Inorganic and Analytical Chemistry, University of Debrecen, Egyetem tér 1, Debrecen, H-4032, Hungary.|Department of Organic and Macromolecular Chemistry, Ghent University, Ghent, Belgium.
authors.forename: Krisztina
authors.initials: K
authors.lastname: Fehér
authors.affiliation: Laboratory of Structural Chemistry and Biology, Institute of Chemistry, Eötvös Loránd University, Pázmány Péter sétány 1/A, Budapest, H-1117, Hungary.
authors.forename: Andrea
authors.initials: A
authors.lastname: Bodor
authors.affiliation: Department of Medical Chemistry, University of Szeged, Dóm tér 8, Szeged, H-6720, Hungary.
authors.forename: Kinga
authors.initials: K
authors.lastname: Rákosi
authors.affiliation: Department of Medical Chemistry, University of Szeged, Dóm tér 8, Szeged, H-6720, Hungary.
authors.forename: Gábor K
authors.initials: GK
authors.lastname: Tóth
authors.affiliation: Department of Inorganic and Analytical Chemistry, University of Debrecen, Egyetem tér 1, Debrecen, H-4032, Hungary.
authors.forename: Katalin E
authors.initials: KE
authors.lastname: Kövér
authors.affiliation: Department of Biophysics and Cell Biology, University of Debrecen, Egyetem tér 1, Debrecen, H-4012, Hungary.|MTA-DE Cell Biology and Signaling Research Group, University of Debrecen, Debrecen, Egyetem tér 1, H-4032, Hungary.
authors.forename: Gyorgy
authors.initials: G
authors.lastname: Panyi
authors.affiliation: MTA-DE-NAP B Ion Channel Structure-Function Research Group, RCMM, University of Debrecen, Debrecen, Egyetem tér 1, H-4032, Hungary.|Department of Biophysics and Cell Biology, University of Debrecen, Egyetem tér 1, Debrecen, H-4012, Hungary.
authors.forename: Zoltan
authors.initials: Z
authors.lastname: Varga
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects; D051664:Kv1.3 Potassium Channel / Q000378:metabolism*; D008214:Lymphocytes / Q000187:drug effects / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D012604:Scorpion Venoms / Q000138:chemical synthesis / Q000737:chemistry* / Q000302:isolation & purification / Q000633:toxicity*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013997:Time Factors
chemical_list: D051664:Kv1.3 Potassium Channel; D012604:Scorpion Venoms