title: Ultrasensitive pharmacological characterisation of the voltage-gated potassium channel K(V)1.3 studied by single-molecule fluorescence microscopy.
abstract: The determination of pharmacologically relevant constants is crucial in order to understand the effects of compounds interacting with various membrane receptors. In this report we study a venom component of the Central American scorpion Centruroides limbatus, a short peptide termed hongotoxin(1) (HgTX(1)), which specifically binds to the voltage-gated potassium channel K(V)1.3 at a molecular stoichiometry of 1:1. A toxin analogue (HgTX(1)-A19C) was subjected to fluorescence labelling studies with Cy5. Utilising an ultrasensitive microscopic method (single-dye tracing; SDT) we were able to directly visualise HgTX(1)-A19C-Cy5 binding to the voltage-gated potassium channel K(V)1.3 on Jurkat cells at the single molecule level. For the first time, this approach allowed the determination of both the dissociation constant (K(D)) and the off-rate (k(off)) of HgTX(1)-A19C-Cy5 on living cells. In order to validate this novel approach, the data obtained with SDT were correlated to radioligand binding studies performed under identical conditions using a radioiodinated HgTX(1) analogue.
authors.affiliation: Institute for Biophysics, University of Linz, Altenbergerstrasse 69, 4040 Linz, Austria.
authors.forename: Günter
authors.initials: G
authors.lastname: Freudenthaler
authors.forename: Markus
authors.initials: M
authors.lastname: Axmann
authors.forename: Hansgeorg
authors.initials: H
authors.lastname: Schindler
authors.forename: Bernt
authors.initials: B
authors.lastname: Pragl
authors.forename: Hans-Günther
authors.initials: HG
authors.lastname: Knaus
authors.forename: Gerhard J
authors.initials: GJ
authors.lastname: Schütz
mesh_terms: D001667:Binding, Competitive / Q000187:drug effects; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007457:Iodine Radioisotopes; D019169:Jurkat Cells; D051664:Kv1.3 Potassium Channel; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods*; D009154:Mutation; D009498:Neurotoxins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D015221:Potassium Channels / Q000235:genetics / Q000378:metabolism*; D024642:Potassium Channels, Voltage-Gated; D011869:Radioligand Assay; D012604:Scorpion Venoms / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D014162:Transfection
chemical_list: D007457:Iodine Radioisotopes; C496973:KCNA3 protein, human; D051664:Kv1.3 Potassium Channel; D009498:Neurotoxins; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D012604:Scorpion Venoms; C110392:hongotoxin 1