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Histochemistry and Cell Biology

, Volume 117, Issue 3, pp 197–202 | Cite as

Ultrasensitive pharmacological characterisation of the voltage-gated potassium channel KV1.3 studied by single-molecule fluorescence microscopy

  • Günter Freudenthaler
  • Markus Axmann
  • Hansgeorg Schindler
  • Bernt Pragl
  • Hans-Günther Knaus
  • Gerhard J. Schütz
Original Paper

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 hongotoxin1 (HgTX1), which specifically binds to the voltage-gated potassium channel KV1.3 at a molecular stoichiometry of 1:1. A toxin analogue (HgTX1-A19C) was subjected to fluorescence labelling studies with Cy5. Utilising an ultrasensitive microscopic method (single-dye tracing; SDT) we were able to directly visualise HgTX1-A19C-Cy5 binding to the voltage-gated potassium channel KV1.3 on Jurkat cells at the single molecule level. For the first time, this approach allowed the determination of both the dissociation constant (KD) and the off-rate (koff) of HgTX1-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 HgTX1 analogue.

Jurkat cells Voltage-gated potassium channel KV1.3 Fluorescence microscopy Single-molecule microscopy Hongotoxin Pharmacology 

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Copyright information

© Springer-Verlag 2002

Authors and Affiliations

  • Günter Freudenthaler
    • 1
  • Markus Axmann
    • 1
  • Hansgeorg Schindler
    • 1
  • Bernt Pragl
    • 2
  • Hans-Günther Knaus
    • 2
  • Gerhard J. Schütz
    • 1
  1. 1.Institute for Biophysics, University of Linz, Altenbergerstrasse 69, 4040 LinzAustria
  2. 2.Institute for Biochemical Pharmacology, University of Innsbruck, Peter-Mayr-Strasse 1, 6020 InnsbruckAustria

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