Ultrasensitive pharmacological characterisation of the voltage-gated potassium channel K(V)1.3 studied by single-molecule fluorescence microscopy

Histochemistry and Cell Biology
Günter FreudenthalerG J Schütz

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.

Citations

Jan 12, 2013·Analytical and Bioanalytical Chemistry·Kseniya S KudryashovaAlexey V Feofanov
Jul 19, 2008·Methods : a Companion to Methods in Enzymology·Stefan Wieser, Gerhard J Schütz
Jan 3, 2007·Biochemical and Biophysical Research Communications·Christian LegrosPierre E Bougis
Jul 13, 2004·Current Opinion in Immunology·Peter Friedl
May 4, 2004·Trends in Pharmacological Sciences·K George ChandyMichael D Cahalan
Feb 16, 2016·Biochemistry. Biokhimii︠a︡·A I KuzmenkovA A Vassilevski
Dec 17, 2002·Histochemistry and Cell Biology·Esther Asan
Jan 4, 2003·The Journal of Biological Chemistry·Christine BeetonK George Chandy
Sep 1, 2011·The Journal of Physical Chemistry. B·Morteza KhabiriRüdiger Ettrich

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