Journal
HISTOCHEMISTRY AND CELL BIOLOGY
Volume 117, Issue 3, Pages 197-202Publisher
SPRINGER
DOI: 10.1007/s00418-001-0374-y
Keywords
Jurkat cells; voltage-gated potassium channel Kv1.3 fluorescence microscopy; single-molecule microscopy; hongotoxin; pharmacology
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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, (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.
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