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