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

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 _V1.3 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 _V1.3 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.