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

G Freudenthaler, M Axmann, H Schindler, B Pragl, HG Knaus, GJ Schütz

Research output: Contribution to journalArticlepeer-review

23 Citations (Scopus)

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 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.

Original languageEnglish
Pages (from-to)197-202
Number of pages6
JournalHistochemistry and Cell Biology
Volume117
Issue number3
DOIs
Publication statusPublished - Mar 2002

Keywords

  • Fluorescence microscopy
  • Hongotoxin
  • Jurkat cells
  • Pharmacology
  • Single-molecule microscopy
  • Voltage-gated potassium channel K 1.3
  • Cell Line
  • Potassium Channels, Voltage-Gated
  • Iodine Radioisotopes
  • Jurkat Cells
  • Humans
  • Scorpion Venoms/genetics
  • Neurotoxins/genetics
  • Dose-Response Relationship, Drug
  • Kv1.3 Potassium Channel
  • Microscopy, Fluorescence/instrumentation
  • Potassium Channels/genetics
  • Transfection
  • Binding, Competitive/drug effects
  • Mutation
  • Radioligand Assay

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