Co‐staining of KCa3.1 Channels in NSCLC Cells with a Small‐Molecule Fluorescent Probe and Antibody‐Based Indirect Immunofluorescence

Autor: Sarah Maskri, Etmar Bulk, Oliver Koch, Kathrin Brömmel, Zoltán Pethő, Bernhard Wünsch, Albrecht Schwab, Thomas Budde, Marius Rieke
Jazyk: angličtina
Rok vydání: 2020
Předmět:
Boron Compounds
Patch-Clamp Techniques
KCa3.1 channel
01 natural sciences
Biochemistry
Stain
Antibodies
chemistry.chemical_compound
Mice
Very Important Paper
fluorescent probes
Drug Discovery
Acetamides
co-staining
Potassium Channel Blockers
Animals
Humans
General Pharmacology
Toxicology and Pharmaceutics
non-small cell lung cancer cells
Fluorescent Antibody Technique
Indirect
Fluorescent Dyes
Pharmacology
senicapoc derivatives
biology
Full Paper
Staining and Labeling
010405 organic chemistry
Chemistry
Organic Chemistry
Trityl Compounds
Full Papers
Intermediate-Conductance Calcium-Activated Potassium Channels
Fluorescence
Small molecule
Potassium channel
0104 chemical sciences
Staining
010404 medicinal & biomolecular chemistry
Docking (molecular)
A549 Cells
Biophysics
biology.protein
Molecular Medicine
Antibody
BODIPY
Protein Binding
Zdroj: Chemmedchem
ISSN: 1860-7187
1860-7179
Popis: The Ca2+ activated potassium channel 3.1 (KCa3.1) is involved in critical steps of the metastatic cascade, such as proliferation, migration, invasion and extravasation. Therefore, a fast and efficient protocol for imaging of KCa3.1 channels was envisaged. The novel fluorescently labeled small molecule imaging probes 1 and 2 were synthesized by connecting a dimethylpyrrole‐based BODIPY dye with a derivative of the KCa3.1 channel inhibitor senicapoc via linkers of different length. Patch‐clamp experiments revealed the inhibition of KCa3.1 channels by the probes confirming interaction with the channel. Both probes 1 and 2 were able to stain KCa3.1 channels in non‐small‐cell lung cancer (NSCLC) cells following a simple, fast and efficient protocol. Pre‐incubation with unlabeled senicapoc removed the punctate staining pattern showing the specificity of the new probes 1 and 2. Staining of the channel with the fluorescently labeled senicapoc derivatives 1 or 2 or with antibody‐based indirect immunofluorescence yielded identical or very similar densities of stained KCa3.1 channels. However, co‐staining using both methods did not lead to the expected overlapping punctate staining pattern. This observation was explained by docking studies showing that the antibody used for indirect immunofluorescence and the probes 1 and 2 label different channel populations. Whereas the antibody binds at the closed channel conformation, the probes 1 and 2 bind within the open channel.
Open vs. closed: The KCa3.1 channel is involved in critical steps of the metastatic cascade. Analyzing KCa3.1 channel expression has predictive power with respect to patient survival. Novel fluorescently labeled small‐molecule imaging probes were able to stain KCa3.1 channels in NSCLC cells selectively and efficiently. Co‐staining using antibody‐based indirect immunofluorescence did not lead to the expected overlapping punctate staining pattern. This observation was explained by docking studies showing that the antibody and the novel imaging probes label different channel populations, closed and open channels, respectively.
Databáze: OpenAIRE
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