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

The Ca(2+)activated potassium channel 3.1 (K(Ca)3.1) 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 K(Ca)3.1 channels was envisaged. The novel fluorescently labeled small molecule imaging probes1and2were synthesized by connecting a dimethylpyrrole-based BODIPY dye with a derivative of the K(Ca)3.1 channel inhibitor senicapoc via linkers of different length. Patch-clamp experiments revealed the inhibition of K(Ca)3.1 channels by the probes confirming interaction with the channel. Both probes1and2were able to stain K(Ca)3.1 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 probes1and2. Staining of the channel with the fluorescently labeled senicapoc derivatives1or2or with antibody-based indirect immunofluorescence yielded identical or very similar densities of stained K(Ca)3.1 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 probes1and2label different channel populations. Whereas the antibody binds at the closed channel conformation, the probes1and2bind within the open channel.