Small‐molecule probes for the in vitro imaging of KCa3.1 channel‐expressing cells were developed. Senicapoc, showing high affinity and selectivity for the KCa3.1 channels, was chosen as the targeting component. BODIPY dyes 15–20 were synthesized and connected by a CuI‐catalyzed azide–alkyne [3+2]cycloaddition with propargyl ether senicapoc derivative 8, yielding fluorescently labeled ligands 21–26. The dimethylpyrrole‐based imaging probes 25 and 26 allow staining of KCa3.1 channels in NSCLC cells. The specificity was shown by removing the punctate staining pattern by pre‐incubation with senicapoc. The density of KCa3.1 channels detected with 25 and by immunostaining was identical. The punctate structure of the labeled channels could also be observed in living cells. Molecular modeling showed binding of the senicapoc‐targeting component towards the binding site within the ion channel and orientation of the linker with the dye along the inner surface of the ion channel.

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.

Small-molecule probes for the in vitro imaging of K Ca 3.1 channel-expressing cells were developed. Senicapoc, showing high affinity and selectivity for the K Ca 3.1 channels, was chosen as the targeting component. BODIPY dyes 15-20 were synthesized and connected by aC u I -catalyzed azidealkyne [3+ +2]cycloaddition with propargyl ether senicapoc derivative 8,yielding fluorescently labeled ligands 21-26.The dimethylpyrrole-based imaging probes 25 and 26 allow staining of K Ca 3.1 channels in NSCLC cells.T he specificity was shown by removing the punctate staining pattern by preincubation with senicapoc.T he density of K Ca 3.1 channels detected with 25 and by immunostaining was identical. The punctate structure of the labeled channels could also be observed in living cells.M olecular modeling showed binding of the senicapoc-targeting component towards the binding site within the ion channel and orientation of the linker with the dye along the inner surface of the ion channel.Supportinginformation and the ORCID identification number(s) for the author(s) of this article can be found under: https://doi.

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Expression of the Ca2+ activated potassium channel 3.1 (KCa3.1) channel (also known as the Gàrdos channel) is dysregulated in many tumor entities and has predictive power with respect to patient survival.