Fluorescent Probes to Image the KCa3.1 Channel in Tumor Cells

<b>Background/Objectives</b>: The Ca²⁺-activated K⁺ channel K_Ca3.1 is not only involved in physiological processes such as immune reactions and control of vascular tone, but is highly expressed in various tumor entities. Thus, imaging of K_Ca3.1 channels comes into focus for the localization of high channel density, i.e., for tumor diagnosis. In particular, the physicochemical properties of the fluorescent probes should be improved compared to existing probes. <b>Methods</b>: The small molecule inhibitor of the K_Ca3.1 channel, senicapoc, was used as a warhead and was coupled with different fluorescent dyes. After synthesis of the novel probes, their physicochemical properties (lipophilicity, photophysical properties) and their ability to image K_Ca3.1 channels in A549-3R lung tumor cells were determined. <b>Results</b>: In order to increase the polarity and quantum yield of reported fluorescent probes, three strategies were followed: (1) An F-atom at the B-atom of bodipy-labeled senicapoc derivatives 9a, 9b, and 15a was replaced by a OCH₃ moiety, which decreased the logP value by one log-unit. (2) The <i>p</i>-phenylene moiety of the linker was replaced by an aliphatic tetramethylene linker decreasing the lipophilicity by 0.3-0.5 log-units. (3) Instead of bodipy dyes, fluorescein was coupled with the senicapoc warhead resulting in very polar probes 21a and 21b with low logP values of 1.5 and 1.3, respectively. Introduction of an ethyl moiety at the bodipy core increased the quantum yield, which resulted in the best punctate staining pattern of fixed and living A549-3R lung tumor cells with the ethylbodipy-labeled senicapoc derivative 10b. The specificity was shown by various control experiments. Co-staining with 10b and an antibody did not result in overlapping signals. <b>Conclusions</b>: The well-balanced lipophilicity and fluorescent quantum yield render the ethylbodipy-labeled senicapoc derivative 10b a very good probe to image selectively K_Ca3.1 ion channels in fixed and living tumor cells. It was hypothesized that the antibody binds selectively at the closed channel (58.5%), whereas the senicapoc-bodipy conjugate <b>10b</b> binds selectively at the open channel (41.5%). The ratio 58.5:41.5 reflects the ratio of the ion channel in closed and open conformations.