Visualization of KCa3.1 Channels in Tumor Cells by Optimized Senicapoc-Bodipy Conjugates

Abstract

Upregulation of K_Ca3.1 channels was observed in highly aggressive tumor cells, such as non small cell lung cancer cells of the A549 line. In order to visualize K_Ca3.1 channels in these cells, novel fluorescent probes with increased polarity were designed. Key step of the synthesis was a 1,3-dipolar cycloaddition of senicapoc propargyl ether 4 with various azide substituted bodipy dyes. Due to their reduced lipophilicity and promising photophysical properties, the senicapoc-bodipy conjugates 7a (logP = 4.3) and 16 (logP = 4.4) were able to stain K_Ca3.1 ion channels in fixed, living, and permeabilized A549–3R tumor cells. The apparent size of the observed fluorescent dots indicates labeling of single K_Ca3.1 channels. The recorded density is in good accordance with literature values. The specificity of K_Ca3.1 labeling by the senicapoc-bodipy conjugates 7a and 16 was shown with HEK293 cells, blocking experiments and azide precursors. Subsequent staining of K_Ca3.1 ion channels with hydroxyphenyl derivative 16 and antibodies did not lead to overlapping (yellow) dots, as different states of the ion channel were stained by 16 (open state) and antibody (closed state). In patch clamp experiments, both senicapoc-bodipy conjugates 7a and 16 reduced the current density, although less efficiently than senicapoc. MD simulations showed weaker interactions of the amide moiety of 16 with Thr250, explaining the lower channel inhibition of the open-pore blocker 16 compared to senicapoc (1). Due to their optimal imaging properties, high specificity, balanced lipophilicity/hydrophilicity, and sufficient water solubility, senicapoc-bodipy conjugates 7a and 16 represent innovative diagnostic tools to image K_Ca3.1 channels.