Cell-based fluorescence screen for K⁺ channels and transporters using an extracellular triazacryptand-based K⁺ sensor

K⁺ channels and K⁺-coupled membrane transporters are important targets for drug discovery. We previously developed a triazacryptand (TAC)-based K⁺ sensor, TAC-Red, and demonstrated its utility to image K⁺ waves in mouse brain in vivo (Padmawar et al. Nat. Methods. 2005, 2, 825-827). Here, we synthesized a green-fluorescing dextran conjugate of TAC-bodipy ("TAC-Lime_dex") for use as an extracellular K⁺ sensor and demonstrated its utility in measuring K⁺ transport across cell membranes. TAC-Lime_dex fluorescence increased by 50% with increasing [K⁺] from 0 to 2 mM and was insensitive to [Na⁺], [Cl^-], or pH. K⁺ efflux from cells was quantified from increasing extracellular TAC-Lime_dex fluorescence following cell immersion in K⁺-free buffer. In HT-29 cells, K⁺ efflux was 2.0 ± 0.1 μmol/cm²/s, increasing 8-fold following K⁺ channel activation by ATP; the increase in K⁺ efflux was inhibited by a K⁺ channel blocker or by preventing cytoplasmic calcium elevation. Electroneutral K⁺/Cl^- cotransport was demonstrated in SiHa cells, in which K⁺ efflux was increased 3-fold by hypotonic challenge; the increase in K⁺ efflux was fully inhibited by a K⁺/Cl^- transport blocker. K⁺ efflux measurements were adapted to a commercial fluorescence platereader for automated screening. The fluorescence-based K⁺ transport assay largely replaces assays requiring radioactive rubidium and is suitable for high-throughput identification of K⁺ transport modulators.