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 (TAG)-based K(+) sensor, TAG-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(dax)) 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-Limedex fluorescence following cell immersion in K(+)-free buffer. In HT-29 cells, K(+) efflux was 2.0 +/- 0.1 mu mol/cm(2)/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.