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-Limedex") for use as an extracellular K+ sensor and demonstrated its utility in measuring K+ transport across cell membranes. TAC-Limedex 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 micromol/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.
Namkung
Wan
W
Department of Medicine, University of California, San Francisco, California 94143-0521, USA.
Padmawar
Prashant
P
Mills
Aaron D
AD
Verkman
A S
AS
D001681:Biological Assay / Q000379:methods*; D001692:Biological Transport; D001896:Boron Compounds / Q000138:chemical synthesis* / Q000737:chemistry; D003911:Dextrans / Q000138:chemical synthesis* / Q000737:chemistry; D005456:Fluorescent Dyes / Q000138:chemical synthesis* / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000138:chemical synthesis / Q000737:chemistry; D019073:HT29 Cells; D006801:Humans; D049408:Luminescent Agents / Q000138:chemical synthesis / Q000737:chemistry; D015394:Molecular Structure; D011188:Potassium / Q000378:metabolism*; D015221:Potassium Channels / Q000032:analysis* / Q000378:metabolism; D012995:Solubility; D014867:Water / Q000737:chemistry
D001896:Boron Compounds; D003911:Dextrans; D005456:Fluorescent Dyes; D049408:Luminescent Agents; D015221:Potassium Channels; D014867:Water; D049452:Green Fluorescent Proteins; D011188:Potassium