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

Area of Science:

• Biochemistry
• Cell Biology
• Pharmacology

Background:

• Potassium (K+) channels and transporters are crucial drug targets.
• Previous development of a triazacryptand (TAC)-based K+ sensor (TAC-Red) for in vivo imaging.
• Need for improved methods to quantify K+ transport across cell membranes.

Purpose of the Study:

• To synthesize and validate a novel green-fluorescing extracellular K+ sensor, TAC-Limedex.
• To demonstrate the utility of TAC-Limedex for measuring K+ efflux and transport in cell-based assays.
• To adapt the assay for high-throughput screening.

Main Methods:

• Synthesis of TAC-Limedex, a dextran conjugate of TAC-bodipy.
• Characterization of TAC-Limedex fluorescence response to varying K+ concentrations, Na+, Cl-, and pH.
• Quantification of K+ efflux from HT-29 and SiHa cells using TAC-Limedex.
• Pharmacological manipulation of K+ channels and transporters.

Main Results:

• TAC-Limedex showed a 50% fluorescence increase with 0-2 mM K+ and was insensitive to other ions and pH.
• K+ efflux in HT-29 cells increased 8-fold upon ATP-induced K+ channel activation, inhibited by blockers.
• Electroneutral K+/Cl- cotransport in SiHa cells showed a 3-fold increase in K+ efflux under hypotonic conditions, fully inhibited by a blocker.
• Assay adapted to a fluorescence plate reader for automated screening.

Conclusions:

• TAC-Limedex is a sensitive and specific extracellular K+ sensor for cell membrane transport studies.
• The fluorescence-based assay effectively quantifies K+ efflux and transporter activity.
• This method offers a high-throughput alternative to radioactive assays for identifying K+ transport modulators.