Development of a BRET based chloride biosensor for high throughput screening of KCC2 modulators

Area of Science:

• Neuroscience
• Biochemistry
• Molecular Biology

Background:

• Chloride ion transport is crucial for neuronal action potentials and physiological processes.
• Existing FRET-based chloride imaging lacks high-throughput screening capabilities for drug discovery.
• Novel methodologies are needed to accelerate the identification of chloride transport modulators.

Purpose of the Study:

• To develop a novel BRET-based biosensor for high-throughput screening of chloride ion transport.
• To enable real-time kinetic measurement of chloride channel and transporter modulators in living cells.
• To validate the biosensor's utility with known modulators of WNK, KCC2, and NKCC1.

Main Methods:

• Development of a BRET-based biosensor (Glorider) by fusing a chloride-sensitive GFP variant to NanoLuciferase.
• Utilizing the Glorider biosensor for kinetic measurements in living cells.
• Assessing the effects of WNK, KCC2, and NKCC1 modulators, including KCC2 agonists.

Main Results:

• Successfully developed and validated the Glorider BRET-based biosensor.
• Demonstrated real-time kinetic measurement of chloride transport modulators.
• Showcased the biosensor's applicability to WNK, KCC2, and NKCC1 modulators in living cells.

Conclusions:

• The Glorider biosensor offers a novel, high-throughput screening method for chloride transport.
• This BRET-based approach facilitates real-time kinetic analysis of drug effects.
• Glorider advances drug discovery for conditions involving chloride ion dysregulation.