Screening fluorescent voltage indicators with spontaneously spiking HEK cells

Area of Science:

• Neuroscience
• Molecular Biology
• Biophysics

Background:

• Developing fluorescent voltage indicators is crucial for neuroscience.
• Patch-clamp characterization limits the throughput of voltage indicator development.
• Novel methods are needed for rapid screening of voltage-sensitive dyes.

Purpose of the Study:

• To develop a high-throughput screening method for voltage-sensitive dyes.
• To identify novel voltage-sensitive fluorescent proteins with enhanced properties.

Main Methods:

• Engineered HEK cells stably expressing NaV 1.3 and KIR 2.1 channels to generate spontaneous action potentials.
• Utilized a standard fluorescence microscope for electrode-free screening.
• Screened a library of archaerhodopsin 3 (Arch) mutants in the engineered cells.

Main Results:

• Successfully established a line of non-fluorescent HEK cells with spontaneous electrical activity.
• Developed a rapid, electrode-free screening platform for voltage-sensitive dyes.
• Identified two Arch mutants exhibiting superior voltage sensitivity compared to existing indicators.

Conclusions:

• The developed HEK cell line and screening platform significantly accelerate the discovery of novel voltage indicators.
• This advancement facilitates the development of improved tools for neuroscience research.
• The identified Arch mutants represent promising candidates for future voltage imaging applications.