Mechanism of ArcLight derived GEVIs involves electrostatic interactions that can affect proton wires

Area of Science:

• Biophysics
• Molecular Biology
• Neuroscience

Background:

• Genetically encoded voltage indicators (GEVIs) like ArcLight utilize fluorescent proteins (FPs) for voltage sensing.
• Intermolecular electrostatic interactions between FPs mediate voltage-dependent optical signals.

Purpose of the Study:

• To improve the voltage sensitivity of ArcLight and its derivatives.
• To investigate the mechanisms underlying voltage-dependent fluorescence changes.
• To explore wavelength-specific responses and potential applications in cellular electrophysiology.

Main Methods:

• Random mutagenesis of FPs to introduce external charges.
• Voltage-clamp fluorometry with alternating excitation wavelengths (390 nm and 470 nm).
• Analysis of mutant responses to varying membrane potentials.

Main Results:

• A negative charge mutation increased signal >10-fold.
• Repositioning the charge reversed signal polarity, indicating "hot spots" of interaction.
• Mutants exhibited wavelength-dependent, ratiometric signals with opposing polarities.
• Specific mutation (E222H) showed voltage-dependent fluorescence increase at 390 nm, affecting the proton wire.

Conclusions:

• External electrostatic pathways in FPs influence fluorescence in a wavelength-specific manner.
• Mutated FPs offer novel ways to probe chromophore fluorescence and conformational changes.
• These engineered sensors provide new tools for mapping cellular electrical activity.