V220 Mutants of Ciona Voltage-Sensing Domain Based Genetically Encoded Fluorescent Voltage Sensors

Abstract

Genetically-encoded fluorescent voltage sensors enable the optical reporting of changes in membrane potentials. These sensors consist of a voltage-sensing domain of voltage-sensing phosphatase (usually from Ciona intestinalis Voltage-Sensing phosphatase) fused to a fluorescent protein (Super Ecliptic pHlorin A227D). To optically differentiate action potentials from sub-threshold synaptic activity, residues in the voltage-sensing domain were mutated. Surprisingly one mutation, V220T in the S4 domain shifted the voltage response to more negative potentials, since this residue does not interact with the positively charged residues in S4 responsible for gating currents. To fine tune the voltage sensitivity of this probe, we mutated the V220 position extensively testing the remaining 19 amino acids. We found that tyrosine, phenylalanine, tryptophan, aspartate, threonine, and proline shift the voltage response substantially to more negative potentials enabling optical signals from hyper-polarizing voltages. Polar amino acids (glutamine, asparagine, alanine, and serine) vastly reduce the optical signal. Hydrophobic residues leucine, isoleucine, methionine, cysteine do not shift the voltage sensitivity to more negative potentials. We combined another mutant D164N (S2 domain) capable of shifting the voltage sensitivity to left with V220F and P. This combination further shifts the voltage response to more negative potentials and shallows the curve. These results suggest that the V220 residue interacts with the plasma membrane and could be mutated in conjunction with other mutations to generate probes capable of differentiating action potentials from sub-threshold synaptic activity, and hyper-polarizing inhibitory activity.