Optically Tracing Electrical Synapses with Proton Channel-Based Voltage Sensing Protein

Abstract

The field of optogenetics has extensively improved over the last couple years allowing optical detection of single action potentials [CITE PAPERS]. In order to improve voltage sensors, a search strategy using the highly conserved S2 trans-membrane motif sequence identified 8 novel voltage sensing proteins. These proteins were fused to the fluorescent super ecliptic pHluorin A227D and transfected to HEK293 cells. The voltage sensing domain of a proton channel from liver fluke resulted in a sensor, Pado, which gave large optical signal and also responded to the induced pH change in the cell. This proton channel exhibits inhibition by extracellular Zn2+ and shifted response curves in differing pH environments. Higher buffering of the internal solution resulted in diminished pH dependent fluorescence change. With the ability to controllably manipulate pH of a cell, gap junctions can be traced through a network of HEK cells. Pado also provides a tool to screen fps for pH and voltage related activities. Ultimately Pado would make it possible to map electrical synapses and voltage activity simultaneously.