Pado, a Novel Fluorescent Voltage-Sensing Protein, Identified by a Highly Conserved Motif in the S2 Trans-Membrane Segment

Abstract

A highly conserved sequence motif in the S2 trans-membrane segment of the voltage-sensing domain was used to search and identify novel voltage-sensing proteins. This motif pulled down in silico proteins with homology to voltage-gated calcium, potassium, sodium and proton channels, as well as voltage-sensing phosphatases in addition to several proteins of unknown function. To test for voltage-sensitive optical responses we fused the putative voltage-sensing domain from 8 novel proteins to the fluorescent protein super ecliptic pHlorin A227D. The cytosolic amino terminus of the Ciona voltage sensing phosphatase was used to improve plasma membrane expression. The voltage-gated proton channel from liver fluke, which we call Pado (파도), gave a large optical signal (>10%) in response to a 200mV depolarization in HEK293. Furthermore, a cation current was detected above a threshold of about 180mV which is associated with an increase in the baseline fluorescence. Inhibition with Zn2+ inhibits the movement of S4 and caused a substantial decrease in the optical signal upon membrane depolarization. Decreasing the extracellular pH resulted in a reduced current and also reduced the optical signal upon membrane depolarization. These results suggest that the voltage-gated current is due to the voltage-gated proton channel and that the corresponding change in internal pH affects the fluorescent intensity of Pado (파도). This search criterion is capable of identifying novel voltage-gated proteins that can be used to generate voltage-sensing probes.