Abstract
Zahra is a fluorescent protein (FP) voltage sensor of membrane potential that exhibits fast kinetics (Baker et al., 2012). Archlight is another FP voltage sensor that gives a large signal in response to membrane depolarizations (Jin et al., 2012). Zahra utilizes the voltage-sensing domain from the Danio rerio voltage-sensing phosphatase (VSP), while Archlight is based on the voltage-sensing domain from the Ciona intestinealis VSP. In an attempt to make Archlight faster we aligned the transmembrane domains from several VSP homologs. Some of the conserved residues that were charged or polar were also conserved in the transmembrane domains of voltage-gated potassium channels, voltage-gated sodium channels, voltage-gated calcium channels, and voltage-gated proton pumps. Mutagenesis of these residues affected the speed of the fluorescent response, the size of the fluorescent response, and the voltage-dependence of the fluorescent response. We have designed mutant modules of each transmembrane domain so that we may tweak this electrical lattice to yield a genetically-encoded sensor that exhibits large fluorescent changes with fast kinetics. The best sensor at the time of this abstract gives a 5% fractional fluorescent change in response to 100 mV depolarization with a tauon of <2.6 ms and a tauoff <2.4 ms.This work was supported by the World Class Institute (WCI) Program of the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology of Korea (MEST) (NRF Grant Number:WCI 2009-003) and by US NIH Grant DC005259.
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