Supplementary Figure 2: Improving the voltage sensitivity of FlicR1 on the screening platform.

(a) Alignment of amino acid sequence of the FlicR1 fluorophore cp-mApple with mApple sequence¹ and the sequence of R-GECO FP², which is a cp-mApple variant. (b) Alignment of amino acid sequences of cp-mApple in FlicR1 with the sequences of mApple, mRuby3 and RCaMP FP³, which is a cp-mRuby variant. Boxes indicate residues in the FP barrel which were targeted for saturation mutagenesis in FlicR1 based on sequence and/or structural homology with key residues that improved performance of R-GECO or RCaMP^2,3. (b) Top, Schematic of the FlicR2 construct fused to a nuclear localization signal (NLS)-tagged blue fluorescent protein (BFP) reporter. FlicR2 and NLS-BFP were interspersed by a T2A peptide (scissor). Mutations in the FP that improved voltage sensitivity are shown. Bottom, crystal structures of CiVSD (PDB 4G80) and cp-mApple in R-GECO1 (PDB 4I2Y) in a fusion configuration. S1-4 indicate transmembrane helices of the VSD. (c) Epifluorescence image of HEK cells expressing FlicR2 (n = 12 wells, 3 cultures). (d) Fluorescence responses from a FlicR2-transfected HEK cell held at –70 mV to depolarizing and hyperpolarizing voltage steps (∆20 mV). (e) Fluorescence-voltage (∆F/∆V) plots for FlicR1 and FlicR2 in HEK cells (n = 4 and 6 cells, respectively). Values represent mean ± s.e.m. (f) Epifluorescence images of FlicR2-electroporated pyramidal neurons (left) and magnified images of a soma (right) in an acute brain slice (n = 8 neurons). 1. Shaner, N. C. et al. Improving the photostability of bright monomeric orange and red fluorescent proteins. Nat. Methods 5, 545–551 (2008). 2. Zhao, Y. et al. An expanded palette of genetically encoded Ca²⁺ indicators. Science 333, 1888–1891 (2011). 3. Akerboom, J. et al. Genetically encoded calcium indicators for multi-color neural activity imaging and combination with optogenetics. Front. Mol. Neurosci. 6, 2 (2013).