Extended Data Fig. 1: Optimization of cpGFP insertion site into OX2R.
From: A genetically encoded sensor for in vivo imaging of orexin neuropeptides
a, Sequence alignment of transmembrane domains (TM) 5 and 6 from OX2R, OX1R and DRD1 indicating the insertion site for the cpGFP module from dLight1. Color code is based on percent identity between orexin receptors and DRD1. b, Representative images showing membrane expression profile of sensor prototypes based on either the OX1 or OX2 receptor in HEK293T cells. c, Membrane expression and fluorescence response to 10 µM OXA of the OX1 and OX2 sensor prototypes shown in a-b. p = 0.3510, for the membrane expression and p = 1.109 × 10−7 for the fluorescence response. n = 15 cells from 3 independent experiments. d-i, cpGFP Insertion point optimization on the TM6 and TM5 of OX2R. The variant showing best membrane expression and response to 10 µM OXA was called OxLight0.1. d, Schematic representation of TM6 insertion site optimization. e, Representative images of TM6 insertion sites variants. f, Quantification of fluorescence responses to 10 µM OXA and surface expression from variants shown in e. p = 0.0047 for OX2 prototype response compared to OxLight0.1. n = 12 and 16 cells for OX2 prototype and OxLight0.1 or ≥ 5 cells for all other variants. g, Schematic representation of TM5 insertion site optimization. h, Representative images of TM5 insertion sites variants. i, Quantification of responses to 10 µM OXA and surface expression from variants shown in h for all variants. j-k, In vitro characterization of OxLight0.1 in HEK293T cells. j, Representative images of OxLight0.1 pre and post addition of 10 µM OXA. k, Response of OxLight0.1 to sequential addition of OXA or OXB followed by dual orexin receptor antagonist EMPA. n = 26 and 31 cells for OXA + EMPA and OXB + EMPA, respectively. All data are shown as mean ± s.e.m. All scale bars, 10 µm. All statistical tests performed using two-tailed student’s t-test with Welch’s correction.
