Extended Data Fig. 3: Biophysical characterization of FcR1 and comparison to bacteriorhodopsin.
(a) Photocurrent of FcR1 wild-type without N and C terminal modifications measured using two-electrode voltage clamp in two Xenopus laevis oocytes. (b) Schematics of DNA constructs used for two-electrode voltage clamp analysis in X. laevis oocytes. Orange, amino acids 1–105 of rat gastric H+ /K+ -ATPase β-subunit fragment (β-linker); sky blue, amino acids 1–10 of Coccomyxa subellipsoidea rhodopsin (CsR); bluish green, N-terminally truncated FcR1 (FcR1Δ27); yellow, enhanced yellow fluorescent protein (eYFP); gray, FLAG-tag (FLAG). (c) Photocurrents of different FcR1 expression constructs. Statistics are displayed as mean ± s.d. (n = 4) together with individual data points. (d) FcR1 and bacteriorhodopsin (BR) photocurrents with and without additional all-trans retinal (Ret). +Ret (orange), incubation of oocytes in ND96 buffer containing 1 µM Ret. –Ret (green), no additional Ret in ND96 buffer. +1dpi Ret (light blue), 1 µM Ret was added to ND96 buffer one day post oocyte injection (1 dpi). Photocurrent was measured at a membrane potential of -30 mV in Ori NMG pH 7.5 buffer. A 532 nm green light laser was used for illumination. Statistics are displayed as mean ± s.d. (n = 4). (e) Representative green light-induced photocurrent traces of FcR1 R118H mutant in three different extracellular buffers: NMG Asp pH 7.7, NMG pH 7.6 and Ori BaCl2 pH 7.6. Currents were recorded at incremental membrane potential steps of 30 mV from -120 mV to +30 mV.
