Figure 1.

Design of a light-gated Ca²⁺-permeant cation channel. (A) Photocurrents of Xenopus oocytes expressing the bovine olfactory cyclic nucleotide gated channel (OLF) and the photoactivatable adenylyl cyclase (bPAC) and various fusion constructs. Total cRNA amounts were adjusted to keep the copies of injected OLF constant. Ratios of cRNA mixtures are indicated. Experiments were performed employing blue light illumination (0.2 s, 1 mW/mm², 473 nm, n = 6). (B) cAMP production of different OLF and bPAC fusion constructs or mixes; blue light (473 nm, 0.3 mW/mm²); n = 3 experiments, each with 6 oocytes; error bars = SD. (C) Schematic of the designed OLF (T537S) and bPAC fusion construct. T, plasma membrane trafficking signal; Ex, ER export signal. (D) Fluorescence picture of Xenopus oocyte expressing OLF-T-YFP-bPAC-Ex. (E) Example photocurrent of OLF-T-YFP-bPAC-Ex and 1:1 mixture of OLF and bPAC. Holding potential −60 mV; illumination with 473 nm blue light, 1 mW/mm², red dashed arrow indicates 0 nA. (F) Kinetics of photocurrents in oocytes expressing OLF-T-YFP-bPAC-Ex and 1:1 mixture of OLF and bPAC. Light intensity was adjusted to evoke currents of ~0.6 μA. n = 3, error bars = SD. (G) Example OLF-T-YFP-bPAC-Ex photocurrent traces from 1 oocyte induced by 1 s 473 nm light of different intensities, ~15 min recovery time in the dark for each illumination. (H) OLF-T-YFP-bPAC-Ex photocurrents induced by light of different intensities. n = 4, error bars = SD. (I) A current recording trace from an oocyte expressing OLF-T-YFP-bPAC-Ex with 1 s blue light (473 nm, 550 μW/mm²) illumination and switching bath solutions containing different cations (115Na 2Ba²⁺: 115 mM NaCl, 2 mM BaCl₂; 80Ba²⁺: 80 mM BaCl₂; 80Ca²⁺: 80 mM CaCl_2. All buffers contain 1 mM MgCl₂, 5 mM HEPES and pH adjusted to 7.6). Orange line indicates basal current, arrows indicate light pulse, ~15 min recovery time in the dark between each illumination.