Synthesized genes encoding KnRh3 and CrChR2 (1-315 amino acids) were subcloned into a phKR2-3.0-eYFP plasmid (gifted by Dr. H. Yawo (University of Tokyo, Japan)) using an In-Fusion HD cloning kit (Takara Bio, Shiga, Japan). Length variants of KnRh3 (amino acid length of 272, 280, 290, 300, 310, 317, 321, 397, 697, and 831) were created by using appropriate PCR primers. For immunostaining experient, the plasmid pKnRh3(697 amino acids)-3.0-eYFP was inserted N-QKLISEEDL-C (10 amino acids, c-Myc epitope tag) in the C-terminal of eYFP using inverse PCR. Site-directed mutagenesis was performed using a QuikChange site-directed mutagenesis kit (Agilent, CA, USA). All the constructs were verified by DNA sequencing (Fasmac Co., Ltd. Kanagawa, Japan).
Mammalian cell culture
Electrophysiological assays and immunostaining of KnRh3 and CrChR2 were performed on ND7/23 cells, which are hybrid cell lines derived from neonatal rat dorsal root ganglia neurons fused with mouse neuroblastoma. ND7/23 cells were grown on a coverslip in Dulbecco's modified Eagle’s medium (DMEM; Wako, Osaka, Japan) supplemented with 2.0 mM of all-trans retinal and 5% fetal bovine serum, and under a 5% CO2 atmosphere at 37°C. The expression plasmids were transiently transfected by using the FuGENE HD transfection Reagent (Promega, Fitchburg, WI, USA) according to the manufacturer’s instructions. Electrophysiological recordings were then conducted 24-36 h after transfection. Successfully transfected cells were identified by eYFP fluorescence under a microscope prior to measurements.
Immunostaining of mammalian cells
ND7/23 cells were cultured on glass coverslips. Cells expressing KnRh3 or KnRh3 bearing the c-Myc epitope tag at the C-terminus were fixed in 4% paraformaldehyde phosphate buffer solution for 15 min at room temperature. Cells were then washed with phosphate-buffered saline (PBS) for three times. When necessary, cells were permeabilized with 0.5% Triton X-100 for 15 min at room temperature. Cells were treated with blocking buffer consisting of 3% goat serum for 60 min at room temperature. After blocking, the cells were incubated with rabbit anti–c-Myc primary antibody (C3956; Sigma-Aldrich, St. Louis, MO, USA) at a 1:500 dilution for 60 min at room temperature, then washed with PBS for three times and labeled with goat anti-rabbit IgG secondary antibody Alexa Fluor 594 (A-11037; Thermo Fisher Scientific, Waltham, MA, USA) at a 1:200 dilution for 2 h at room temperature. After a final wash with PBS, the coverslips were mounted on glass slides with ProLong Diamond Antifade Mountant (Thermo Fisher Scientific).
Live cultured KnRh3 bearing the c-Myc epitope tag at the C-terminus expressing-cells were washed with PBS. The cells were moved from the culture medium to serum-free DMEM and incubated with rabbit anti–c-Myc primary antibody at a 1:500 dilution for 60 min under a 5% CO2 atmosphere at 37°C. The cells were washed with PBS twice before fixing with 4% paraformaldehyde phosphate buffer solution for 15 min at room temperature. The cells were washed with PBS three times before labeling with goat anti-rabbit IgG secondary antibody Alexa Fluor 594 at a 1:200 dilution for 2 h at room temperature. After a final wash with PBS, the coverslips were mounted on glass slides with ProLong Diamond Antifade Mountant.
Fluorescent images were acquired with a confocal microscopy (LSM880, CarlZeiss, Jena, Germany) equipped with ×40 objective lens.
All experiments were carried out at room temperature (22 ± 2°C). Photocurrents were recorded by an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA, USA) under a whole-cell patch clamp configuration14. Data were filtered at 5 kHz and sampled at 20 kHz (Digdata1550, Molecular Devices, Sunnyvale, CA, USA) and stored in a computer (pClamp10.7, Molecular Devices). Pipette resistance was 3–6 MΩ. The standard internal pipette solution for the whole-cell voltage clamp contained (in mM) 126 NaAsp, 0.5 CaCl2, 2 MgCl2, 5 EGTA, 25 HEPES, 12.2 NMG, and adjusted to pH 7.4 by citric acid. The standard extracellular solution for the whole-cell voltage clamp contained (in mM) 150 NaCl, 1.8 CaCl2, 1 MgCl2, and 10 HEPES, 10 NMG, 5 glucose, and adjusted to pH 7.4 by NMG. Internal and external solutions for ion selectivity are shown in Table 1. The liquid junction potential was calculated and compensated by pClamp 10.7 software. Time constants were determined by a single exponential fit unless indicated otherwise.
Measurment solution component for ion selectivity
For whole-cell voltage clamp, irradiation at 480 nm was carried out using collimated LED (parts No. LCS-0470-03-22, Mightex, Toronto, Canada) controlled by computer software (pCLAMP10.7, Molecular Devices). Light power was measured directly by an objective lens of a microscope by a power meter (LP1, Sanwa Electric Instruments Co., Ltd., Tokyo, Japan). All action spectra were measured at the same light intensity in the range of 410 nm to 650 nm by a xenon light source OSG (Hamamatsu photonics, Hamamatsu, Japan).
Statistical validation of the data
Data in the text and figures are expressed as mean ± SEM and were evaluated with the Mann-Whitney U test for statistical significance, unless noted otherwise. Means were judged as statistically insignificant when P > 0.05.