Patch-Clamp Analysis of Ciliary Membrane Currents

Ciliary membrane currents were recorded from single ORCs with a WC recording configuration (Hamill et al., 1981 (link)) under the voltage-clamp mode (V_h = −54 mV) as previously described (Takeuchi and Kurahashi, 2002 (link); 2019 (link)). The culture dish was mounted on the stage of laser scanning microscopy (LSM, Axiovert 510 system; Carl Zeiss Microimaging GmbH). Patch pipettes (resistance, 10–15 MΩ) were made from borosilicate tubing with filaments (outer diameter, 1.2 mm; World Precision Instruments) using a two-stage vertical patch electrode puller (PP-830; Narishige). The recording pipette was filled with Cs⁺ solution containing (in mM) 119 CsCl, 1 CaCl₂, 5 EGTA, 10 HEPES, and 0.001% phenol red (pH 7.4 adjusted using CsOH), as well as 1 mM caged cAMP (catalog number 116810; Calbiochem; Merck Millipore) and 50 µM Fluo-4 (F14200; Invitrogen; Thermo Fisher Scientific). Current signals were I-V converted using a 200B amplifier (Molecular Devices LLC), and data were sampled using pCLAMP ver.10 (Molecular Devices LLC) at 10 kHz, after being low-pass filtered at 2 kHz. For curve drawings of the membrane current, some data were low-pass FFT-filtered at 0.02 kHz. Care was taken to avoid saturation of response, particularly when evaluating adaptation. All experiments were performed at room temperature (23–25°C).