Tw150f 4

The Axopatch 200B Patch Clamp Amplifier (Axon Instruments, Foster City, CA, USA) was used for whole-cell mode patch clamping to record K⁺ currents from single HCFs. The K⁺ currents were filtered at 2 kHz and digitized at 10 kHz. pCLAMP 9.0 software (Axon Instruments) was used for data acquisition and analysis of the whole-cell currents. The recording patch pipettes were prepared from filament-containing borosilicate tubes (TW150F-4; World Precision Instruments, Sarasota, FL, USA) using a two-stage microelectrode puller (PC-10; Narishige, Tokyo, Japan) and were fire-polished using a microforge (MF-830; Narishige). The pipetted material exhibited a resistance of 2–3 MΩ. All electrophysiological experiments were carried out at room temperature. The bath solution to record delayed rectifier K⁺ currents (I_K) contained 150 mM NaCl, 5.4 mM KCl, 1 mM CaCl₂, 2 mM MgCl₂, 10 mM glucose, and 5 mM HEPES (pH adjusted to 7.35 with NaOH). The pipette solution contained 130 mM KCl, 1 mM CaCl₂, 2 mM MgCl₂, 10 mM HEPES, 10 mM EGTA, and 2 mM Mg-ATP (pH adjusted to 7.3 with KOH). All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). To record only the I_K of the cells, 100 nM iberiotoxin (a specific large-conductance calcium-activated K⁺ channel blocker) was added to the bath solution and 10 mM EGTA was added to the pipette solution during the experiments.