2.4. Whole-Cell Patch-Clamp Recordings

Whole-cell voltage- and current-clamp recordings were performed at 24–28°C to measure currents and action potentials, respectively, using an Axopatch 200B amplifier (Axon Instruments, Union City, CA, USA). The patch pipettes were pulled from borosilicate capillaries (Chase Scientific Glass Inc., Rockwood, CA, USA). When filled with the pipette solution, the resistance of the pipettes was 4-5 MΩ. The recording chamber (volume 300 μL) was continuously superfused (2-3 mL/min). Series resistance was compensated for (>80%), and leak subtraction was performed. Data were low-pass filtered at 2 kHz and sampled at 10 kHz. pClamp8 (Axon Instruments) software was used for experiments and analysis. The pipette solution for voltage-clamp experiments contained (in mM) 135 CsCl, 30 CsOH, 2 Mg-ATP, 5 EGTA, and 10 HEPES, adjusted to pH 7.4 with CsOH, with an osmolarity of 295–300 mOsm. In some cases, guanosine 5′-[β-thio] diphosphate trilithium salt (GDPβ-S, 2.5 mM) was included in the intracellular solution to block G protein-coupled receptors (GPCRs). The extracellular solution for voltage-clamp experiments contained (in mM) 140 NaCl, 5 KCl, 1 MgCl₂, 10 HEPES, 10 glucose, and 2 EGTA, adjusted to pH 7.4 with NaOH, with an osmolarity of 300–310 mOsm. Voltage-clamp experiments were performed at a holding potential of −60 mV. The pipette solution for current-clamp experiments contained (in mM) 145 K-gluconate, 2 MgCl₂, 1 CaCl₂, 10 EGTA, 5 HEPES, and 5 K₂ATP, adjusted to pH 7.3-7.4 with KOH, with an osmolarity of 300 mOsm. The extracellular solution for current-clamp experiments contained (in mM) 140 NaCl, 5 KCl, 2 CaCl₂, 1 MgCl₂, 10 HEPES, and 10 glucose, adjusted to pH 7.4 with NaOH, with an osmolarity of 300–310 mOsm. IC₅₀ values were calculated by normalizing peak current amplitudes at different drug concentrations to the value obtained for the control solution. Furthermore, the data were fitted to the Hill equation [12].