Patchliner octo

An automated patch-clamp system (Patchliner Octo®, Nanion Technologies, Münich, Germany) was utilized as per standard procedure detailed by the manufacturer and as described previously (Haythornthwaite et al., 2012 (link)). Up to eight cells were measured simultaneously using the Patchliner Octo®, which is equipped with two EPC-10 Quadro patch-clamp amplifiers (HEKA Elektronik, Reutlingen, Germany). The external solution contained 4 mM KCl (Sigma), 140 mM NaCl (Sigma), 2 mM CaCl₂ (Sigma), 1 mM MgCl₂ (Sigma), 10 mM HEPES (Sigma), and 5 mM glucose (Sigma), at pH 7.4. The internal solution (60 mM KF, 50 mM KCl, 20 mM EGTA, 10 mM NaCl, 10 mM HEPES, and 1 mM MgCl₂; Nanion) was supplemented with 3 mM MgCl₂ (Sigma), 0.2 mM GTP (Sigma), and 3 mM ATP (Sigma), adjusted to pH 7.2. A seal-enhancer solution (3 mM KCl, 80 mM NaCl, 35 mM CaCl₂, 10 mM MgCl₂, and 10 mM HEPES [pH 7.4]; Nanion) was used and subsequently changed into external solution upon the establishment of the whole-cell configuration. Patch-ControlHT software (Nanion Technologies, version 12.0.1f9) and Patchmaster software (HEKA Elektronik, version 2×90.4 beta) were utilized for data acquisition. Data were recorded at a sampling rate of 20 kHz (at room temperature) and filtered at 5 kHz.

Cells were harvested and recorded according to the manufacturer’s standard procedures (Obergrussberger et al. 2014 (link)). In brief, CHO cells, which were chosen as cellular hosts since they are suitable for voltage clamp analysis (Scheffel et al. 2018b (link); Scheffel et al. 2018a (link)), were detached as described in the section “Cell culture” and washed with 2 mM EDTA in PBS pH 7.4. Electrophysiological whole-cell recordings were conducted using Nanion’s Patchliner (Patchliner Octo, Nanion Technologies), an automated patch-clamp system with eight amplifier channels due to two HEKA EPC10 Quadro Amplifiers. All currents were elicited from single cells using a medium resistance NPC-16 borosilicate chip 1.8–3 MΩ (071102 by Nanion Technologies) at a holding potential of −70 mV. Buffer solutions external solution 083001, internal solution 083007, and seal solution 083012 (all by Nanion Technologies) were used. A fresh stock solution of 10 mM nicotine was used for each analysis. For receptor activation, 15 μL of 70 μM nicotine was applied for 132 ms at a rate of 114 μL/s, directly followed by 200 μL of external buffer solution for washout. The sampling rate was set to 50 kHz and filtered at 2.9 kHz.

Automated electrophysiological recordings in CHO-mTRPA1 cells was performed using Patchliner Octo (Nanion Technologies, Munich, Germany) fitted with two EPC-10 quadro amplifiers (HEKA Elektronik GmBH, Lambrecht, Germany) using medium resistance (1.8–3 MΩ) NPC-16 chip (Nanion Technologies). All experiments were performed at 25 °C using the following solutions (in mM): intracellular—10 CsCl, 110 CsF, 20 EGTA, 10 HEPES, 0.3 GTP, 5 ATP, pH 7.2; extracellular—160 NaCl, 4 KCl, 1 MgCl₂, 2 CaCl₂, 5 glucose, 10 HEPES, pH 7.4. Cells were held at 0 mV and mTRPA1 currents were elicited by 250 ms steps first to −60 mV and subsequently to +60 mV, at the end of which steady-state currents were measured. Data were collected at a sampling rate of 10 kHz and filtered at 3.33 kHz. Data were acquired using the Patchmaster software (HEKA Elektronik GmBH, Lambrecht, Germany) and analyzed offline using IGOR-Pro (WaveMetrics, Inc. Portland, OR, USA) and Origin 9 (OriginLab Corporation).