Axopatch 1d amplifier

Electrophysiology experiments were performed in a modified Tyrode’s solution (containing, in mmol/L: 133 NaCl, 5 KCl, 1 NaH₂PO₄, 10 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 10 glucose, 1.8 CaCl₂, 1 MgCl₂, pH 7.4 with NaOH) at 36 ± 1 °C. Patch pipettes were pulled from borosilicate glass using a P80 micropipette puller (Sutter Instruments). Pipettes were filled with an intracellular solution containing, in mmol/L: 120 aspartic acid, 20 KCl, 10 HEPES, 10 NaCl, 5 glucose, 5 Mg.ATP, 0.05 Fluo-4 pentapotassium salt, with KOH added to adjust to pH 7.2. Tip resistance was typically 1.6–2.0 MΩ when filled with this solution. Membrane potential and currents were recorded using an Axopatch 1D amplifier (Molecular Devices), Power1401 digitizer (Cambridge Electronic Design), and Signal data acquisition software (version 6.04, Cambridge Electronic Design). Cell membrane capacitance was measured by step depolarizations to −75 mV from a holding potential of −80 mV for 25 ms. Series resistance was compensated by ∼70%. Liquid junction potential (10 mV) was subtracted from recordings.

Whole-cell recordings were created at room temperature (22 °C) from immortalized podocytes as described in detail previously [9 (link),25 (link),26 (link)] using fire-polished borosilicate glass microelectrodes (4–6 MΩ) and an Axopatch 1D amplifier (Molecular Devices, Foster City, CA, USA). The bath was perfused at a constant flow rate (0.3 mL/min) and TRPC6 channels were activated by a bath application of a hypotonic stretch solution as described previously [9 (link)]. Macroscopic currents were monitored during 2.5-s voltage ramps from −80 mV to +80 mV from a holding potential of −40 mV and currents at +80 mV were quantified for statistical analysis. We have previously shown that the stretch-evoked cation currents are eliminated by TRPC6 knockdown [9 (link)] and by application of agents such as SAR-7334 that selectively inhibit TRPC6 [27 (link)].

The inner segments of SPs and LPs lying close to the slice surface (Figure 1A) were visually targeted with 5–6 MOhm pipettes pulled with a P-97 (Sutter Instruments, Novato, CA) from borosilicate glass capillaries (1B120F-4, WPI, Sarasota, FL) and filled with a solution containing (in mM) 90 Kaspartate, 20 K₂SO₄, 15 KCl, 10 NaCl, 5 K₂Pipes, as well as 0.5 mg ml⁻¹ Lucifer yellow, and corrected to a pH of 7.20 with KOH/HCl. The backfilling solution also contained 0.4 mg ml⁻¹ Amphotericin B (item no. 11636, Cayman, Ann Arbor, MI) pre-dissolved in dimethyl sulfoxide (DMSO) at 60 mg ml⁻¹. Based on an analysis of the liquid junction and Donnan potentials when using this solution and recording photoreceptors (Cangiano et al., 2012 (link)), we report uncorrected values of membrane potential. Recordings were made with an Axopatch 1D amplifier, low-pass filtered at 500 Hz and acquired at 5 KHz with a Digidata 1320 and pClamp 9 software (Molecular Devices, Sunnyvale, CA).

Myristoylated Gα_o1 (hence referred to as myrGα_o) was obtained from Calbiochem and used for most experiments; myrGα_o-Q205L and its control myrGα_o were prepared as described below; GTPγS was obtained from Sigma (Sigma-Aldrich, St Louis, MO); phosducin was a gift from Dr. Vadim Arshavsky (Duke University); and His₆-Gα_o was a gift from Dr. Richard Neubig (University of Michigan).
Current recordings in the US were obtained with an Axopatch 1D amplifier (Molecular Devices) and in China with an EPC−10 patch clamp amplifier (HEKA, Lambrecht, Germany). Membrane potentials were corrected for liquid junction potential calculated to be ~15 mV. Cells were discarded if the baseline current in the dark exceeded −100 pA at a holding potential of −60 mV. Voltage command generation and data acquisition were accomplished with Clampex (Molecular Devices) or PatchMaster (HEKA). Cells were voltage clamped at −60 mV and the holding current and light-evoked current responses were compared over time for control and different dialyzed reagents. It has been reported that clamping a cell at +50 mV may extend the recording time because less calcium-dependent desensitization occurs63 (link). However, in our hands, such clamping did not prove beneficial, and we preferred to make the measurements under more physiologically-relevant voltages.