Pclamp v10

The signals were monitored and stored online at 10 kHz in an ASUS VivoBook Flip 14 laptop computer (TP412FA-0131A10210U; ASUS, Tainan, Taiwan) equipped with a Digidata 1440A interface (Molecular Devices). During the measurements with analog-to-digital and digital-to-analog conversion, the latter device was controlled using pCLAMP v10.7 software (Molecular Devices) run under Microsoft Windows 10 (Redmond, WA, USA). The laptop computer was put on the top of an adjustable Cookskin stand (Ningbo, Zheijiang, China) to allow efficient manipulation during the recordings. Through digital-to-analog conversion, the pCLAMP-created voltage-clamped protocols with varying rectangular or ramp waveforms were specifically designed and suited for determining the steady-state or instantaneous relationship pf current versus voltage (I–V) and voltage-dependent hysteresis of the current involved.

The signals, comprising potential and current tracings, were monitored on an HM-507 oscilloscope (Hameg, East Meadow, NY, USA) and digitally stored online at 10 kHz in an ASUS VivoBook Flip 14 laptop computer (TP412FA-0131A10210U; ASUS, Tainan, Taiwan) equipped with a 12-bit resolution Digidata 1440A interface (Molecular Devices). During the measurements with either analog-to-digital or digital-to-analog conversion, the latter device was controlled using pCLAMP v.10.7 software (Molecular Devices) run on Microsoft Windows 10 (Redmond, WA, USA). A laptop computer was put on the top of an adjustable Cookskin stand (Ningbo, Zheijiang, China) to allow efficient manipulation during the recordings.

Whole-cell currents from cultured rat cortical neurons were recorded using a MultiClamp 700B patch-clamp amplifier, low-pass filtered at 400 Hz, and digitized at the acquisition rate of 20 kilosamples per second using Digidata 1440A and pCLAMP v10.6 software (Molecular Devices). Solution exchange was performed by means of a fast solution application system as described earlier (Sibarov et al., 2015 (link)). The external bathing solution contained (in mM): 144 NaCl; 2.8 KCl; 1 or 2 CaCl₂; 10 HEPES, at pH 7.2–7.4, osmolarity 310 mOsm. The pipette solution contained (in mM): 120 CsF, 10 CsCl, 10 EGTA, and 10 HEPES, osmolarity 300 mOsm, with pH adjusted to 7.4 with CsOH. Patch pipettes of 4–6 MΩ were pulled from Sutter BF150-89-10 borosilicate glass capillaries. Experiments were performed at room temperature (22–25°C). Currents were recorded on neurons voltage clamped at −70 mV. Data are reported without corrections for liquid junction potential, which was −11 mV in our experiments. NMDAR currents were elicited by 100 μM NMDA co-applied with 30 μM glycine as a co-agonist. Methyl-β-cyclodextrin (MβCD, 1.5 mM) application for 5 min was used to extract plasma membrane cholesterol.

Whole-cell currents from cultured rat cortical neurons were recorded using a MultiClamp 700B patch-clamp amplifier, low-pass filtered at 400 Hz, and digitized at the acquisition rate of 20 kilosamples per second using Digidata 1440A and pClamp v10.6 software (Molecular Devices). Solution exchange was performed by means of a fast perfusion system as described earlier [21 (link)]. The external bathing solution contained (in mM): 144 NaCl; 2.8 KCl; 1 or 2 CaCl₂; 10 HEPES, at pH 7.2–7.4, osmolarity 310 mOsm. The pipette solution contained (in mM): 120 CsF, 10 CsCl, 10 EGTA, and 10 HEPES, osmolarity 300 mOsm, with pH adjusted to 7.4 with CsOH. Patch pipettes of 4–6 MΩ were pulled from Sutter BF150-89-10 borosilicate glass capillaries. Experiments were performed at room temperature (22–25 °C). Currents were recorded on neurons voltage clamped at −70 mV. Data are reported without corrections for liquid junction potential, which was −11 mV in our experiments. NMDAR currents were elicited by 100 µM NMDA co-applied with 30 µM glycine as a co-agonist.

Whole-cell currents were recorded from cultured rat cortical neurons using a MultiClamp 700B patch-clamp amplifier. Recordings were low-pass filtered at 400 Hz and digitized at the acquisition rate of 20,000 samples per second by Digidata 1440A controlled by pClamp v10.6 software (Molecular Devices). Solution exchange was performed by means of a fast solution application system as described earlier (Sibarov et al., 2015 (link)). Unless otherwise specified, the external bathing solution contained (in mM): 144 NaCl; 2.8 KCl; 1.0 CaCl₂; 10 HEPES, at pH 7.2–7.4, osmolarity 310 mOsm. The intrapipette solution contained (in mM): 120 CsF, 10 CsCl, 10 EGTA, and 10 HEPES, osmolarity 300 mOsm, with pH adjusted to 7.4 with CsOH. Patch pipettes of 4–6 MΩ were pulled from Sutter BF150-89-10 borosilicate capillaries. Experiments were performed at 22–25°C. Under control conditions, neurons were voltage clamped at −70 mV. Data are reported without corrections for liquid junction potential, which was measured as −11 mV. To activate NMDARs, 100 µM NMDA was always co-applied with 10 µM glycine as co-agonist. Some experiments were performed on neurons loaded with BAPTA. To load BAPTA, cortical neuron cultures were incubated in a bathing solution containing 5 µM BAPTA-AM for 1 h.

Methods for conventional whole-cell recordings from podocytes were carried out by standard methods that have been described in detail previously (Anderson et al., 2013 (link), 2014 (link); Roshanravan and Dryer, 2014 (link)). Recordings were made with an Axopatch 1D amplifier (Molecular Devices) and analyzed using pClamp™ v10 software (Molecular Devices). For analyses of currents through TRPC6, the bath solution contained 150 mM NaCl, 5.4 mM CsCl, 0.8 mM MgCl₂, 5.4 mM CaCl₂, and 10 mM HEPES, pH 7.4. Pipette solutions contained 10 mM NaCl, 125 mM CsCl, 6.2 mM MgCl₂, 10 mM HEPES, and 10 mM EGTA, pH 7.2. The presence of Cs⁺ in bath and pipette solutions, and the absence of K⁺ in those solutions, precludes current flowing through KCa1.1 channels that are also expressed in these cells (Kim et al., 2008 (link)). The relatively high Ca²⁺ concentration in the bath solution markedly increases stability of whole cell recordings. In experiments on TRPC6, currents were periodically evoked by 2.5-s ramp voltage commands (−80 mV to +80 mV) from a holding potential of −40 mV. Whole cell current amplitudes were quantified at +80 mV. Recording pipettes in all experiments had resistances of 2–5 MΩ, and it was possible to maintain stable recordings while compensating up to 80% of this series resistance.