Axopatch 1d

Single whole cell patch electrode voltage clamp experiments were performed on neonatal mouse ventricular myocytes (NMVMs) using conventional procedures with an Axon Instruments Axopatch 1D or 200B patch clamp amplifier, Digidata 1320A or 1440 A/D converter, and pClamp8.2 or 10.1 software (Molecular Devices, San Jose, CA, USA). Transient (peak) and steady state outward potassium (I_K,to and I_K,ss) currents were recorded during 1 sec voltage steps from a holding potential (V_h) of −100 to +60 mV in 10 mV increments. Voltage-gated sodium currents (I_Na) were elicited from a V_h of −120 mV during voltage steps from −90 to +50 mV in 5 mV increments for 150 ms using reduced NaCl solutions. For the I_Na inactivation protocol, V was −120 mV and the prepulse voltage increased from −130 to −30 mV in +5-mV increments for 150 ms followed by a 30-ms activation step to −40 mV [13 (link)]. Late I_Na protocol was measured as the average current from 100 to 150 ms of the activating voltage steps or were recorded during a ramp from −80 to +10 mV in 0.1 mV/4 ms steps from a V_h of −60 mV [13 (link),28 (link)].

Cells were plated on 5×5 mm glass coverslips coated with poly-D-lysine, and coverslips were transferred to the recording chamber of the microscope. For the single channel recording, an Axon200B patch-clamp amplifier was used to record the channel current. Amiloride-sensitive ENaC single channel and multichannel activities were measured at −60 mV holding potential. Pipette solution contained (in mM): 140 LiCl, 2 MgCl₂ and 10 HEPES, pH 7.4; bath solution contained (in mM): 140 NaCl, 5 KCl, 1.8 CaCl₂, 1.5 MgCl₂, 10 HEPES (pH 7.4). Gap-free single-channel current data from gigaohm seals were acquired and subsequently analyzed with an Axopatch 1D (Axon Instruments) patch-clamp amplifier interfaced via a Digidata 1440A (Axon Instruments) to a computer running the pClamp 10.2 (Axon Instruments). The channel open probability (P_o) was calculated from the channel number (N) and NP_o (a product of channel number and open probability), which was calculated from data samples of 60-s duration in the steady state as follows: where t_i is the fractional open time spent at each of the observed current levels.
In multichannel cell attached mode net current (in pA) is measured in individual patches at specific holding potentials in cell attached mode. The measured pA signal in this case reflects the sum of all the channel activities in one patch.

Ionic currents were recorded using the whole-cell patch clamp technique as previously described (Han et al., 2014 (link)) using Axopatch1D, Digidata 1322A, and pClamp 9 (Axon Instruments) for data amplification, acquisition and analysis. Currents were elicited by a protocol of depolarizing potentials of −130 mV to 50 mV in 10 mV increments from a holding potential of −80 mV. Current densities were measured as the peak current for each potential pulse. Currents were normalized to the cell capacitance and expressed in pA/pF.

An Axopatch −1D (Axon Instruments: Molecular Devices LLC, CA, USA) amplifier and Axopatch 10.3 software were used for recording and data analysis. Patch electrode resistance was set at 4–6 MΩ and room temperature was 22 ± 1 °C. During the recording, cells were held at −50 mV, then, the voltage was gradually raised to +60 mV in 10 mV increments with 300 ms pulse. The bath solution included the following components: 137 mM TEA-Cl, 1 mM MgCl₂, 2 mM CaCl₂, 10 mM HEPES, 10 mM glucose (pH 7.4, adjusted with TEA-OH). For the pipette the solution contained: 111 mM CsCl, 20 mM TEA-Cl, 10 mM glucose, 10 mM HEPES, 14 mM EGTA, 5 mM MgATP (pH 7.2, adjusted with CsOH). The I_Cal currents were recorded in the absence and presence of indicated concentrations of emetine or analog in three independent experiments for each drug.

Cells were seeded on polylysine-coated 16 mm coverslips. Whole-cell currents were recorded at RT using borosilicate patch pipettes of 3–5 MΩ resistance. Electrical signals were amplified using Axopatch 1D (Axon Instruments), and data were captured using a Digidata 1440A (Molecular Devices) interfaced to a computer. The membrane potential was held at 0 mV, and currents were measured in response to 1,050 ms voltage ramps from –150 to +150 mV every 5 seconds.