Barium currents (IBa) through voltage-gated Ca2+ channels were recorded at 22–25 °C by patch-clamp [18 (link)] using an Axopatch 200A patch clamp amplifier (Axon Instruments, Foster City) 24–48 h after transfection. To avoid calcium-dependent inactivation, barium was used as charge carrier. The extracellular bath solution (in mM: BaCl2 20, MgCl2 1, HEPES 10, choline-Cl 140) was titrated to pH 7.4 with sodium hydroxide. Patch pipettes with resistances of 1 to 4 MΩ were made from borosilicate glass (Clark Electromedical Instruments, UK) and filled with pipette solution (in mM: CsCl 145, MgCl2 3, HEPES 10, EGTA 10), titrated to pH 7.25 with CsOH. All data were digitized using a DIGIDATA 1200 interface (Axon Instruments, Foster City), smoothed by means of a four-pole Bessel filter and saved to disc. One hundred-megasiemen current traces were sampled at 10 kHz and filtered at 5 kHz. Leak currents were subtracted digitally using the average values of scaled leakage currents elicited by a 10-mV hyperpolarizing pulse or electronically by means of an Axopatch 200 amplifier (Axon Instruments, Foster City). Series resistance and offset voltage were routinely compensated for. The pClamp software package (Version 10.0 Axon Instruments, Inc.) was used for data acquisition and preliminary analysis. Microcal Origin 7.0 was used for analysis and curve fitting.
Axopatch 200
Manufactured by Molecular Devices
Sourced in United States
The Axopatch 200 is a laboratory instrument designed for electrophysiological recording and analysis. It is a highly sensitive and versatile patch-clamp amplifier that can be used to measure electrical signals from a variety of biological samples, including cells, tissues, and neurons.
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Clampfit software, by Molecular Devices (1 mentions)
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22 protocols using axopatch 200
1
Barium Current Recordings from Voltage-Gated Channels
2
Patch-clamp recording of nuclear pores
3
Electrophysiological Characterization of cGMP-gated Ion Channels
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The functionality of all constructs was verified by exposure to 2 mM cGMP (saturating concentration) and then recording the channel current using electrophysiological measurement in the excised patch configuration (see Supplementary Fig. 1a–g ). cGMP-gated currents in a voltage-clamp condition were recorded using a patch-clamp amplifier (Axopatch 200, Axon Instruments Inc., Foster City, CA, USA) 2–6 days after RNA injection at room temperature (20–24 °C), using borosilicate glass pipettes with resistances of 2–5 MΩ. The perfusion system allowed a complete solution change in <1 s. During the experiments, oocytes were kept in Ringer’s solution containing the following (in mM): 110 NaCl, 2.5 KCl, 1 CaCl2, 1.6 MgCl2 and 10 HEPES-NaOH (pH 7.4 buffered with NaOH). The Standard solution on both sides of the membrane consisted of (in mM) 110 NaCl, 10 HEPES and 0.2 EDTA (pH 7.4 buffered with NaOH). We used Clampex 10.0, Clampfit 10.1 and SigmaPlot 9.0 for data acquisition and analysis. Data are usually given as the mean±s.e.m. We attempted SMFS only in oocytes in which the measured cGMP-activated current was larger than 1 nA at ±100 mV.
4
Measurement of Kv1.5-mediated Currents in J774.1 and HL-1 Cells
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Kv1.5-mediated currents corresponding to Ikur in J774.1 and HL-1 cells were measured at 37 °C using whole-cell patch-clamp techniques with an Axopatch-200 amplifier (Axon Instruments, USA). The procedures for the current measurement were essentially the same as described previously [6 (link)]. The extracellular solution contained (mM): NaCl 140, KCl 4, CaCl2 1.8, MgCl2 0.53, NaH2PO4 0.33, glucose 5.5, and HEPES 5, with a pH adjusted to 7.4. The internal pipette solution contained (mM) K-aspartate 100, KCl 20, CaCl2 1, Mg-ATP 5, EGTA 5, HEPES 5, and creatine phosphate dipotassium salt 5 (pH 7.2 with KOH). The currents were elicited every 6-s by 500-ms test pulses ranging from − 60 to + 40 mV ( in 10 mV increments) with a holding potential of − 60 mV. DPO-1 (1 μM) was added to the bath solution to block Kv1.5 channel currents. The DPO-1 sensitive currents were obtained by digitally subtracting the current traces in the presence of DPO-1 from those in the absence of DPO-1. Action potentials (APs) of HL-1 cells were elicited at 0.5 Hz by 5-ms square current pulses of 1nA, and sampled at 20 kHz. AP durations (APDs) were measured at 20%, 50% and 90% repolarization (APD20, APD50 and APD90), respectively.
5
Patch Clamp Analysis of Ion Channel Kinetics
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Electrophysiological recording was performed using a patch clamp amplifier (Axopatch 200, Axon Instruments, Union City, CA, USA). Whole-cell currents were recorded after canceling the capacitive transients. Single-channel and whole-cell currents were analyzed with the pCLAMP program (Version 9, Axon). Pipette and bath solutions contained (mM): 150 KCl, 1 MgCl2, 5 ethylene glycol tetraacetic acid (EGTA), and 10 hydroxyethyl piperazineethanesulfonic acid (HEPES ) (pH 7.3), and bath solution for recording of whole-cell current contained (mM): 135 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 5 glucose, and 10 HEPES (pH 7.3). The pH was adjusted to desired values with HCl or KOH. To obtain IC50 and half maximal effective concentration (EC50) values for dose-dependent inhibition and activation, respectively, data were averaged and then fit with a standard sigmoid function.
6
Outward Current Characterization in Cells
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All experiments were performed at room temperature in whole-cell mode. Patch pipettes had resistances of 2–5 MΩ. The recordings were made with an Axopatch 200 amplifier (Axon Instruments, Burlingame, CA, USA). Stimulation of currents and data analysis were conducted with the software package ISO2. To reduce the contribution of BK channels, 10−7 mol/L iberiotoxin, a specific inhibitor of these channels, was added to the bath solution [34 (link)]. Initial experiments had shown that neither 1 µmol/L glibenclamide, an inhibitor of ATP-sensitive potassium channels, nor 10 µmol/L barium, an inhibitor of inward rectifying potassium channels, produced any effect on the outward current of these cells.
7
Single-channel Patch-clamp Recording Protocol
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Electrophysiological data were recorded using a patch-clamp amplifier (Axopatch 200, Axon Instruments, Union City, CA, USA). Single-channel currents were digitized using a digital data recorder (VR10, Instrutech, Great Neck, NY, USA) and were stored on videotape. The recorded signals were filtered at 2 kHz using an 8-pole Bessel filter (−3 dB; Frequency Devices, Haverhill, MA, USA) and transferred to a computer using the Digidata 1322A interface (Axon Instruments) at a sampling rate of 20 kHz. Threshold detection of channel openings was set at 50%. The single-channel current tracings shown in the figures were filtered at 2 kHz. In experiments using cell-attached patches and excised patches, pipette and bath solutions contained: 150 mM KCl, 1 mM MgCl2, 5 mM EGTA, and 10 mM HEPES (pH = 7.3). The pH was adjusted to the desired values using HCl or KOH. The voltage clamp experiment was performed at room temperature (22–25 °C).
8
Whole-cell patch-clamp recording protocol
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Electrophysiological recording was performed using a patch clamp amplifier (Axopatch 200, Axon Instruments, Union City, CA, USA). Pipette tip resistances were 4~6 MΩ. Wholecell current was recorded in response to a voltage ramp (–120 to +60 mV; 865 ms duration) from a holding potential of –80 mV in physiological solution containing 5 mM KCl. Currents were filtered at 2 kHz, and the currents measured at +60 mV were obtained and analyzed. Bath solution contained (mM): 135 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 5 glucose and 10 HEPES (pH 7.4), and pipette solutions contained (mM): 150 KCl, 1 MgCl2, 5 EGTA and 10 HEPES (pH 7.3). The pH was adjusted to desired values with HCl or NaOH. All experiments were performed at ~25℃.
9
Patch-clamp recording of nuclear pores
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For whole-cell recordings, electrodes were with a final resistance of 5–10 MΩ when filled with pipette solution (in mM): 120.0 potassium gluconate, 15.0 HEPES, 2.2 CaCl2, 1.0 MgCl2, 4.0 EGTA, and 4.0 HEDTA, pH7.35, 305mOsm. Currents were recorded using an Axopatch 200B (Axon Instruments, Foster City, CA) amplifier at a sampling rate of 1–5 kHz and leak-subtracted on line. Series resistance never exceeded 25 MΩ and was compensated. Data were acquired and analyzed with pClamp9 (Axon Instruments). Single-channel currents were recorded from nuclei positively labeled with ethidium homodimer-1 by using standard patch-clamp recording procedures24 (link). Currents were recorded in symmetrical 135 mM K using a patch-clamp amplifier (Axopatch 200; Axon Instruments) at a sampling rate of 5 kHz and filtered at 1 kHz filter. Experiments were performed at RT. High resistance seals were formed (5–7 GΩ), indicating minimal contamination by the endoplasmic reticulum (ER) membrane24 (link). Under these circumstances, it has been proposed that nuclear pores would be occluded or nonconducting because of experimental conditions or lack of cytosolic factors6 (link),39 (link),53 (link). PCLAMP software was used to measure the current magnitude and duration.
10
Measuring cGMP-gated Currents in Excised Patches
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cGMP-gated currents from excised patches were recorded with a patch-clamp amplifier (Axopatch 200; Axon Instruments Inc., Foster City, CA, USA), 2–6 days after RNA injection, at room temperature (20–24 °C)30 (link). The perfusion system allowed a complete solution change in less than 0.1 seconds10 (link),27 (link). Macroscopic and single-channel current recordings were obtained with borosilicate glass pipettes which had resistances of 2–5 MOhm in symmetrical standard solution. The standard solution on both sides of the membrane consisted of (in mM) 110 NaCl, 10 Hepes and 0.2 EDTA (pH 7.4). Solutions were buffered with tetramethylammonium hydroxide at the desired pH. When the cation X+ was used as the charge carrier, NaCl in the standard solution on both sides of the membrane patch was replaced by an equimolar amount of the cation X+. We used Clampex version 10.0 for data acquisition. Recordings to perform noise analysis were low-pass filtered at 10 kHz. All other macroscopic current recordings were low-pass filtered between 1 to 10 kHz.
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