MscL protein purification and reconstitution into soybean azolectin liposomes were described previously (Nomura et al., 2012 (link)). All results were obtained with proteoliposomes at the protein: lipid ratio of 1:200 (wt/wt). Channel activities of the wild-type and mutant MscL were examined in inside-out liposome patches using patch-clamp technique. Borosilicate glass pipettes (Drammond Scientific Co, Broomall, PA) were pulled using a Narishige micropipette puller (PP-83; Narishige, Tokyo, Japan). Pipettes with resistance of 2.5–4.9 MΩ were used for the patch-clamp experiments. Pipette and bath solution contained 200 mM KCl, 40 mM MgCl2, and 5 mM HEPES (pH 7.2 adjusted with KOH). The current was amplified with an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA), filtered at 2 kHz and data acquired at 5 kHz with a Digidata 1440A interface using pCLAMP 10 acquisition software (Molecular Devices, Sunnyvale, CA) and stored for analysis. Negative pressure (suction) was applied to the patch pipettes using a syringe and was monitored with a pressure gauge (PM 015R, World Precision Instruments, Sarasota, FL).
Pclamp 10 acquisition software
Manufactured by Molecular Devices
Sourced in United States
PClamp 10 is a data acquisition and analysis software for electrophysiology experiments. It provides tools for recording, visualizing, and analyzing electrical signals from cells and tissues.
Automatically generated - may contain errors
Lab products found in correlation
Axopatch 200b amplifier, by Molecular Devices (5 mentions)
Clampfit 10, by Molecular Devices (3 mentions)
Digidata 1440a interface, by Molecular Devices (2 mentions)
Pm 015r, by World Precision Instruments (2 mentions)
P 1000 flaming brown micropipette puller, by Sutter Instruments (2 mentions)
Axopatch 200b patch clamp amplifier, by Molecular Devices (2 mentions)
Digidata 1322a, by Molecular Devices (2 mentions)
Digidata 1440a, by Molecular Devices (2 mentions)
Multiclamp 700b, by Molecular Devices (2 mentions)
Matlab r2015a, by MathWorks (1 mentions)
Matlab, by MathWorks (1 mentions)
Pp 83, by Narishige (1 mentions)
Digidata 1550b, by Molecular Devices (1 mentions)
Digidata 1440, by Molecular Devices (1 mentions)
Te2000 s microscope, by Nikon (1 mentions)
Origin 8, by OriginLab (1 mentions)
X cite 120, by Nikon (1 mentions)
Mf 83, by Narishige (1 mentions)
P 97 puller, by Sutter Instruments (1 mentions)
13 protocols using pclamp 10 acquisition software
1
MscL Protein Purification and Reconstitution
2
Voltage-clamp recordings of iPSC-derived neurons
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Voltage‐clamp recordings were obtained from iPSC‐derived neurons using an Axopatch 200B amplifier (Molecular Devices, San Jose, CA). For the current clamp recordings 130mM of KCl, 1 mM of MgCl2, 5mM of MgATP, 10mM of HEPES, and 0.5mM of EGTA (pH 7.3) solution was used. Recordings were obtained at room temperature, with a sampling rate of 5KHz, using the pClamp 10 acquisition software (Molecular Devices). Data were analysed using MatLab R2015A (The MathWorks, Inc., Natick, MA).
3
Whole-cell Patch-clamp Recording Protocol
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Whole-cell currents were recorded in a conventional whole-cell patch-clamp configuration. Patch electrodes were pulled from Borosilicate glass tubes using a P-1000 Flaming/Brown micropipette puller (Sutter Instrument, Novato, CA, USA) and fire polished using a microforge (Narishge, MF-830) to reach a resistance of 3–6 MΩ. Cells were placed in a bath solution with the following composition: (in m m ) NaCl 140, MgCl2 1.2, CaCl2 1.8, HEPES 10, glucose 10, pH 7.4 (using 1 M NaOH). The pipette solution used had the following composition: (in m m ) CsCl 140, MgCl2 2, HEPES 10, EGTA 5, glucose 10, Na-ATP 5, pH 7.35 (using 1 m CsOH). All data were acquired and digitized using Axopatch 200B patch clamp amplifier, Axon Instruments Digidata 1550B analog-digital converter, and pClamp 10 acquisition software (Molecular Devices, CA, USA). Data were sampled at 10 kHz, filtered at 2 kHz (low-pass Bessel filter), and sampled at 20 kHz. Membrane currents were recorded by applying 940-ms voltage ramps from −120 to +30 mV. Using a voltage clamp protocol, the command voltage ramped up from −100 to +100 mV over 940 ms from a zero-mV holding potential.
4
Electrophysiological Recording and Analysis
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Data were recorded using an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA) in series with a secondary instrumentation amplifier (NPI electronics GmbH, Tamm, Germany). Recordings were lowpass filtered (10 kHz), highpass filtered (1 Hz) and digitized (50 Hz) using a 1322A Digidata (Molecular Devices, Sunnyvale, CA) and pClamp 10 acquisition software (Molecular Devices, Sunnyvale, CA, USA). Data were reanalyzed offline using Clampfit 10.3 (Molecular Devices, Sunnyvale, CA) and custom written scripts in Matlab (Mathworks, Natwick, MA). Data pooled across slices are expressed as the mean ± SEM, effects of the conditioning stimulation were measured at 50–60 min after the induction. Statistical significance was determined using an appropriate parametric test with an alpha value of 0.05.
5
Patch-clamp Analysis of MscL and MscS Channels
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An aliquot of proteoliposomes (1–3 μl) was introduced into the recording chamber. Channel activity of WT MscL and mutant proteins (+2G and +5G) were co-reconstituted with the WT MscS channel and examined in inside-out azolectin liposome patches using the patch-clamp technique43 (link). Borosilicate glass pipettes (Drammond Scientific Co, Broomall, PA) were pulled using a micropipette puller (PP-83; Narishige, Tokyo, Japan). Pipettes with resistance of 3.0–5.0 MΩ were used for the patch-clamp experiments. Pipette and bath solution contained 200 mM KCl, 40 mM MgCl2 and 5 mM HEPES (pH 7.2 adjusted with KOH). In the case of spheroplast patching, the bath solution was supplemented with 400 mM sucrose. The current was amplified with an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA), filtered at 5 kHz and data acquired at 20 kHz with a Digidata 1440A interface using pCLAMP 10 acquisition software (Molecular Devices) and stored in a personal computer. Negative pressure was applied manually with a syringe or High-Speed Pressure Clamp-1 apparatus (HSPC-1; ALA Scientific Instruments, Farmingdale, NY) and was monitored with a pressure gauge (PM 015R, World Precision Instruments, Sarasota, FL).
6
Glycinergic Synaptic Current Recordings
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Whole-cell patch-clamp recordings were performed at room temperature in voltage-clamp mode using a Multiclamp 700B controlled by pClamp 10 acquisition software (Molecular Devices). Currents were filtered at 2–3 kHz and sampled at 10 kHz using a Digidata 1440 A (Molecular Devices). Patch pipettes (4–6 MΩ) were filled with internal solution containing 70 mM CsCl, 70 mM caesium methanesulphonate, 1 mM EGTA, 1 mM MgCl2, 4 mM Mg-ATP and 10 mM HEPES, adjusted to pH 7.4 with CsOH. Spontaneous glycinergic currents were recorded at a holding potential VH = −60 mV in external solution containing 137 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 20 mM glucose and 10 mM HEPES, pH 7.4. AMPARs, NMDARs and GABAARs were blocked with 5 µM NBQX, 50 µM D-APV and 5 µM gabazine. In an independent set of experiments (data in Fig. 4F) 0.5 µM tetrodotoxin (TTX) was added to the external solution to isolate miniature currents (mIPSCs) and recordings were done at 32 °C. Spontaneous and miniature glycinergic currents were detected using the Clampfit template procedure. For each cell a sliding template was created by averaging 15–20 events selected by eye, and detection of synaptic currents was done on the basis of closeness of fit to the template. Overlapping events were excluded from analysis.
7
Electrochemical Characterization of Lipid Bilayers
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Electrodes (Ag/AgCl) were prepared as previously described2 (link) using silver wires with diameters of 1.5 mm and 100 μm for the ground electrode and the droplet electrode, respectively, which were immersed in sodium hypochlorite (4%) overnight to form a AgCl coating. The ground electrode was inserted into the agarose reservoir (3.25% wt/vol). The droplet electrode was coated in agarose (3.25% wt/vol in experiment buffer) and inserted into the droplet upon bilayer formation using a micromanipulator. Both electrodes were connected to a Bilayer Clamp Amplifier (BC-535, Warner Instruments). The current was filtered at 1 kHz and acquired at 5 kHz with a Digidata 1440 A interface using pCLAMP 10 acquisition software (Molecular Devices, Sunnyvale, CA) and analyzed. Current traces for figures have subsequently been smoothed using a box algorithm (10 points).
8
Larval Neuromuscular Junction Electrophysiology
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Wandering third-instar larvae were chosen for electrophysiology. Larvae were dissected in a modified HL3 saline: NaCl (70 mM), KCl (5 mM), MgCl2 (10 mM), NaHCO3 (10 mM), sucrose (115 mM = 3.9%), trehalose (4.2 mM = 0.16%), HEPES (4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid; 5.0 mM = 0.12%), and CaCl2 (0.5 mM). Sharp electrode recordings were taken from muscle 6 of abdominal segment A2 or A3, as previously described (Davis et al., 1998 (link); Frank et al., 2006 (link)). Data were collected using an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA), digitized using a Digidata 1440A data acquisition system (Molecular Devices), and recorded with pCLAMP 10 acquisition software (Molecular Devices). For presynaptic nerve stimulation, a Master-8 pulse stimulator (A.M.P. Instruments, Jerusalem, Israel) and an ISO-Flex isolation unit (A.M.P. Instruments) were used to deliver 1-ms suprathreshold stimuli to the appropriate segmental nerve. The average spontaneous miniature EPSP (mEPSP) amplitude was quantified by measuring the amplitude of ∼100–200 individual spontaneous release events per NMJ. The average evoked EPSP amplitude was calculated for each NMJ. Quantal content (QC) was determined for each NMJ by calculating the ratio of average EPSP and average mEPSP amplitudes. QC was corrected for nonlinear summation as described (Martin, 1955 (link)).
9
Whole-Cell Patch-Clamp Cation Current Recording
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Cation currents were recorded in conventional whole-cell patch-clamp configuration. Patch electrodes were pulled from borosilicate glass tubes using a P-1000 Flaming/Brown micropipette puller (Sutter Instrument, Novato, CA, USA) and fire polished using a microforge (Narishge, MF-830) to reach a resistance of 3-6 MΩ. Cells were placed in a bath solution with the following composition: (in mM) NaCl 140, MgCl 2 1.2, CaCl 2 1.8, HEPES 10, Glucose 10, pH 7.4 (using 1M NaOH). The pipette solution used had the following composition: (in mM) CsC1 140, MgCl 2 2, HEPES 10, EGTA 5, Glucose 10, Na-ATP 5, pH 7.35 (using 1M CsOH). Membrane currents were recorded using a voltage clamp protocol where the command voltage ramped up from -100 mV to +100 mV over 940 ms from a zero-mV holding potential. All data were acquired and digitized using Axopatch 200B patch clamp amplifier, Axon Instruments Digidata 1550B analog-digital converter, and pClamp 10 acquisition software (Molecular Devices, CA, USA). Data were filtered at 2 kHz (low pass Bessel filter) and sampled at 20 KHz.
10
HEK293 Transfection and Electrophysiology
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HEK293 cells (ATCC CRL-1573) at the passages 25 -32 were maintained in Dulbecco's Modified Eagle Medium with 10% Fetal Bovine Serum and 1% glutamine. Cells were incubated at 37°C in 5% CO 2 and 95% atmospheric air. Prior to experiments, cells were transfected with rat GluN1-1a (U08262.1), GluN2A (M91561.1), or mutants as indicated, and GFP cDNA at a 1:1:1 ratio using polyethylenimine 35 . The transfected cells were grown for 24 -48 h in medium supplemented with 10 mM Mg 2+ to prevent excitotoxicity. All mutations were introduced by using the QuickChange method (Stratagene, La Jolla, CA) and verified by DNA sequencing. ) and written into digital files with pClamp 10 acquisition software (Molecular Devices, Sunnyvale, CA). Clamped cells were extracellularly perfused with solutions using BPS-8SP solenoid-valve perfusion system (ALA Scientific Instruments, Westbury, NY). Free Ca 2+ concentrations were calculated with the software MAXC (www.maxchelator.stanford.edu).
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