Computational protocol: Mechanosensitivity of nicotinic receptors

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Protocol publication

[…] In most cases, the cell-attached mode was used in single-channel recordings. All experiments were at room temperature. The recording was conducted with Axopatch 200B patch-clamp amplifier with associated software (Axon Instruments/Molecular Devices, Union City, CA). Currents were typically digitized at 10 kHz, macroscopic records were filtered at 2 kHz. Data were analyzed with Clampfit (version 10.0; Axon Instruments) and Sigmaplot (Systat Software, Inc., Chicago, IL) software. All data are presented as mean ± SEM.For recording from Xenopus muscle cells, Ringer solution (110 mM NaCl, 1 mM KCl, 1.8 mM CaCl2, and 8 mM HEPES, pH 7.4) was used in the pipette and bath. For recording from mammalian cells (C2C12 myotubes and HEK293T cells), the pipette solution contained 150 mM KCl, 2.5 mM MgCl2, 0.2 mM EGTA, and 10 mM HEPES, pH 7.4, and the bath solution contained 150 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM glucose, and 10 mM HEPES, pH 7.4. For inside-out patch-clamp experiments on HEK293T cells, the first solution containing high KCl was used in the pipette and in the bath.To stretch the membrane patch while recording, negative pressure was manually applied with a 1-mL syringe attached to the pipette via silicone tubing and a three-way stopcock. Pressures were monitored with a pressure gauge (PM01R, World Precision Instruments, Sarasota, FL) and displayed by Axon pClamp10 software. The pressure trace is shown in figures together with current recordings with the baseline showing the atmospheric pressure (negative pressure = 0 mmHg) and downward steps showing the magnitude of the negative pressure. A scale for the negative pressure is included in each figure where current recordings are shown. The patch diameter was approximately 1 μm. The value of NPo was calculated by using “single-channel search” function in the Clampfit software. For data obtained from the same cell, paired t test was used whereas unpaired t test was adopted for analyzing data collected from a group of cells. […]

Pipeline specifications

Software tools SigmaPlot, pCLAMP
Applications Miscellaneous, Patch-clamp
Organisms Xenopus laevis
Chemicals Acetylcholine, Lysophosphatidylcholines