Oc 725c amplifier

All ionic currents were recorded using the Shaker-IR construct where residues 6–46 were deleted to remove N-type inactivation (Hoshi et al., 1990 (link)). For experiments with the tarantula toxin GxTx1E, the toxin-sensitive ShakerΔ5 construct (L327I, A328F, V330T, V331E and A332S) was used (Milescu et al., 2013 (link)). GxTx1E toxin was synthesized on an ABI peptide synthesizer using Fmoc chemistry, refolded in vitro and purified as previously described (Gupta et al., 2015 (link)). Experiments with AHA-modified channel were performed after preincubating the oocytes in 4 mM AHA (prepared in ND96 from a 100 mM stock in ddH₂O) overnight, followed by cRNA injection. Oocytes were injected with 50 nl of channel RNA (5–10 ng/μl) in the absence or presence of AHA and studied after 1–4 days to allow for expression at 17°C in ND96 solution. All the recordings were performed using the two-electrode voltage-clamp recording technique (OC-725C amplifier; Warner Instruments, Hamden, CT) using a 150 μl recording chamber. Data were filtered at 1 kHz and digitized at 5–10 kHz using Digidata 1321A interface board and pCLAMP 10 software (Molecular Devices, Sunnyvale, CA). Microelectrode resistances were 0.2–0.8 MΩ when filled with 3 M KCl. The external recording solution contained (in mM): 50 KCl, 50 NaCl, 10 HEPES, pH 7.6 with NaOH at room temperature (~22°C).

After injection with 50 nl TRPV2 cRNA (500 ng/μl), oocytes were incubated at 17 °C for 4–6 days. Ionic currents were recorded using an OC-725C amplifier (Warner Instruments) and digitized using Digidata 1440 (Axon Instruments). The recording bath solution contained 96 mM NaCl, 2 mM KCl, 1 mM MgCl₂, 1.8 mM CaCl₂ and 5 mM HEPES at pH 7.4 (adjusted with NaOH). Oocytes were voltage-clamped at −60 mV, and current data was obtained every 1 s. Chloramine T (ChT; MP Biomedicals) was dissolved in the bath solution. For heat stimulation, bath solution heated with a temperature controller (TC-344B; Warner Instruments) was applied by perfusion. For poking assay, mechanical stimulation was applied using a tungsten needle (outer diameter, 40 μm) mounted on the manual manipulator (NARISHIGE NMN-21). The needle set to 40° from the horizontal plane into the moving parts of manipulator. The tip of the needle was positioned through the rotation of dials so that it just attached to the cell membrane. After that, we rotated the dials (Z-axis) of manipulator (2.5 rotation). The needle was then moved toward the oocyte as the 8 μm step. It takes for 30 s that the needle moved 8 μm.

Before recording, oocytes were either Na⁺-loaded, to saturate intracellular-facing Na⁺-binding sites, or Na⁺-depleted (to remove most intracellular Na⁺). Na⁺ depletion was done in either a K⁺-loading or an NMG⁺-loading solution. These procedures were done by a 1-h incubation in a solution containing (in mM) 90 cation [either NaOH (Na⁺-loading), KOH (K⁺-loading), or NMG⁺ (NMG⁺-loading)], 20 tetraethylammonium-OH, 0.2 EGTA, and 40 HEPES (pH 7.2 with sulfamic acid), supplemented with 10 μM ouabain. The extracellular solution contained (also in mM): 133 methane-sulfonic acid (MS), 10 HEPES, 5 Ba(OH)₂, 1 Mg(OH)₂, 0.5 Ca(OH)₂, 125 NaOH (Na⁺ solution). External K⁺ was added from a 450 mM K⁺-MS stock. Ouabain was directly added to the extracellular solution. Its solubility above 10 mM was achieved by warming the solution & vortexing on the day of the experiment. These solutions were allowed to cool to room temperature before oocyte perfusion.
Two-electrode voltage clamp was performed at room temperature (21–23 °C), with an OC-725C amplifier (Warner Instruments), a Digidata 1440 A/D board, a Minidigi 1A, and pClamp 10 software (Molecular Devices). Signals were filtered at 2 kHz and digitized at 10 kHz. Resistance of both microelectrodes (filled with 3M KCl) was 0.5–1 MΩ.

For two electrode voltage clamping‎ (TEVC), oocytes were impaled with two 3M KCl-filled capillary Ag/AgCl electrodes with resistances in the 0.2–1.0 MΩ range (Gonzalez-Perez et al., 2010 (link)). Current recordings were performed using an OC-725C amplifier (Warner Instruments) through a PCI-6035 interface (National Instruments) under the command of pCLAMP software (Molecular Devices). Normal recording solution consisted of (in mM) 100 NaCl, 2.5 KCl, 1.8 CaCl₂, 1 MgCl₂, and 10 HEPES-NaOH (pH 7.4). TRPV1 channel activation was determined by measuring the current amplitude elicited by a depolarizing voltage pulse to 30 mV before and after the addition of the compound to the external solution. The maximal channel activation was tested by adding a saturating concentration of capsaicin (50 µM), and channel activation at each concentration was normalized by the maximal TRPV1 response in the presence of capsaicin. Five independent experiments were performed for each compound and averages are expressed with their respective Standard Error (SE).