Heka epc 10 amplifier

Current responses from GlyR-transfected HEK293 cells were measured at room temperature (21–23°C) at a holding potential of −50 mV using a HEKA EPC10 amplifier (HEKA Electronics, Lambrecht, Germany) controlled by Pulse software (HEKA Electronics). Recording pipettes were pulled from borosilicate glass (World Precision Instruments, Berlin, Germany) using a Sutter P-97 horizontal puller (Sutter, Novato, CA, USA). Solutions were applied using an Octaflow system (NPI electronics, Tamm, Germany). The external buffer consisted of 135 mM NaCl, 5.5 mM KCl, 2 mM CaCl₂, 1.0 mM MgCl₂, and 10 mM Hepes (pH adjusted to 7.4 with NaOH); the internal buffer was 140 mM CsCl, 1.0 mM CaCl₂, 2.0 mM MgCl₂, 5.0 mM EGTA, and 10 mM Hepes (pH adjusted to 7.2 with CsOH). Glucose (Sigma-Aldrich, Munich, Germany) was added to the growth medium on the day before experiments to give a pre-exposure to 50 mM of Glucose for 16–20 h. Dose-response data were fitted to the Hill equation (see Supplementary Material) to determine EC₅₀ and IC₅₀. Significance of differences between EC₅₀ values were determined using one-way ANOVA with p ≤ 0.05 (*) and p ≤ 0.01 (**) taken as significant.

Cells were transfected with either GlyR-α3L-eGFP, GlyR-α3K-eGFP or GlyR-α3K-mCherry. Recordings were performed at room temperature in voltage-clamp mode using a HEKA EPC10 amplifier (HEKA Electronics, Lambrecht, Germany) controlled by HEKA acquisition software. Patch pipettes (3–4 MΩ) were filled with internal solution containing 120 mM CsCl, 2 mM Na₂ATP, 2 mM MgATP, 10 mM EGTA and 10 mM HEPES, adjusted to pH 7.2 with CsOH. The standard external solution (SES) had a composition of 150 mM NaCl, 5.4 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 10 mM glucose and 10 mM HEPES. Glycinergic currents were recorded at a holding potential $V_H=-60\mathrm{mV}$ . Different glycine concentrations in SES including 20 µM, 50 µM, 100 µM, 200 µM, and 500 µM were applied for 10 s. For dose–response curves in whole cell configuration, glycine was applied using a Warner SF77B fast step superfusion system that allowed an exchange time of < 20 ms (Warner Instruments LLC, Hamden, CT, USA). Maximum current amplitude was measured using FitMaster software (HEKA Electronics). The EC₅₀ was calculated by plotting the normalized current as a function of concentration and fitting the data with the Hill equation (GraphPad Prism, La Jolla, CA, USA). For desensitization analysis, the decaying current phase was fitted using a mono-exponential in FitMaster software (HEKA Electronics, Lambrecht, Germany).