Two-Electrode Voltage-Clamp Recordings in Oocytes

Membrane current recordings were performed at 21–25°C, 16–72 h after proteoliposome injection, using a high compliance two-microelectrode voltage-clamp system (TurboTEC-10CD, npi Tamm, Germany). The recording methodology has been described previously (Morales et al., 1995; Alberola-Die et al., 2016). Briefly, oocytes were placed in a 150 μl recording chamber and continuously superfused with normal frog Ringer’s solution (NR: 115 mM NaCl, 2 mM KCl, 1.8 mM CaCl₂, 5 mM HEPES, pH 7.0) supplemented with 0.5 μM atropine sulfate (ANR) to block any muscarinic response (Kusano et al., 1982). The membrane potential was held at -60 mV, unless otherwise stated. ACh and other tested drugs were diluted in ANR solution and oocytes were superfused with them at a flow rate of 13–17 ml min^-1. Membrane currents elicited by ACh (I_ACh) either alone or co-applied with DMA, were low-pass filtered at 30–1000 Hz and, after sampling at fivefold the filter frequency (Digidata series 1200 and 1440A; Axon Instruments, Foster City, CA, USA), recorded on two PC-computers, using the WCP v. 3.2.8 package developed by J. Dempster (Strathclyde Electrophysiology Software, University of Strathclyde, Scotland, UK) and AxoScope v. 10.0.0.60 (Molecular Devices Corporation, Sunnyvale, CA, USA).