4.2. Two-Electrode Voltage-Clamp Recordings in Oocytes

Membrane currents were recorded 16–72 h after proteoliposome injection, as previously reported [21,23]. Briefly, oocytes were continuously superfused with normal frog Ringer’s solution (115 mM NaCl, 2 mM KCl, 1.8 mM CaCl₂, 5 mM HEPES, pH 7.0) supplemented with 0.5 μM atropine sulfate (normal Ringer with atropine, ANR) to block any muscarinic response [43]. The membrane potential was held at −60 mV, unless otherwise stated. Membrane currents elicited by ACh, either alone or co-applied with Pm, were low-pass filtered at 30–1000 Hz and, after sampling at fivefold the filter frequency (Digidata series 1440A and 1550; Axon Instruments, Foster City, CA, USA), recorded on two PC-computers, using the WCP v. 4.8.6 package developed by J. Dempster (Strathclyde Electrophysiology Software, University of Strathclyde, Glasgow, UK) and AxoScope v. 10.0.0.60 (Molecular Devices Corporation, Sunnyvale, CA, USA).