Two-Electrode Voltage-Clamp (TEVC) Measurements on Xenopus laevis Oocytes Expressing Human nAChR

Defolliculated Xenopus laevis oocytes were obtained from Ecocyte Bioscience (Castrop-Rauxel, Germany). Oocytes from at least two different Xenopus laevis individuals were used in all experimental groups. Oocytes were stored in Ringer’s solution (ORi) containing (in mM) 90 NaCl, 1 KCl, 2 CaCl₂, 5 HEPES, 2.5 pyruvate, 20 mg/ml penicillin, and 25 mg/ml streptomycin (pH 7.4) at 16°C. All chemicals used for ORi preparation were purchased from Fluka (Deisenhofen, Germany), except for HEPES, penicillin, and streptomycin (Sigma-Aldrich). Plasmid DNAs encoding the human CHRNA9 and CHRNA10 as well as the 43 kDa receptor-associated protein of the synapse (RAPSN) were obtained from Eurofins Genomics (Ebersberg, Germany) and capped cRNA was synthesized as described before (31). Human CHRNA7 encoding cRNA was kindly provided by G. Schmalzing (Department of Molecular Pharmacology, RWTH Aachen University, Aachen, Germany) and synthesized as described before (33). cRNA was dissolved in nuclease-free water and injected into oocytes in a volume of 50.6 nl using a microinjector (Nanoject, Drummond Scientific, Broomall, PA, USA). Oocytes were injected with cRNA encoding CHRNA7, CHRNA9, and CHRNA10 nAChR subunits (16, 19, and 19 ng/oocyte, respectively) along with 5 ng cRNA encoding RAPSN, and oocytes were incubated at 16°C for 3–5 days. In control experiments, 50.6 nl of nuclease-free water was injected.

In TEVC measurements, oocytes were perfused (gravity driven) with ORi without pyruvate and antibiotics (pH 7.4). Intracellular borosilicate microelectrodes were filled with 1 M KCl solution and the membrane voltage was clamped to –60 mV using a TEVC amplifier (Warner Instruments, Hamden, CT, USA). Low-pass filtered transmembrane currents (1,000 Hz, Frequency Devices 902, Haverhill, MA, USA) were recorded using a strip chart recorder (Kipp & Zonen, Delft, The Netherlands). For experiments examining the inhibition and recovery from inhibition of choline-gated currents in presence and absence of eCRP (5 µg/ml), oocytes were injected with a 1:1 ratio of cRNA encoding human CHRNA9 and CHRNA10 in a 1:1 ratio, incubated at 17°C for 2–3 days and measured as described before (31).