*3.9. Oocyte Two-Electrode Voltage Clamp Recording and Data Analysis*

Electrophysiological recordings were carried out 2–5 days post cRNA microinjection. Two-electrode voltage clamp recordings of *X. laevis* oocytes expressing human nAChRs were performed at room temperature (21–24 ◦C) using a GeneClamp 500B amplifier and pClamp9 software interface (Molecular Devices, Sunnyvale, CA, USA) at a holding potential −80 mV. Voltage-recording and current-injecting electrodes were pulled from GC150T-7.5 borosilicate glass (Harvard Apparatus, Holliston, MA) and filled with 3 M KCl, giving resistances of 0.3–1 MΩ. Due to the Ca2+ permeability of *α*9*α*10 nAChRs, 100 μM BAPTA-AM incubation was carried out before recording to prevent the activation of *X. laevis* oocyte endogenous Ca2+-activated chloride channels. Oocytes expressing h*α*9*α*10 nAChRs were perfused with ND115 solution containing (in mM): 115 NaCl, 2.5 KCl, 1.8 CaCl2, and 10 HEPES at pH 7.4, whereas oocytes expressing all other nAChR subtypes were perfused with ND96 solution using a continuous Legato 270 push/pull syringe pump perfusion system (KD Scientific, Holliston, MA, USA) at a rate of 2 mL/min in an OPC-1 perfusion chamber of <20 μL volume (Automate Scientific, Berkeley, CA, USA).

Initially, oocytes were briefly washed with ND115/ND96 solution, followed by 3 applications of ACh at a half-maximal excitatory ACh concentration (EC50) for the nAChR subtypes (3 μM for h*α*4*β*2, 5 μM for h*α*1*β*1*γδ* and h*α*1*β*1*εδ*, 6 μM for h*α*3*β*2 and h*α*9*α*10, 100 μM for h*α*7 and 300 μM for h*α*3*β*4) [27]. Washout with bath solution was performed for 3 min between ACh applications. Oocytes were incubated with compounds for 5 min with the perfusion system turned off, followed by co-application of ACh and compound with flowing bath solution. All compound solutions were prepared in ND115/ND96 + 0.1% bovine serum albumin (BSA). Incubation with 0.1% BSA was performed to ensure that the BSA and the pressure of the perfusion system had no effect on nAChRs. Peak current amplitudes before (ACh alone) and after (ACh + compound) compound incubation were measured using Clampfit version 10.7.0.3 software (Molecular Devices, Sunnyvale, CA, USA), where the ratio of ACh + compound-evoked current amplitude to ACh alone-evoked current amplitude was used to assess the activity of the compounds at the nAChRs. All electrophysiological data were pooled (n = 6–11) and represent means ± standard deviation (SD). Data analysis was performed using GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA).
