All acquired electrophysiological data were analysed with Clampfit 10.7 (Molecular Devices, Sunnyvale, CA, USA) and GraphPad Prism 8 (GraphPad Software, La Jolla, CA, USA) and from all experiments, peak currents from BAPTA-AM-incubated oocytes were measured after application of drugs. For the auxiliary protein (RIC-3) test, the group mean current of oocytes co-injected with Xla-ric-3 and Tsu-acr-16-like cRNAs in response to 100 μM ACh was set to 100%, and all other responses were normalized to this. The relative means were statistical analysed using a non-parametric Kruskal-Wallis Test and P < 0.05 was considered significant. For the drug-potency-test, peak currents of drugs were normalized to the peak current measured in the presence of 100 μM ACh and was expressed as mean ± SEM. Data was tested for normality using the D’Agostino-Pearson normality test. Drug-group means were statistical analysed using One-Way ANOVA with a Turkey’s Multiple Comparison Test where P < 0.05 was considered significant.

For the dose-response relationships, and for each experiment, 6 oocytes were exposed to ACh (300 μM) for 10 s, and all other drug responses in the same experiment, were normalized to the mean response of these controls. The normalized current as a function of drug concentration allowed fitting the dose-response curves with a Hill equation, using nonlinear regression analysis with a variable slope model in GraphPad Prism 8. The following equation was used:

where Irel is the mean relative current, Imax, is the relative current obtained at saturating agonist concentration, Imin is the relative current obtained at agonist concentration 0 μM, EC50 is the concentration of agonist resulting in 50% of the maximal current response, [D] is the drug concentration and nH is the Hill coefficient. Imax, EC50 and nH were fitted as free parameters whereas Imin, was constrained to 0.

For the antagonist test with α-BTX, the current response of α-BTX and ACh in the continued presence of α-BTX (second application) was normalized to the first response at 100 μM ACh (first application) which was set to 1. The group mean of the α-BTX response were statistical analysed using One-Way ANOVA with a Dunnett’s test.

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