Electrophysiological Two-Electrode Voltage Clamp Assay.

Female Xenopus laevis frogs were purchased from Nasco (Fort Atkinson, WI). Oocyte harvesting was performed according to protocol approved by the University of Arizona IACUC. Electrophysiological measurements were performed using the AM2 gene out of the A/California/07/2009 as previously described (Balannik et al., 2010; Musharrafieh et al., 2018). In brief, mRNA coding A/California/07/2009 AM2 and its variants were synthesized via an mMessage and mMachine T7 kit (Invitrogen) according to manufacturer protocol. Ten to 50 ng of mRNA was injected into each oocyte. Twenty-four to 72 hours after mRNA injection, electrophysiological two-electrode voltage clamp (TEVC) recordings were performed. Oocytes were constantly held at −20 mV, and current was recorded at various testing conditions. The detailed EC₅₀ measurement was described by Jing et al. (2008). At least eight concentrations covering the EC₅₀ value of each compound were applied in the TEVC recording. At least three oocytes were recorded at each concentration. The percentage of remaining channel current after application of compounds was plotted against the compound concentration with dose-response function in GraphPad Prism 5.0.

The AM2 channel–specific conductance was measured as described by Musharrafieh et al. (2018). In brief, the membrane currents of individual oocytes expressing A/California/07/2009 AM2 or its mutants were first recorded with TEVC electrophysiology technique. Eight to 10 oocytes expressing AM2 or L46P mutant recorded with different currents were saved and fixed with 2% paraformaldehyde for 30 minutes. Fixed whole oocytes were immunostained with primary 14C2 (anti-AM2; gift from Dr. Robert A. Lamb, Northwestern University, Evanston, IL) monoclonal antibody and secondary Alexa Fluor 546–labeled goat anti-mouse IgG (Molecular Probes, Inc., Medford, OR). A ZOE fluorescent Cell Imager (Bio-Rad) was used to acquire fluorescence images, and about one-half of the surface of each oocyte was imaged. Photoshop was used to quantify the fluorescence intensity by measuring the gray-scale value. As a control for the autofluorescence signal from the yolk, uninjected oocytes were measured. The relative specific activity of AM2 was obtained by taking the slope of the linear regression curve generated from plotting the whole-cell current against the AM2 expression level (detected by immunofluorescence) for each oocyte.

The L46P mutation was introduced using site-directed mutagenesis (QuikChange Site-Directed Mutagenesis Kit; Agilent, Santa Clara, CA).