Two-electrode voltage clamp recording and analysis

Currents from expressed channels 1–3 days post-injection were recorded in two-electrode voltage clamp (Dagan TEV-200, HEKA ITC and Patchmaster software). Oocytes were held at −80 mV and perfused continuously with buffer (ND96) or buffer containing PTX, GABA, or DZ. PTX was diluted from a 1 M stock solution in DMSO. DZ was diluted from a 10 mM stock solution in DMSO. Fresh PTX and DZ stock solutions were tested several times with no change in results. GABA was dissolved directly from powder. A microfluidic pump (Elveflow OB1 MK3+) and rotary valve (Elveflow MUX Distributor) provided consistent and repeatable perfusion and solution exchange across experiments, which limited solution exchange variability to primarily differences between oocytes only. Ten second pulses of PTX, GABA, or DZ were followed by 3–6 min in buffer to allow currents to return to baseline. Recorded currents were analyzed with custom scripts in MATLAB (Mathworks). Recordings of concentration–response relationships were bookended by pulses of PTX to correct for any drift or rundown during the experiment. Briefly, currents were baseline subtracted to correct for drift and then scaled by a linear fit to the peak of each PTX response to correct for rundown (Figure 2—figure supplement 1). The amount of current rundown was variable across oocytes, with no clear relation to specific constructs. Concentration–response relationships were fit to the Hill equation:

 where I is the peak current response, X is ligand concentration, EC50 is the concentration eliciting a half-maximal response, and n is the Hill slope.