All GIRK channels were expressed and purified in P. pastoris as described previously15, 32. Briefly, the highest-expressing clone was grown in BMGY medium and induced in BMM medium containing 1% methanol. Cells were harvested, resuspended in buffer (50 mM HEPES, pH 7.4; 150 mM KCl; 1 mM TCEP; 1 mM AEBSF and Complete EDTA-free protease inhibitor tablets (Roche)), dripped into liquid nitrogen, and placed at −80 °C. Frozen cells were lysed in a Mixer Mill (Retsch) 5-times for 3 minutes at 25 Hz and solubilized in 50 mM HEPES, pH 7.35; 150 mM KCl; 1 mM TCEP; 1 mM AEBSF; 3% (w/v) n-Dodecyl-β-D-maltoside (DDM; Anatrace) and Complete ULTRA EDTA-free protease inhibitor tablets (Roche) with gentle stirring at 4 °C. Unsolubilized material was separated by centrifugation at 40,000 × g for 40 min at 4 °C. The supernatant was injected onto a HISTrap HP column (GE Healthcare) equilibrated in wash buffer (50 mM HEPES, pH 7.0; 150 mM KCl; 0.4% DDM; 20 mM imidazole) connected to an ÄKTA pure (GE Healthcare) chromatography system and eluted in buffer containing 300 mM imidazole.
