KcsA was expressed in E. coli and purified on Ni2+ affinity columns as described (Heginbotham et al. 1997; MacKinnon et al. 1998). The purified channel was eluted in 400 mM imidazole at 1–5 mg/ml protein concentration quantified by the extinction coefficient at 280 nm (Heginbotham et al. 1998). Immediately after purification, KcsA was reconstituted into liposomes at room temperature as follows. A micellar solution of phospholipids (7.5 mg/ml POPE, 2.5 mg/ml POPG) and 34 mM CHAPS in reconstitution buffer (450 mM KCl/10 mM HEPES/4 mM N-methylglucamine, pH 7.0) was prepared as described (Heginbotham et al. 1998), and KcsA protein was added to final concentrations of 2.5–10 μg/ml, according to the number of channels per liposome desired. After 20–30 min incubation, 400 μl of the mixture was passed down a 20–ml Sephadex G-50 (fine) column equilibrated with reconstitution buffer. Liposomes eluted in the void volume with a dilution of approximately threefold and were stored in 75-μl aliquots at −80°C for up to 3 mo.