Expression of functional KcsA channels by coupled IVTT. (A) Assembly of KcsA channels during IVTT. Products of IVTT were separated in a 12.5% SDS polyacrylamide gel supplemented with 10 mM KCl. Lane 1, WT KcsA; lane 2, E71A KcsA. The proteins were labeled with [³⁵S]methionine and visualized by exposure of the dried gel to x-ray film. (B–D) Representative single-channel recordings of KcsA purified by SDS-PAGE. (B) WT KcsA in POPE/POPG (3:1) bilayers at +150 mV. (C) WT KcsA in DPhPC bilayers at ±100 mV. (D) E71A KcsA in DPhPC bilayers at ±100 mV. The measurements were made in 200 mM KCl, buffered with 10 mM HEPES, pH 7.0, in the cis chamber and 200 mM KCl, buffered with 10 mM succinic acid, pH 4.0, in the trans chamber. The traces were digitally filtered at 1 kHz for display.