Concurrent In Vitro Synthesis and Functional Detection of Nascent Activity of the KcsA Channel under a Membrane Potential.

Processes involved in the functional formation of prokaryotic membrane proteins have remained elusive. Here, we developed a new in vitro membrane protein expression system to detect nascent activities of the KcsA potassium channel in lipid bilayers under an applied membrane potential. The channel was synthesized using a reconstituted Escherichia coli-based in vitro transcription/translation system (IVTT) in a water-in-oil droplet lined by a membrane. The synthesized channels spontaneously incorporated into the membrane even without the translocon machinery (unassisted pathway) and formed functional channels with the correct orientation. The single-channel current of the first appearing nascent channel was captured, followed by the subsequent appearance of multiple channels. Notably, the first appearance time shortened substantially as the membrane potential was hyperpolarized. Under a steadily applied membrane potential, this system serves as a production line of membrane proteins via the unassisted pathway, mimicking the bacterial synthetic membrane.