In bulla functional channel expression systems that mimic bacterial synthetic membranes

A functional characterization of channel proteins has been performed using planar lipid bilayers as the following procedure. For bacterial channels, such as the KcsA potassium channel, channel proteins were synthesized in Escherichia coli, followed by solubilization, purification, and incorporation into liposomes. Similarly, channel proteins were synthesized using an in vitro transcription/translation kit in the presence of liposomes. Then, these liposome-incorporated channels were served for electrophysiological recordings after liposome fusion into a preformed planar lipid bilayer. Here, we established a straightforward method for concurrent channel synthesis and functional measurement using a water-in-oil bubble bilayer system. Channel proteins were synthesized in vitro within a water-in-oil bubble, having a lipid bilayer at the contact with another bubble (in bulla synthesis). The channels were spontaneously incorporated into the lipid bilayer under application of the membrane potential, and we successfully detected nascent channel activities. This way our experiment has mimicked bacterial synthetic membrane in the presence of a resting membrane potential. Technical details for establishing the in bulla expression system are described.