Extracellular vesicle fusion-mediated compartmentalized membrane protein reconstitution toward intracellular signaling coupling.

Membrane proteins play essential roles in drug targeting and biological signal regulation, and planar lipid bilayer systems provide a powerful platform for single-molecule electrophysiological analyses. However, conventional reconstitution methods suffer from low incorporation efficiency and random orientation of proteins. We developed a novel reconstitution platform that exploits the membrane fusion capability of extracellular vesicles (EVs) to incorporate membrane proteins into planar lipid bilayers with high efficiency and preferential orientation. Using EVs derived from HEK293 cells expressing GABAA receptors, we confirmed receptor localization on EV membranes and observe GABA-responsive single-channel currents after fusion with planar lipid bilayers. These currents were inhibited by treatment with bicuculline and abolished by trypsin, demonstrating successful reconstitution of functional GABAA receptors. Further, the overall success rate of channel current detection was calculated irrespective of the EV volume added and the type of channel current detected (single-channel or multichannel). Channel currents were detected in 78 of 88 independent experiments, yielding a detection success rate of 88.64%. GABA responses were detected only when GABA was added to the chamber corresponding to the extracellular side, indicating preferential receptor orientation and successful reconstruction of membrane compartmentalization. Intracellular addition of protein kinase A enhanced the single-channel conductance and channel open time, whereas extracellular addition had no effect. This platform provides a versatile foundation for membrane protein analysis, drug discovery, and the engineering of functional biomimetic membranes, with the potential to pave the way for membrane protein-based bioelectronics.