We describe nanotube-vesicle networks with reconstituted membrane protein from cells and with interior activity defined by an injection of microparticles or molecular probes. The functionality of a membrane protein after reconstitution was verified by single-channel ion conductance measurements in excised inside-out patches from the vesicle membranes. The distribution of protein, determined by fluorescence detection, in the network membrane was homogeneous and could diffuse via a nanotube connecting two vesicles. We also show how injecting small unilamellar protein-containing vesicles can differentiate the contents of individual containers in a network. The combination of membrane activity and interior activity was demonstrated by ionophore-assisted accumulation, and internal Calcium Green-mediated detection, of Ca2+ within a single network container. This system can model a variety of biological functions and complex biological multicompartment structures and might serve as a platform for constructing complex sensor and computational devices.