Transmembrane proteins (TMPs) regulate processes occurring at the cell surface and are essential gatekeepers of information flow across the membrane. TMPs are difficult to study, given the complex environment of the membrane and its influence on protein conformation, mobility, biomolecule interaction, and activity. For the first time, we create mammalian biomembranes supported on a transparent, electrically conducting polymer surface, which enables dual electrical and optical monitoring of TMP function in its native membrane environment. Mammalian plasma membrane vesicles containing ATP-gated P2X2 ion channels self-assemble on a biocompatible polymer cushion that transduces the changes in ion flux during ATP exposure. This platform maintains the complexity of the native plasma membrane, the fluidity of its constituents, and protein orientation critical to ion channel function. We demonstrate the dual-modality readout using microscopy to characterize protein mobility by single-particle tracking and sensing of ATP gating of P2X2 using electrical impedance spectroscopy. This measurement of TMP activity important for pain sensing, neurological activity, and sensory activity raises new possibilities for drug screening and biosensing applications.

Transmembrane proteins represent a major target for modulating cell activity, both in terms of therapeutics drugs and for pathogen interactions. Work on screening such therapeutics or identifying toxins has been severely limited by the lack of available methods that would give high content information on functionality (ideally multimodal) and that are suitable for high-throughput. Here, we have demonstrated a platform that is capable of multimodal (optical and electronic) screening of ligand gated ion-channel activity in human-derived membranes. The TREK-1 ion-channel was expressed within supported lipid bilayers, formed via vesicle fusion of blebs obtained from the HEK cell line overexpressing TREK-1. The resulting reconstituted native membranes were confirmed via fluorescence recovery after photobleaching to form mobile bilayers on top of films of the polymeric electroactive transducer poly(3,4-ethylenedioxythiophene) polystyrenesulfonate (PEDOT:PSS). PEDOT:PSS electrodes were then used for quantitative electrochemical impedance spectroscopy measurements of ligand-mediated TREK-1 interactions with two compounds, spadin and arachidonic acid, known to suppress and activate TREK-1 channels, respectively. PEDOT:PSS-based organic electrochemical transistors were then used for combined optical and electronic measurements of TREK-1 functionality. The technology demonstrated here is highly promising for future high-throughput screening of transmembrane protein modulators owing to the robust nature of the membrane integrated device and the highly quantitative electrical signals obtained. This is in contrast with live-cell-based electrophysiology assays (e.g., patch clamp) which compare poorly in terms of cost, usability, and compatibility with optical transduction.

Membrane biosensors that can rapidly sense pathogen interaction and disrupting agents are needed to identify and screen new drugs to combat antibiotic resistance. Bioelectronic devices have the capability to read out both ionic and electrical signals, but their compatibility with biological membranes is somewhat limited. Supported lipid bilayers (SLBs) have served as useful biomimetics for a myriad of research topics involving biological membranes. However, SLBs are traditionally made on inert, rigid, inorganic surfaces. Here, we demonstrate a versatile and facile method for generating SLBs on a conducting polymer device using a solvent-assisted lipid bilayer (SALB) technique. We use this bioelectronic device to form both mammalian and bacterial membrane mimetics to sense the membrane interactions with a bacterial toxin (α-hemolysin) and an antibiotic compound (polymyxin B), respectively. Our results show that we can form high quality bilayers of both types and sense these particular interactions with them, discriminating between pore formation, in the case of α-hemolysin, and disruption of the bilayer, in the case of polymyxin B. The SALB formation method is compatible with many membrane compositions that will not form via common vesicle fusion methods and works well in microfluidic devices. This, combined with the massive parallelization possible for the fabrication of electronic devices, can lead to miniaturized multiplexed devices for rapid data acquisition necessary to identify antibiotic targets that specifically disrupt bacterial, but not mammalian membranes, or identify bacterial toxins that strongly interact with mammalian membranes.

Adipose-derived stem cells (ASCs) are abundantly present in the mammary microenvironment and can promote breast cancer malignancy by differentiating into myofibroblasts. However, it remains largely unclear which role tumor-derived extracellular vesicles (TEVs) play in this process. Here, we used microfabricated, type I collagen-based 3-D tissue culture platforms to investigate the effect of breast cancer cell-derived TEVs on ASCs myofibroblast differentiation and consequential changes in extracellular matrix remodeling and vascular sprouting. TEVs collected from MDA MB-231 human metastatic breast cancer cells (MDAs) promoted ASC myofibroblast differentiation in both 2-D and 3-D culture as indicated by increased alpha smooth muscle actin (α-SMA) and fibronectin (Fn) levels. Correspondingly, TEV-treated ASCs were more contractile, secreted more vascular endothelial growth factor (VEGF), and promoted angiogenic sprouting of human umbilical vein endothelial cells. These changes were dependent on transforming growth factor beta (TGF-β)-related signaling and tumor cell glutaminase activity as their inhibition decreased TEV-related myofibroblastic differentiation of ASCs and related functional consequences. In summary, our data suggest that TEVs are important signaling factors that contribute to ASC desmoplastic reprogramming in the tumor microenvironment, and suggest that tumor cell glutamine metabolism may be used as a therapeutic target to interfere with this process.

Reconstituted supported lipid bilayers (SLB) are widely used as in vitro cell-surface models because they are compatible with a variety of surface-based analytical techniques. However, one of the challenges of using SLBs as a model of the cell surface is the limited complexity in membrane composition, including the incorporation of transmembrane proteins and lipid diversity that may impact the activity of those proteins. Additionally, it is challenging to preserve the transmembrane protein native orientation, function, and mobility in SLBs. Here, we leverage the interaction between cell plasma membrane vesicles and polyelectrolyte brushes to create planar bilayers from cell plasma membrane vesicles that have budded from the cell surface. This approach promotes the direct incorporation of membrane proteins and other species into the planar bilayer without using detergent or reconstitution and preserves membrane constituents. Furthermore, the structure of the polyelectrolyte brush serves as a cushion between the planar bilayer and rigid supporting surface, limiting the interaction of the cytosolic domains of membrane proteins with this surface. Single particle tracking was used to analyze the motion of GPI-linked yellow fluorescent proteins (GPI-YFP) and neon-green fused transmembrane P2X2 receptors (P2X2-neon) and shows that this platform retains over 75% mobility of multipass transmembrane proteins in its native membrane environment. An enzyme accessibility assay confirmed that the protein orientation is preserved and results in the extracellular domain facing toward the bulk phase and the cytosolic side facing the support. Because the platform presented here retains the complexity of the cell plasma membrane and preserves protein orientation and mobility, it is a better representative mimic of native cell surfaces, which may find many applications in biological assays aimed at understanding cell membrane phenomena.