RESUMEN
Membrane proteins are among the most difficult to study as they are embedded in the cellular membrane, a complex and fragile environment with limited experimental accessibility. To study membrane proteins outside of these environments, model systems are required that replicate the fundamental properties of the cellular membrane without its complexity. We show here a self-assembled lipid bilayer nanoarchitecture on a solid support that is stable for several days at room temperature and allows the measurement of insect olfactory receptors at the single-channel level. Using an odorant binding protein, we capture airborne ligands and transfer them to an olfactory receptor from Drosophila melanogaster (OR22a) complex embedded in the lipid membrane, reproducing the complete olfaction process in which a ligand is captured from air and transported across an aqueous reservoir by an odorant binding protein and finally triggers a ligand-gated ion channel embedded in a lipid bilayer, providing direct evidence for ligand capture and olfactory receptor triggering facilitated by odorant binding proteins. This model system presents a significantly more user-friendly and robust platform to exploit the extraordinary sensitivity of insect olfaction for biosensing. At the same time, the platform offers a new opportunity for label-free studies of the olfactory signaling pathways of insects, which still have many unanswered questions.
RESUMEN
The plasma membrane protects the interiors of cells from their surroundings and also plays a critical role in communication, sensing, and nutrient import. As a result, the cell membrane and its constituents are among the most important drug targets. Studying the cell membrane and the processes it facilitates is therefore crucial, but it is a highly complex environment that is difficult to access experimentally. Various model membrane systems have been developed to provide an environment in which membrane proteins can be studied in isolation. Among them, tethered bilayer lipid membranes (tBLMs) are a promising model system providing a solvent-free membrane environment which can be prepared by self-assembly, is resistant to mechanical disturbances and has a high electrical resistance. tBLMs are therefore uniquely suitable to study ion channels and charge transport processes. However, ion channels are often large, complex, multimeric structures and their function requires a particular lipid environment. In this paper, we show that SthK, a bacterial cyclic nucleotide gated (CNG) ion channel that is strongly dependent on the surrounding lipid composition, functions normally when embedded into a sparsely tethered lipid bilayer. As SthK has been very well characterized in terms of structure and function, it is well-suited to demonstrate the utility of tethered membrane systems. A model membrane system suitable for studying CNG ion channels would be useful, as this type of ion channel performs a wide range of physiological functions in bacteria, plants, and mammals and is therefore of fundamental scientific interest as well as being highly relevant to medicine.
Asunto(s)
Canales Iónicos , Técnicas Electroquímicas , Canales Iónicos/química , Membrana Dobles de Lípidos/química , Microscopía de Fuerza Atómica , AMP Cíclico/metabolismo , Bacterias/química , Bacterias/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismoRESUMEN
Membrane proteins are among the most important drug targets. To improve drug design, it is critical to study membrane proteins. However, due to the myriad roles fulfilled by the cellular membrane, it is a highly complex environment and challenging to study. Tethered membranes reproduce the basic physicochemical properties of the cellular membrane without its inherent complexity. The high electrical resistance and stability makes them ideal to study membrane proteins, particularly ion channels. However, due to the close proximity of the membrane to the support and the reduced fluidity and high packing density, they are unsuitable to study larger membrane proteins. We present here a tethered membrane system which adresses these challenges, allowing the functional reconstitution of the odorant receptor coreceptor from Drosophila melanogaster, a tetrameric ionotropic receptor was incorporated and its sensitivity to various ligands was examined via electrochemical impedance spectroscopy and atomic force microscopy.