Functional Reconstitution of Dopamine D2 Receptor into a Supported Model Membrane in a Nanometric Confinement.

Dopamine D2 receptor (D2R), a G-protein-coupled receptor (GPCR), plays critical roles in neural functions and represents the target for a wide variety of drugs used to treat neurological diseases. However, its fundamental physicochemical properties, such as dimerization and affinity to different lipid environments, remain unknown. Here, reconstitution and characterization of D2R in a supported model membrane in nanometric confinement are reported. D2R is expressed in Chinese hamster ovary (CHO) cells and transferred into the supported model membrane as cell membrane blebs. D2R molecules are reconstituted with an elevated density in the cleft between the substrate and poly(dimethylsiloxane) (PDMS) elastomer. Reconstituted D2R retains the physiological functions, as evaluated from its binding to an antagonist and dimerization lifetime. The transient dimer formation of D2R, similar to the live cell, suggests that it is an innate property that does not depend on the cellular structures such as actin filaments. Although the mechanism of this unique reconstitution process is currently not fully understood, the finding points to a new possibility of using a nanometric space (<100 nm thick) as a platform for reconstituting and studying membrane proteins under the quasi-physiological conditions, which are difficult to be created by other methods.

Self-Spreading of Phospholipid Bilayer in a Patterned Framework of Polymeric Bilayer.

Phospholipid bilayers spontaneously spread on a hydrophilic substrate such as glass in aqueous solution due to the energetic gain of surface wetting. This process (self-spreading) was utilized to form a patterned model biological membrane containing reconstituted membrane proteins. A mechanically stable framework of a polymerized lipid bilayer was first generated by the lithographic polymerization of a diacetylene phospholipid. Then, natural lipid membranes (fluid bilayers) were introduced into the channels between polymeric bilayers by the self-spreading from a phospholipid reservoir. The spreading velocity could be fitted into a slope of -0.5 in a double logarithmic plot versus time due to the balance between the spreading force and resistive drag. The preformed polymeric bilayer accelerated the spreading by the energetic gain of covering hydrophobic edges with a lipid bilayer. At the same time, the domains of the polymeric bilayer obstructed spreading, and the spreading velocity linearly decreased with their fractional coverage. Above the critical coverage of ca. 50%, self-spreading was completely blocked (percolation threshold) and the fluid bilayer was confined in the polymer-free regions. Nonspecific adsorption of lipids onto the surface of polymeric bilayers was negligible, which enabled a heightened signal-to-background ratio in the reconstitution and observation of membrane proteins. Self-spread bilayers had a higher density of lipids than those formed by the spontaneous rupture of vesicles (vesicle fusion), presumably due to the continual supply of lipid molecules from the reservoir. These features give the self-spreading important advantages for preparing patterned model membranes with reconstituted membrane proteins.