Reconstitution of homomeric GluA2(flop) receptors in supported lipid membranes: functional and structural properties.

AMPA receptors (AMPARs) are glutamate-gated ion channels ubiquitous in the vertebrate central nervous system, where they mediate fast excitatory neurotransmission and act as molecular determinants of memory formation and learning. Together with detailed analyses of individual AMPAR domains, structural studies of full-length AMPARs by electron microscopy and x-ray crystallography have provided important insights into channel assembly and function. However, the correlation between the structure and functional states of the channel remains ambiguous particularly because these functional states can be assessed only with the receptor bound within an intact lipid bilayer. To provide a basis for investigating AMPAR structure in a membrane environment, we developed an optimized reconstitution protocol using a receptor whose structure has previously been characterized by electron microscopy. Single-channel recordings of reconstituted homomeric GluA2(flop) receptors recapitulate key electrophysiological parameters of the channels expressed in native cellular membranes. Atomic force microscopy studies of the reconstituted samples provide high-resolution images of membrane-embedded full-length AMPARs at densities comparable to those in postsynaptic membranes. The data demonstrate the effect of protein density on conformational flexibility and dimensions of the receptors and provide the first structural characterization of functional membrane-embedded AMPARs, thus laying the foundation for correlated structure-function analyses of the predominant mediators of excitatory synaptic signals in the brain.

AFM observation of single, functioning ionotropic glutamate receptors reconstituted in lipid bilayers.

BACKGROUND: Ionotropic glutamate receptors (iGluRs) are responsible for extracellular signaling in the central nervous system. However, the relationship between the overall structure of the protein and its function has yet to be resolved. Atomic force microscopy (AFM) is an important technique that allows nano-scale imaging in liquid. In the present work we have succeeded in imaging by AFM of the external features of the most common iGluR, AMPA-R (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor), in a physiological environment. METHODS: Homomeric GluR3 receptors were over-expressed in insect cells, purified and reconstituted into lipid membranes. AFM images were obtained in a buffer from membranes immobilized on a mica substrate. RESULTS: Using Au nanoparticle-conjugated antibodies, we show that proteins reconstitute predominantly with the N-terminal domain uppermost on the membrane. A tetrameric receptor structure is clearly observed, but it displays considerable heterogeneity, and the dimensions differ considerably from cryo-electron microscopy measurements. CONCLUSIONS: Our results indicate that the extracellular domains of AMPA-R are highly flexible in a physiological environment. GENERAL SIGNIFICANCE: AFM allows us to observe the protein surface structure, suggesting the possibility of visualizing real time conformational changes of a functioning protein. This knowledge may be useful for neuroscience as well as in pharmaceutical applications.

Electrically conducting, ultra-sharp, high aspect-ratio probes for AFM fabricated by electron-beam-induced deposition of platinum.

We report on the fabrication of electrically conducting, ultra-sharp, high-aspect ratio probes for atomic force microscopy by electron-beam-induced deposition of platinum. Probes of 4.0 ±1.0 nm radius-of-curvature are routinely produced with high repeatability and near-100% yield. Contact-mode topographical imaging of the granular nature of a sputtered gold surface is used to assess the imaging performance of the probes, and the derived power spectral density plots are used to quantify the enhanced sensitivity as a function of spatial frequency. The ability of the probes to reproduce high aspect-ratio features is illustrated by imaging a close-packed array of nanospheres. The electrical resistance of the probes is measured to be of order 100 kΩ.