Imaging the Nicotinic Acetylcholine Receptor in Reconstituted Membranes by Atomic Force Microscopy

Abstract

Several recent studies have suggested that the nicotinic acetylcholine receptor (nAChR) segregates lipids into domains in reconstituted bilayers. These studies, however, lack direct evidence (e.g., microscopic images) to show domain formation. Atomic force microscopy (AFM) has been used extensively to image both lipid domains and proteins in membranes, but has not been applied extensively to reconstituted membrane proteins due to the lack of available protocols for preparing suitable planar bilayers on AFM supports. The aim of the work presented in this thesis was to image the nAChR in planar reconstituted membranes by AFM. I developed a novel method for reconstituting the nAChR in POPC (POPC-nAChR) to generate vesicles with high lipid-to-protein (L:P) ratios (i.e., greater than 100:1 w/w). Freeze-thaw cycles are required to improve vesicle homogeneity. The high L:P vesicles must also be isolated from protein-free vesicles using sucrose density gradients. Finally, the preparation of planar bilayers from the high L:P ratio proteoliposomes requires an appropriate sample load and incubation time on a defined area of mica surface (the solid AFM support) and an appropriate level of calcium. AFM images of a POPC-nAChR bilayers show a number of features that protrude out of the bilayer with an average height of 3 nm and diameter of 4 – 9 nm, which is appropriate for the dimensions of the cytoplasmic side of the nAChR. My results thus represent the first AFM images of the nAChR in a reconstituted membrane environment. Now that the key parameters governing nAChR reconstitution and planar bilayer preparation for AFM imaging are understood, they will undoubtedly be useful for reconstituting and imaging the nAChR in more complex bilayers.