Fluorescence Studies of the Acetylcholine Receptor: Structure and Dynamics in Membranes and Cells

The nicotinic acetylcholine receptor (AChR) is the archetype member of the superfamily of ligand-gated ion channels that mediate fast intercellular communication in response to endogenous neurotransmitters. Here I review a series of biophysical studies on the AChR protein, with particular focus on the interactions of the macromolecule with its lipid microenvironment. Fluorescence recovery after photobleaching and phosphorescence anisotropy studies of the membrane-embedded AChR have contributed to our understanding of the translational and rotational dynamics of this protein in synthetic lipid bilayers and in the native membrane. Electron spin resonance studies led to the discovery of a lipid fraction in direct contact with the AChR with rotational dynamics 50-fold slower than that of the bulk lipids. This lipid belt region around the AChR molecule has since been intensively studied with the aim to define its possible role in the modulation of receptor function. The polarity and molecular dynamics of solvent dipoles—mainly water—in the vicinity of the lipids in the AChR membrane have been studied exploiting the amphiphilic fluorescent probe Laurdan's exquisite sensitivity to the phase state of the membrane, and Förster-type resonance energy transfer (FRET) was introduced to characterize the receptor-associated lipid microenvironment. FRET was used to discriminate between the bulk lipid and the lipid belt region in the vicinity of the protein. Further refinement of this topographical information was provided by the parallax method using phospholipid spin labels. The AChR-vicinal lipid is in a liquid-ordered phase and exhibits a higher degree of order than the bulk bilayer lipid. Changes in FRET efficiency induced by fatty acids, phospholipid, and cholesterol also led to the identification of discrete sites for these lipids on the AChR protein. I also illustrate the extension of Laurdan fluorescence studies to intact living cells heterologously expressing AChR in a brief section devoted to recent studies using two-photon fluorescence microscopy. The spatial resolution afforded by the two-photon optical sectioning of the cell in combination with the advantageous spectroscopic properties of Laurdan are exploited to obtain information on the physical state of the lipid environment of the membrane. Finally, the application of site-specific labeling and steady-state fluorescence spectroscopy to probe the location of AChR membrane-embedded domains is illustrated. The topography of the pyrene-labeled Cys residues in transmembrane domains M1, M4, M1, and M4 with respect to the membrane was determined by differential fluorescence quenching with lipid-resident spin-labeled probes. Cys residues were found to lie in a shallow position. For M4 segments, this is compatible with a linear -helical structure, but not so for M1, for which classical models locate Cys residues at the center of the hydrophobic stretch. The transmembrane topography of M1 can be rationalized on the basis of the presence of a substantial amount of nonhelical structure and/or of kinks attributable to the occurrence of the evolutionarily conserved proline residues. The latter is a striking feature of M1 in the AChR and all members of the rapid ligand-gated ion channel superfamily.