Conformational rearrangement of the nicotinic acetylcholine receptor induced by its lipid environment.

ANTOLLINI, S. S.

Angra dos Reis, Río de Janeiro, Brasil

Congreso; TWAS-ROLAC, 1st Regional Conference of Young Scientists (RCYS); 2006

TWAS-ROLAC

A group of highly hydrophobic molecules, such as steroids and free fatty acids (FFA), act as non-competitive inhibitors of the nicotinic acetylcholine receptor (AChR) by a mechanism still unknown. Also, perturbations of the AChR native lipid environment alter its function. The lipid-protein interface has been considered as a potential locus where the “lipid signal” gates and causes the modulation of the AChR function. A hypothesis proposes that this is possible by inducing a conformational change of the AChR. 1- Using Förster resonance energy transfer (FRET) between the intrinsic fluorescence of membrane-bound T. californica AChR and the fluorescent probe Laurdan, and exploiting the decrease in FRET efficiency (E) caused by these hydrophobic molecules, we identified common sites for steroids and FFA. Furthermore, working with proteinase K and phospholipase A2 we could define the location of the sites at the lipid-protein interface. 2- More recently, we determined that these sites are sensitive to the AChR conformation using the fluorescence probe crystal violet (CrV), which is an AChR ion blocker that has higher affinity for the desensitized conformation than for the resting conformation of the AChR. We found that AChRs located in cholesterol-depleted or cholesterol-enriched Torpedo membranes stay in an intermediate conformational state, and that addition of FFA and steroids to native membranes in the absence of agonist induce the stabilization of the AChR in a conformation no longer affected by agonists, thus demonstrating a direct correlation between AChR lipid environment and conformational states. 3- Working with a synthetic peptide corresponding to the fourth transmembrane domain of the AChR g subunit (gM4-Trp6) we studied the conformational dynamics of a peptide making up the outermost ring of the AChR transmembrane region. The fluorescence studies exploit the intrinsic fluorescence of the only tryptophan residue in this peptide, and combine this information with quenching studies using depth-sensitive phosphatidylcholine spin-labeled probes and acrylamide, polarization of fluorescence, and general polarization of Laurdan. A direct correlation was found between bilayer width and the depth of insertion of Trp6 that we compared with data from MD studies.