Conformational equilibrium changes of the nicotinic acetylcholine receptor induced by steroids and free fatty acids

FERNÁNDEZ NIEVAS, G.; ANTOLLINI, S.S.; BARRANTES, F.J.

Montevideo, Uruguay

Congreso; ICBP-2007 6th International Conference of Biological Physics and 5th Southern Cone Biophysics Congress; 2007

Steroids and free fatty acids (FFA) are non-competitive inhibitors of the nicotinic acetylcholine receptor (AChR). They are purportedly located at the lipid-AChR interface, and their exact mechanism of action is still unknown. We studied the effect of structural different FFA and steroids on the AChR conformational equilibrium. T. californica AChR-rich membranes treated with the hydrophobic inhibitors, in the presence or in the absence of the agonist carbamoylcholine, were then labeled with increasing concentrations of the fluorescence probe, and AChR open channel blocker, Crystal Violet (CrV). We took advantage of the different affinity of this probe for the distinct conformational states of the AChR, displaying higher affinity for the desensitized-state than for the resting-state (low and high KD of CrV, respectively). Parallel membrane fluidity measurements were performed with a polarity sensitive fluorescence probe (Laurdan). Increasing concentrations of steroids (cortisone, hydrocortisone) decreased the KD values in the absence of agonist, without changed it in the presence of agonist. Different cis-unsaturated FFA (cis-u-FFA) produced a similar KD decrease in the absence of agonist than the one produced by the addition of steroids; however, cis-u-FFA caused also an increase of the KD values in the presence of agonist. Both saturated FFA (s-FFA) and trans-unsaturated FFA have no effect on the KD of CrV values. Sequential additions of steroids and cis-u-FFA, indistinctly of the order of addition, changed the KD values in a similar way as the only presence of cis-u-FFA did. Finally, complementary studies with cholesterol-depleted AChR-rich membranes by cyclodextrin treatment showed high KD values, both in the absence and in the presence of agonist. Thus, the presence of these hydrophobic non-competitive inhibitors induces an AChR conformational change far away from its “native resting-conformation”, probably by localizing at sites in the AChR-lipid interface. cis-uFFAs cause also a displacement from the typical agonist induced AChR desensitized-conformation. This latter effect could probably be explained by the membrane fluidity increase caused by the FFA (a similar effect was observed with cholesterol-depleted, and fluidized, membranes). Finally, the prevalent effect of cis-uFFA over steroids suggests that FFAs have higher affinity for these common sites.