Conformational equilibrium changes of AChR induced by steroids and free fatty acids.

FERNÁNDEZ NIEVAS, G. A.; 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. Here, we studied the effect of structurally 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 labeled with increasing concentrations of the fluorescence probe and AChR open channel blocker, crystal violet (CrV). We took advantage of the different affinities of this probe for the conformational states of the AChR: CrV, displays higher affinity for the desensitized (D) than for the resting (R) state. Increasing concentrations of steroids (cortisone, hydrocortisone) decreased the KD values in the absence of agonist, without changes  in the presence of agonist. Different cis-unsaturated FFA (cis-u-FFA) produced a similar decrease in KD in the absence of agonist than the one produced by the addition of steroids; however, cis-u-FFA also caused an increase of the KD in the presence of agonist. Both saturated FFA (s-FFA) and trans-unsaturated FFA had no effect on the KD. Sequential additions of steroids and cis-u-FFA changed the KD values in a similar way as the only presence of cis-u-FFA did. Thus, the presence of hydrophobic non-competitive inhibitors such as FFA or steroids induces an AChR conformation distinct from its native resting-conformation. cis-uFFAs also cause a displacement from the typical agonist-induced D state. This latter effect could probably be explained by the increase in membrane fluidity caused by the FFA. Finally, the prevalent effect of cis-uFFA over steroids indicates that FFAs have higher affinity for these common sites.