5-Doxylstearate-induced displacement of phencyclidine from its low- affinity binding sites on the nicotinic acetylcholine receptor

Fatty acids as well as phencyclidine (PCP) inhibit the ion channel activity of the nicotinic acetylcholine receptor (AChR) by a noncompetitive mechanism. However, the exact localization of the fatty acid binding sites is unknown and, thus, the noncompetitive inhibitory mechanism for these endogenous modulators remains to be elucidated. In an attempt to determine the location of the fatty acid binding sites, we study the mutually exclusive action between 5-doxylstearate (5-SASL), a derivative of the endogenous noncompetitive antagonist (NCA) stearic acid, and other exogenous NCAs. For this purpose, both equilibrium and competitive binding assays using fluorescent and radiolabeled ligands were performed on desensitized AChRs. More specifically, we determined: (i) the effect of 5-SASL on the binding of the exogenous NCA [³H]PCP; (ii) the effect of 5-SASL on the binding of either quinacrine or ethidium, two fluorescent NCAs from exogenous origin; and (iii) the PCP-induced displacement of quinacrine and ethidium from their respective high-affinity binding sites. Our first target (i) is carried out by measuring the [³H]PCP binding in the absence or in the presence of increasing concentrations of 5-SASL. We found that 5-SASL displaces PCP from its low-affinity binding sites. The low-affinity PCP binding sites were pharmacologically characterized by an apparent dissociation contant (K(d)) of 6.1 ± 5.0 μM and a stoichiometry of 3.7 ± 1.5 sites per AChR. The fact that 5-SASL increased the apparent K(d) without changing the number of sites per AChR is indicative of a mutually exclusive action. From these results, an apparent inhibition constant (K(i)) of 75 ± 31 μM for 5-SASL was calculated. In addition, 5-SASL affected neither the apparent K(d) (0.46 ± 0.37 μM) nor the stoichiometry (1.07 ± 0.57 sites per AChR) of the high- affinity PCP binding site. The second objective (ii) is achieved by titrating either quinacrine or ethidium into AChR native membranes in the absence or in the presence of increasing concentrations of 5-SASL. These experiments showed that 5-SASL efficiently increased the apparent K(d) of quinacrine without perturbing the interaction of ethidium with its high-affinity locus. Considering that (a) 5-SASL effectively quenched the AChR-bound quinacrine fluorescence (H. R. Arias, Biochim. Biophys. Acta 1347, 9-22, 1997) and (b) fluorescence-quenching is a short-range process, it is possible to suggest that 5-SASL displaces quinacrine from its high-affinity binding site by a steric mechanism. In this regard, a K(i) of 38 ± 5 μM for 5-SASL was calculated. Concerning the last objective (iii), AChR-bound quinacrine or ethidium was back titrated with PCP. Two PCP K(i) values were obtained by fitting the displacement plots by nonlinear regression with two components. The lowest K(i) values obtained for either quinacrine (0.86 ± 0.37 μM) or ethidium (0.29 ± 0.23 μM) displacement from their respective high-affinity binding sites coincide with the previously determined high-affinity [³H]PCP K(d). In addition, the highest K(i) values obtained for either NCA displacement are in the same concentration range as the observed low-affinity [³H]PCP K(d). Taking into account all experimental data, we reached the following conclusions: (i) fatty acid molecules, or at least 5-SASL, sterically interact with both the PCP low-affinity and the quinacrine high- affinity binding sites; (ii) the low-affinity PCP binding sites, as well as the high-affinity quinacrine locus, are located at the nonannular lipid domain of the AChR; and, finally, (iii) fatty acid molecules are not accessible to the lumen of the ion channel, indicating an allosteric mode of action for fatty acids to inhibit ion flux. Thus, the 5-SASL, the quinacrine high-affinity, and the PCP low-affinity binding sites are all located at overlapping nonannular loci on the muscle-type AChR.