The AChR mediates synaptic transmission at the neuromuscular junction&between certain neurons in the nervous tissue. Defects of peripheral receptors cause various myasthenias, while the neuronal receptors are altered in brains of Alzheimer and Parkinson patients, as well as in smokers. Therefore, understanding the functional architecture of this prototypic ligand-gated ion channel is important for a medical perspective as well as for the field of protein structure. This receptor is a highly plastic protein, where 'competitive inhibition' between two ligands may result from steric as well as from allosteric interactions. A kinetic model was developed that differentiates between these two mechanisms, providing both ligands are available in the radiolabelled form. The specific questions addressed in this part of the project are: Do cembranoids and phencyclidine (PCP) sites physically overlap? Do cembranoids bind to additional sites not shared with PCP? What is the relationship between cembranoid binding sites and those of pentobarbital, quinacrine, QX-222, ethidium bromide, histrionicotoxin and tetracaine? This group of compounds are known to bind to at least three topographically different sites, and preference for each receptor conformation is represented (pentobarbital and tetracaine bind preferentially the resting state; quinacrine and QX-222 prefer the open channel conformation; ethidium bromide stabilizes the desensitized state, as does PCP). Experimental Design and Methods: Specific binding of 3H-cembranoid to the AChR-rich membranes from Torpedo californica electric organ will be measured using a filtration assay [Szczawinska et al., Cell. Molec. Neurobiol., 12, 95-106, (1992)]. 3H-Cembranoid concentration curves and dissociation constants will be determined first. 3H-Cembranoid binding will then be inhibited by increasing the concentration of unlabelled PCP to determine PCP's inhibition constant. Similar experiments will be done with radiolabelled PCP and the other inhibitors mentioned above (commercially available). The parameters determined in these experiments will then be used to distinguish steric from allosteric competition. These studies will be done with the receptor in resting and in desensitized states. Recent Results: A first attempt to prepare tritiated eunicin acetate (3H-EUNA) by acetylation of eunicin with 3H-acetic anhydride rendered a very low yield. Next, eunicin was acetylated with 3H-acetoxyphthalimide of high specific activity [the reagent was prepared by treating an iodinated precursor with tributyltin tritide (S.A., 20 Ci/mmole)]. Tritium NMR spectra confirmed the incorporation of tritium into the EUNA molecule. 3H-EUNA was purified from the acetylation mixture by preparative thin-layer chromatography, which rendered a product with more than 95% purity with a 5% yield. Binding of 3H-EUNA to AChR-rich membranes was significantly higher than binding to the filter, but it contained a large proportion of nonspecific binding (not displaceable by unlabelled cembranoids or PCP). This could result from hydrophobic interaction of 3H-EUNA with other proteins or membrane lipids. Several modifications in the binding assay failed to decrease the non-specific binding. In the meantime we found a cembranoid of tenfold higher affinity, which should decrease the problem of non-specific binding. Pseudoplexauric acid methyl ester (PAME) displaces 3H-PCP from the AChR with a KI of 0.3 mM (this is similar to the KI of PCP itself). We are now determining the conditions for the hydrolysis of PAME to pseudoplexauric acid and studying the stability of the resulting product. Radiolabelling will be achieved by esterification of the acid with 3H-methanol of high specific activity (pure 3H-methanol will be the solvent of the reaction). 3H-PAME will be purified, characterized and studied as was described above for EUNA. Conclusions: A cembranoid, eunicin acetate, was successfully tritiated to high specific activity, characterized by NMR and purified with good yield. This compound was not useful for binding studies due to its relatively low potency and high nonspecific binding. A new, more potent cembranoid, is now available and will be tritiated shortly. We expect significant progress in this project over the next year.
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