Studies on Ligand Binding to the Histrionicotoxin and the Agonist Binding Sites of Membrane Bound Acetylcholine Receptor from Torpedo californica

Chapter I The binding of various alkylguanidines to the histrionicotoxin site of the membrane bound acetylcholine receptor of Torpedo californica was studied. These results, along with data obtained on the binding of several histrionicotoxin derivatives, were used to formulate a model of ligand to binding to the receptor's histrionicotoxin site. An azido derivative of histrionicotoxin was used in an attempt to photoaffinity label the histrionicotoxin site. The effect of the agonist carbamylcholine on incorporation of this label was investigated. Chapter II Proton magnetic resonance was used to monitor binding of choline, a known partial agonist, to acetylcholine receptor-enriched membrane preparations from Torpedo californica electroplax. The interaction between choline and receptor led to a broadening of the resonance of the choline methyl groups and this effect was reversed by α-bungarotoxin, a quasi-irreversible antagonist of the acetylcholine receptor. From the concentration dependence of line broadening the equilibrium dissociation constant for choline was obtained (Kd = 190 ± 65 μM). The temperature dependence of the parameters observed in the choline titrations gave an enthalpy of binding ΔH < 1.5 kcal/mol and allowed estimates for the dissociation rate constant of the receptor-choline complex (kdiss > 1.6 x 103 S1) and the respective activation energy, Ea (kdiss) ≈ 5.5 kcal/mol. The association of other ligands with the membrane-bound receptor could also be studied by observing effects of varying concentrations of such ligands on the choline methyl group linewidth at a constant choline concentration. Chapter III Membrane fragments from the electric organ of Torpedo californica were purified so that the only protein components remaining were the four homologous acetylcholine receptor subunits of 40,000, 50,000, 60,000 and 65,000 daltons. The change in agonist affinity induced by carbamylcholine and the rate of this change, especially in the presence of local anesthetics, were studied and compared to the corresponding properties of membranes containing additional protein components. The results were consistent with the notion that desensitization, as defined by conversion to a state of higher ligand affinity of the acetylcholine receptor, and modulation of the kinetics of this effect by local anesthetics are dependent only on the receptor protein in its native environment.