Fast kinetic studies on the interaction of a fluorescent agonist with the membrane-bound acetylcholine receptor from Torpedo marmorata.

The fluorescent cholinergic effector, [1-(5-dimethylamino naphthalene)sulfonamido] n-hexanoic acid β-(N-trimethylammonium bromide) ethyl ester (Dns-C6-Cho) acts as a potent depolarizing agent or agonist on Electrophorus electricus electroplaque (maximal depolarization 65–70 mV). In vitro, it increases the efflux of 22Na+ from receptor-rich membrane fragments prepared from Torpedo marmorata electric organ. The apparent dissociation constant determined with the two systems is close to 1 μM. Preincubation of the receptor-rich microsacs with Dns-C6-Cho results in a decrease of the amplitude of the permeability response to carbamylcholine. Like the physiological transmitter, acetylcholine, Dns-C6-Cho triggers the two characteristic reactions of ‘activation’ and ‘desensitization’. The equilibrium dissociation constant of Dns-C6-Cho measured by following the decrease of the initial velocity of binding of Naja nigricollisα-[3H]toxin to receptor-rich microsacs is close to 0.01 μM after prolonged incubation. Using the same method, it is found that Dns-C6-Cho triggers, in the range of a few minutes, a time-dependent increased of affinity. The interaction of Dns-C6-Cho with the receptor-rich membranes can be monitored under conditions of energy transfer from proteins (λex= 290 nm) and results in an approximately 10-fold increase of fluorescence intensity at the maximal emission wavelength (λem= 557 nm). Rapid mixing of a suspension of membrane fragments with Dns-C6-Cho gives rise to changes of fluorescence intensity which can be recorded in the time range of seconds to milliseconds. The signals disappear after pre-incubation of the membrane fragments with saturating levels of N. nigricollisα-toxin and therefore are due to the interaction of Dns-C6-Cho with the receptor site. Three major relaxation processes are observed. (a) A ‘rapid’ one, in the millisecond range, is characterized by a linear dependence of its apparent rate constant with Dns-C6-Cho concentration. It is analysed in terms of Dns-C6-Cho binding to receptor sites spontaneously present in a high-affinity state for agonists (Kd∼ 3 nM). These sites represent approximately 20% of the total population of receptor sites and exist prior to agonist binding in the membrane at rest. (b) An ‘intermediate’ relaxation process, in the second–millisecond range, shows a linear dependence of the apparent rate constant with Dns-C6-Cho concentration and is accounted for by a bimolecular binding reaction between Dns-C6-Cho and acetylcholine receptor sites in a state (or states) of lower affinity (∼ 1 μM). (c) Finally, a ‘slow’ relaxation process, in the seconds range, is characterised by an apparent rate of an interconversion between low-affinity and high-affinity states of the receptor. A model with two pre-existing and interconvertible affinity states for the receptor qualitatively and quantitatively accounts for the data, and the corresponding rate constants have been determined; the model also accounts for the complex kinetics monitored upon addition of a non-fluorescent effector to a suspension of receptor-rich membrane fragments equilibrated with Dns-C6-Cho; the possible occurrence of a third state with intermediate affinity is discussed. The slow interconversion from low-affinity to high-affinity states of the receptor is correlated with the physiological process of ‘desensitization’ on the basis of its dependence on agonist concentration and temperature and of the effect of local anesthetics; the features of the intermediate relaxation process and its possible correlation with the ‘activation’ reaction are discussed.