Characteristics of [3H]Hemicholinium-3 Binding to Rat Striatal Membranes: Evidence for Negative Cooperative Site-Site Interactions

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Abstract
The chacteristics of [3H]hemicholinium-3 ([3H]HC-3) interactions with rat striatal membranes were investigated. Under the described assay conditions, [3H]HC-3 binds with a saturable population of membrane binding sites having the following regional distribution: striatum > hippocampus .gtoreq. cerebral cortex > cerebellum. The specific binding of [3H]HC-3 showed an obligatory requirement for NaCl; other halide salts of sodium or KCl failed to substitute for NaCl. The Scatchard tranformation of saturation isotherm data generated a curvilinear plot with high- and low-affinity components of binding. The dissociation of [3H]HC-3 at infinite dilution was also multiexponential. The dissociation could, however, be accelerated if unlabeled HC-3 was included in the diluting buffer, and this increase in dissociation appeared to be dependent on the concentrations of unlabeled HC-3 used, with the maximal increase demonstrable at 100 nM. The dissociations was also dependent on the fractional saturation of binding sites with labeled HC-3, such that, at higher fractional saturation of binding sites, the overall dissociation was faster and the difference in the dissociation observed between "dilution only" and "dilution + unlabeled HC-3" was reduced. This occupany-dependent change in dissociation could also be influenced by temperature and pH. Based on the results of these kinetic studies, the steady-state [3H]HC-3 binding data were analyzed for a homogeneous population of binding sites undergoing site-site interactions of the negative cooperative type. Such an analysis yielded a KD of 9.3 nM for the high-affinity state and a KD of 22.8 nM for the low-affinity state of binding sites, with a Bmax of 434 fmol/mg of protein. Competitive binding studies showed that unlabeled HC-3 was most potent in displacing [3H]HC-3, followed by choline. Other drugs known to have little influence on the synaptosomal sodium-dependent high-affinity choline uptake system (SDHACU) had no significant effect on [3H]HC-3 binding sites. Similarities in ionic dependencies, regional distribituions, and pharmacological selectivites of [3H]HC-3 selectively labels SDHACU sites located on presynaptic cholinergic neurons in rat CNS. We suggest that the two affinity states of [3H]HC-3 binding sites represent the different "functional" states of the SDHACU system. The binding of HC-3 (or choline) with the high-affinity state of the binding sites induces negative cooperatie site-site interactions among the binding sites, resulting in the formation of a low-affinity binding state. Because the affinities of HC-3 and choline for this low-affinity state of the [3H]HC-3 binding sites correspond to the affinities of these agents for the SDHACU system, we also suggest that the low-affinity binding state represents the "functional" form of SDHACU.