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

Authors

  • Tapan K. Chatterjee,

    1. Department of Pharmacology, College of Medicine; and * Department of Medicinal Chemistry, College of Pharmacy, The University of Iowa, Iowa City, Iowa, U.S.A.
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  • Joseph G. Cannon,

    1. Department of Pharmacology, College of Medicine; and * Department of Medicinal Chemistry, College of Pharmacy, The University of Iowa, Iowa City, Iowa, U.S.A.
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  • Ranbir K. Bhatnagar

    Corresponding author
    1. Department of Pharmacology, College of Medicine; and * Department of Medicinal Chemistry, College of Pharmacy, The University of Iowa, Iowa City, Iowa, U.S.A.
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  • This work was presented in part at the 15th Annual Meeting of the Society for Neurosciences, held in Dallas, TX, U.S.A., on October 20–25, 1985.

Address correspondence and reprint requests to Dr. R. K. Bhatnagar at Department of Pharmacology, College of Medicine, The University of Iowa, Iowa City, IA 52242, U.S.A.

Abstract

Abstract: The characteristics 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 ≧ 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 transformation 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 unla-beled 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 dissociation 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 occupancy-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 distributions, and pharmacological selectivi-ties of [3H]HC-3 binding with synaptosomal SDHACU suggest that [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 sjtes 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 cooperative 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 for SDHACU.

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