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

Tapan Kumar Chatterjee, Joseph G. Cannon, Ranbir K. Bhatnagar

Research output: Contribution to journalArticle

25 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)1191-1201
Number of pages11
JournalJournal of Neurochemistry
Volume49
Issue number4
DOIs
StatePublished - Jan 1 1987

Fingerprint

Corpus Striatum
Choline
Rats
Binding Sites
Membranes
Dilution
Cholinergic Neurons
Competitive Binding
Cerebral Cortex
Cerebellum
Cholinergic Agents
Population
Neurons
Isotherms
Labels
Assays
Hippocampus
Buffers
Salts
Sodium

Keywords

  • Choline uptake
  • Negative cooperativity
  • [H]Hemicholinium‐3 binding

ASJC Scopus subject areas

  • Biochemistry
  • Cellular and Molecular Neuroscience

Cite this

Characteristics of [3H]Hemicholinium‐3 Binding to Rat Striatal Membranes : Evidence for Negative Cooperative Site‐Site Interactions. / Chatterjee, Tapan Kumar; Cannon, Joseph G.; Bhatnagar, Ranbir K.

In: Journal of Neurochemistry, Vol. 49, No. 4, 01.01.1987, p. 1191-1201.

Research output: Contribution to journalArticle

Chatterjee, Tapan Kumar ; Cannon, Joseph G. ; Bhatnagar, Ranbir K. / Characteristics of [3H]Hemicholinium‐3 Binding to Rat Striatal Membranes : Evidence for Negative Cooperative Site‐Site Interactions. In: Journal of Neurochemistry. 1987 ; Vol. 49, No. 4. pp. 1191-1201.
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N2 - 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.

AB - 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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