Characterization of [3H]hemicholinium-3 binding associated with neuronal choline uptake sites in rat brain membranes

Brain Res. 1985 Dec 2;348(2):321-30. doi: 10.1016/0006-8993(85)90451-2.


Hemicholinium-3 (HCh-3) is a potent and specific inhibitor of the high-affinity choline transport process (HAChT) localized on cholinergic neurons. In this study, the specific binding of [3H]HCh-3 (120 Ci/mmol) was characterized in crude synaptic membranes prepared from rat brain. The binding of [3H]HCh-3 to forebrain membranes was saturable, reversible and specific with an apparent Kd under optimal conditions of 35 nM and a Bmax of 56 fmol/mg protein. The potency of various HAChT inhibitors correlated with their apparent affinities for the specific [3H]HCh-3 binding site. The specific binding of [3H]HCh-3 exhibited an uneven regional distribution in the adult rat brain that corresponded to the activity of the HAChT in these regions. Transsection of the fornix, which causes a degeneration of the septal hippocampal cholinergic pathway, resulted in comparable reductions of the specific [3H]HCh-3 binding and the specific activity of choline acetyltransferase, a presynaptic marker for cholinergic terminals in the hippocampal formation; the lesion did not affect the specific activity of glutamic acid decarboxylase, a presynaptic marker for GABAergic neurons within the hippocampus. Maximal binding occurred in the presence of 200 mM NaCl: potassium, lithium, rubidium and calcium substituted poorly for sodium; and bromide, fluoride, iodide, sulfate and phosphate were less effective anions than chloride. Increasing concentrations of NaCl increased the affinity of the site for [3H]HCh-3 with no significant effect on the maximal number of sites; the enhancement of affinity was due to a selective slowing of the rate of dissociation of the ligand from its binding site. These findings indicate that [3H]HCh-3 binds to the carrier site mediating the HAChT on cholinergic neurons; thus, this radioligand may be a useful probe for investigating this presynaptic component (HAChT) of cholinergic neurons.

MeSH terms

  • Animals
  • Biological Transport, Active / drug effects
  • Brain / metabolism*
  • Choline / metabolism*
  • Choline O-Acetyltransferase / metabolism
  • Hemicholinium 3 / metabolism*
  • Hemicholinium 3 / pharmacology
  • Hippocampus / metabolism
  • Hydrogen-Ion Concentration
  • Kinetics
  • Male
  • Nerve Crush
  • Neurons / metabolism*
  • Rats
  • Rats, Inbred Strains
  • Receptors, Cholinergic / analysis*
  • Receptors, Cholinergic / drug effects
  • Sodium Chloride / metabolism
  • Subcellular Fractions / metabolism
  • Synaptosomes / metabolism


  • Receptors, Cholinergic
  • Hemicholinium 3
  • Sodium Chloride
  • Choline O-Acetyltransferase
  • Choline