Ca2+‐Dependent, ATP‐Induced Conversion of the [3H]Hemicholinium‐3 Binding Sites from High‐ to Low‐Affinity States in Rat Striatum: Effect of Protein Kinase Inhibitors on This Affinity Conversion and Synaptosomal Choline Transport

Tapan Kumar Chatterjee, Ranbir K. Bhatnagar

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Abstract: Tritium‐labeled hemicholinium‐3 ([3H]HC‐3) was used to characterize the sodium‐dependent high‐affinity choline carrier sites in rat striatal preparations. In an earlier study, we had shown that [3H]HC‐3 labels choline carrier sites with high and low affinities and had suggested that the low‐affinity sites represent “functional” carrier sites. The objective of the present study was to examine the mechanisms involved in the regulation of the two affinity states of [3H]HC‐3 binding. Here, we demonstrate that these two affinity states are totally interconvertible; addition of 0.1 mM ATP in the binding assay medium quantitatively converted all the binding sites to the low‐affinity state, whereas addition of 1 mM β,γ‐methylene 5′‐ATP quantitatively converted all the binding sites to the high‐affinity state. Preincubation of the tissue (for 15 min at 37°C) before the binding assay also converted the binding sites to the high‐affinity state, whereas supplementation of the assay medium with ATP (0.5 mM) again induced expression of the low‐affinity state of the binding sites. This effect of ATP was found to be selective for this nucleotide. Neither ADP (1 mM) nor cyclic AMP could mimic such an effect. Other nucleotide triphosphates–CTP (0.5 mM) and GTP (0.5 mM)–also could not substitute for ATP. GTP, however, caused nearly a 35% reduction in the number of binding sites, accompanying a loss of the low‐affinity component of binding. This effect of GTP was also shared by 5′‐guanylyl‐imidodiphosphate but not by GDP or cyclic GMP. This ATPdependent low‐affinity conversion of [3H]HC‐3 binding sites requires divalent metal ions. Tissue treated with EDTA did not elicit the ATP effect; however, the sensitivity to ATP could be restored fully by supplementation with Ca2+ and Mg2+. Neither a Ca2+/phospholipid‐dependent protein kinase inhibitor (staurosporin) nor Ca2+/calmodulin‐dependent protein kinase inhibitors (trifluoperazine and W‐7) blocked the Ca2+‐dependent ATP‐induced low‐affinity conversion of [3H]HC‐3 binding sites. The low‐affinity conversion could, however, be blocked by gossypol, an agent shown previously to be an inhibitor of certain calcium‐dependent protein kinases. In a similar concentration range, gossypol also inhibited synaptosomal [14C]choline uptake. Staurosporin was inactive as a choline uptake inhibitor, whereas calmodulin antagonists potently inhibited choline uptake with the rank order of calmidazolium > pimozide > trifluoperazine > W‐7 > W‐13 > W‐5 > W‐12. We suggest that the Ca2+‐dependent ATP‐mediated modification of choline carrier sites is important in the activation of the choline carrier. The sensitivity of the synaptosomal choline uptake system to calmodulin inhibitors and the relative insensitivity of [3H]HC‐3 binding parameters to these agents suggest that calmodulin‐dependent regulation of choline uptake is mediated through a site other than [3H]HC‐3 recognition sites or through a certain modification of the carrier sites that is not reflected in the binding site parameters tested.

Original languageEnglish (US)
Pages (from-to)1500-1508
Number of pages9
JournalJournal of Neurochemistry
Issue number5
Publication statusPublished - Jan 1 1990



  • ATP
  • Calmodulin antagonists
  • Ca‐dependent protein kinase
  • Choline uptake
  • [H]Hemicholinium binding affinity

ASJC Scopus subject areas

  • Biochemistry
  • Cellular and Molecular Neuroscience

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