Regulation of hemicholinium-3 sensitive choline uptake in Xenopus laevis oocytes by the second C2 domain of synaptotagmin.
A size-fractionated torpedo electric lobe cDNA library was screened for the neuronal choline transporter by functional expression in oocytes. A clone, TLC2B, was isolated that induced a component of choline uptake that was hemicholinium-3 sensitive and inhibited by the substitution of lithium for sodium at low choline concentrations. However, [3H]choline uptake by both injected and non-injected oocytes were characterized by high affinity constants, suggesting that TLC2B could be affecting a native choline transporter. Indeed, hemicholinium-3 sensitive choline uptake could also be induced by preincubation of non-injected oocytes with a protein kinase C inhibitor, H-7. By sequence analysis and immuno-precipitation, the peptide produced by injection of TLC2B cRNA was identified as a soluble 24 kDa C-terminal fragment of the neuronal protein, synaptotagmin. Full length synaptotagmin was, however, ineffective in the functional test. The peptide encoded by TLC2B corresponds to the second protein kinase C-homologous domain of torpedo synaptotagmin, and like other soluble C2 domain peptides, was capable of calcium-dependent translocation to membranes. Its action on choline uptake in oocytes was, however, abolished by the addition of calcium in the presence of a calcium ionophore. These results suggest that the interaction of certain C2 domains, such as the C-terminal domain of synaptotagmin, with more specific targets may be anulled in the presence of calcium due to its absorption to membrane phospholipids.
D000595:Amino Acid Sequence; D000818:Animals; D002135:Calcium-Binding Proteins; D002794:Choline / Q000378:metabolism*; D018678:Cholinergic Agents / Q000494:pharmacology*; D018076:DNA, Complementary / Q000235:genetics; D005260:Female; D005815:Genetic Code; D006426:Hemicholinium 3 / Q000494:pharmacology*; D008562:Membrane Glycoproteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry*; D009865:Oocytes / Q000187:drug effects* / Q000378:metabolism; D011493:Protein Kinase C / Q000737:chemistry; D017434:Protein Structure, Tertiary; D050857:Synaptotagmins; D014982:Xenopus laevis
D002135:Calcium-Binding Proteins; D018678:Cholinergic Agents; D018076:DNA, Complementary; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D050857:Synaptotagmins; D006426:Hemicholinium 3; D011493:Protein Kinase C; D002794:Choline