REGULATION OF HEMICHOLINIUM-3 SENSITIVE CHOLINE UPTAKE IN XENOPUS-LAEVIS OOCYTES BY THE 2ND C2 DOMAIN OF SYNAPTOTAGMIN

A size-fractionated torpedo electric lobe cDNA library was screened fo r the neuronal choline transporter by functional expression in oocytes . A clone, TLC2B, was isolated that induced a component of choline upt ake that was hemicholinium-3 sensitive and inhibited by the substituti on of lithium for sodium at low choline concentrations. However, [H-3] choline uptake by both injected and non-injected oocytes were characte rized by high affinity constants, suggesting that TLC2B could be affec ting a native choline transporter. Indeed, hemicholinium-3 sensitive c holine uptake could also be induced by preincubation of non-injected o ocytes with a protein kinase C inhibitor, H-7. By sequence analysis an d immuno-precipitation, the peptide produced by injection of TLC2B cRN A was identified as a soluble 24 kDa C-terminal fragment of the neuron al protein, synaptotagmin. Full length synaptotagmin was, however, ine ffective in the functional test. The peptide encoded by TLC2B correspo nds to the second protein kinase C-homologous domain of torpedo synapt otagmin, and like other soluble C2 domain peptides, was capable of cal cium-dependent translocation to membranes. Its action on choline uptak e in oocytes was, however, abolished by the addition of calcium in the presence of a calcium ionophore. These results suggest that the inter action of certain C2 domains, such as the C-terminal domain of synapto tagmin, with more specific targets may be anulled in the presence of c alcium due to its absorption to membrane phospholipids.