Molecular cloning and functional characterization of a neuronal choline transporter from Trichoplusia ni

A cDNA encoding a high-affinity Na+-dependent choline transporter (TrnCHT) was isolated from the CNS of the cabbage looper Trichoplusia ni using an RT-PCR-based approach. The deduced amino acid sequence of the CHT cDNA predicts a 594 amino acid protein of 64.74 kDa prior to glycosylation. TrnCHT has 80%, 79%, 76%, and 58% amino acid identity to putative CHTs from Anopheles gambiae, Drosophila melanogaster and Apis mellifera, and a cloned CHT from Limulus polyphemus, respectively. In situ hybridization of TrnCHT cRNA in whole-mount preparations of caterpillar CNS revealed that TrnCHT mRNA is expressed by hundreds of presumably cholinergic neurons present in both the brain and cortex of all segmental ganglia. Na+-dependent [3H]-choline uptake was induced in Sf9 cells in vitro following infection with a TrnCHT-expressing recombinant baculovirus. Virally induced [3H]-choline uptake was found to approximately equal the endogenous rate of choline uptake in insect cells, seen either after infection with a control virus or in TrnCHT-infected cells exposed to [3H]-choline in the absence of Na+. The Na+-dependent component of [3H]-choline uptake by TrnCHT-infected cells was saturable with a Km for choline transport of 8.4 μM. Several compounds reported to be potent blockers of [3H]-choline uptake by cloned vertebrate choline transporters proved to be relatively weak inhibitors of choline uptake by Sf9 cells expressing TrnCHT. Hemicholinium-3 (Ki=4.1μM) and two oxoquinuclidium analogues of choline, quireston-A (Ki≈10μM) and quireston (Ki≈100μM) inhibited 50% of control uptake only at micromolar concentrations. The endogenous low-affinity Na+-independent uptake of [3H]-choline was also inhibited by high micromolar concentrations of hemicholinium-3.