Molecular cloning of a cDNA for a putative choline co-transporter from Limulus CNS

It is well documented that the sodium dependent, hemicholinium-3 sensitive, high affinity choline co-transporter is rate limiting in the biosynthesis of acetylcholine and is essential to cholinergic transmission. Until recently this transporter had eluded cloning. Okuda et al. (2000. Nature Neurosci. 3, 120-125) recently reported the successful cloning of the choline co-transporter in Caenorhabditis elegans (CHO-1) and rat (CHT1). We report herein the cloning of the choline co-transporter in the horseshoe crab, Limulus polyphemus. Through the use of a series of degenerate primers selected from consensus sequences of CHO-1 and CHT1, we generated two probes that were used to search a Limulus cDNA library produced from central nervous system (CNS) tissue. The full length nucleotide sequence of the Limulus homolog consists of 3368 bp which includes an open reading frame (ORF) that predicts a protein of 579 amino acids and two non-translation regions (NTR), one at the 3' end and the other at the 5' end. The amino acid sequence has 46% identity with rat CHT1 and 50% identity with both CHO-1 in C. elegans and the recently cloned human co-transporter (hCHT; Apparsundaram et al., 2000. Biochem. Biophys. Res. Commun. 276, 862-867; Okuda and Haga, 2000. FEBS Lett. 484, 92-97). Hydropathy plot analysis predicts the Limulus choline co-transporter (LChCoT) to have thirteen transmembrane domains (TMD), with the N-terminus oriented extracellularly and the C-terminus oriented intracellularly. Northern blot analyses using cDNA probes designed from LChCoT cDNA sequences revealed its distribution specifically in central nervous system structures. On the other hand it was not found in non-nervous tissues. The successful cloning of LChCoT, which was shown to be a member of the sodium-dependent glucose transporter family (SLGT), should prove useful in the determination of its physiological regulation, including its intracellular trafficking.