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 recent ly this transporter had eluded cloning. Okuda et al. (2000. Nature Neurosci . 3, 120-125) recently reported the successful cloning of the choline co-tr ansporter in Caenorhabditis elegans (CHO-1) and rat (CHT1). We report herei n 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 u sed to search a Limulus cDNA library produced from central nervous system ( CNS) tissue. The full length nucleotide sequence of the Limulus homolog con sists 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 t he 3 ' end and the other at the 5 ' end. The amino acid sequence has 46% id entity 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. Bi ochem. Biophys. Res. Commun. 276, 862-867; Okuda and Haga, 2000. FEES Lett. 484, 92-97). Hydropathy plot analysis predicts the Limulus choline cotrans porter (LChCoT) to have thirteen transmembrane domains (TMD), with the N-te rminus oriented extracellularly and the C-terminus oriented intracellularly . Northern blot analyses using cDNA probes designed from LChCoT cDNA sequen ces revealed its distribution specifically in central nervous system struct ures. On the other hand it was not found in non-nervous tissues. The succes sful cloning of LChCoT, which was shown to be a member of the sodium-depend ent glucose transporter family (SLGT), should prove useful in the determina tion of its physiological regulation, including its intracellular trafficki ng. (C) 2001 Elsevier Science B.V. All rights reserved.