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In cholinergic neurons, a specific requirement for precursor choline in the biosynthesis of acetylcholine (ACh) is thought to be sustained by a presynaptic, hemicholinium-3 (HC-3)-sensitive choline transporter (CHT). This transporter exhibits micromolar affinity for choline and transport activity is Na(+)- and Cl(-)-dependent. Based on the sequence information available with the recent cloning of rat and human CHTs [Okuda, Haga, Kanai, Endou, Ishihara and Katsura (2000) Nat. Neurosci. 3, 120-125; Apparsundaram, Ferguson, George Jr and Blakely (2000) Biochem. Biophys. Res. Commun. 276, 862-867; Okuda and Haga (2000) FEBS Lett. 484, 92-97], we have identified a murine CHT orthologue (mCHT) by reverse transcriptase-PCR of spinal cord mRNA and confirmed this sequence using assembled mouse genomic DNA. Inferred splice junctions for mCHT exons are conserved with those of hCHT. The mCHT cDNA encodes a protein of 580 amino acids with 93% and 98% amino acid identity to human CHT and rat CHT1, respectively. Hydropathy analysis of the predicted amino acid sequence of mCHT indicates a protein containing 13 transmembrane domains (TMDs), with the N-terminus oriented extracellularly and the C-terminus oriented intracellularly. Northern blot analysis of mouse tissues reveals the expression of mCHT as a single transcript of approximately 5 kb with highest expression in regions that are rich in cholinergic cell bodies, e.g. the spinal cord, brainstem, mid-brain and striatum, whereas hybridization signals are absent in regions lacking cholinergic soma, e.g. the cerebellum and kidney. Expression of mCHT in COS-7 cells results in high-affinity [(3)H]HC-3-binding sites (K(d)=5 nM), and Na(+)- and Cl(-)-dependent HC-3-sensitive choline uptake (K(m)=2 microM), assessed in resealed membrane vesicles. The availability of cloned, functional mCHT and its cognate genomic DNA should prove useful for studies of mCHT regulation and should open possibilities for evaluation of CHT dysfunction in murine models.