Na+-dependent, high-affinity choline uptake in cholinergic neurons is the rate-limiting step in acetylcholine synthesis. Here we report the molecular cloning and functional characterization of the human high-affinity choline transporter (hCHT1). The hCHT1 exhibits significant homology with known members of the Na+-dependent glucose transporter family, but not with members of the neurotransmitter transporter family. The human CHT1 gene is 25 kb in length with 9 exons and was assigned to chromosome II at position IIq11–12. Northern blot analysis showed that a 5.4 kb hCHT1 transcript was expressed exclusively in tissues containing cholinergic neurons. When expressed in Xenopus oocytes, the human clone induced Na+- and Cl−-dependent, high-affinity choline uptake, which was sensitive to the specific inhibitor hemicholinium-3, with a K i of 1.3 nM. The hCHT1-mediated choline uptake increased with increasing concentrations of choline, Na+ and Cl−, with EC50 values of 2.0 μM, 76 mM, and 48 mM, and with apparent Hill coefficients of 1, 2.5 and 2.3, respectively.
