The high affinity choline transporter: topology and choline binding site as studied by site‐directed mutagenesis

Abstract

The high affinity choline transporter (CHT1) is expressed in cholinergic neuron. CHT1 takes up choline and provides it for acetylcholine synthesis. CHT1 has two putative glycosylation sites (N69, N301). We found that N69Q mutant was glycosylated as well as wild type CHT1, but N301Q mutant was not glycosylated and not expressed on the cell surface. We have constructed a number of mutants substituted with cysteine and examined the accessibility of impermeable SH reagent to each cysteine residue in these mutants expressed in HEK293 cells. Obtained results indicated that CHT1 has the extracellular amino terminus and 13 transmembrane segments. Several acidic or basic amino acid residues in the putative third and 12th transmembrane segments were indicated to be involved in the choline uptake activity. In particular, the E451D mutant had approximately five times higher Km for choline uptake and five times lower Kd for HC3 binding as compared with wild type hCHT1, indicating that the E451 residue is involved in recognition of choline and HC3.