Mutational and pH analysis of ionic residues in transmembrane domains of vesicular acetylcholine transporter

This research investigated the roles of 7 conserved ionic residues in the 12 putative transmenibrane domains (TMDs) of vesicular acetylcholine transporter (VAChT). Rat VAChT in wildtype and mutant forms was expressed in PC12(A123.7) cells. Transport and ligand binding were characterized at different pH values using filter assays. The ACh binding site is shown to exhibit high or low affinity (K-d values are approximate to 10 and 200 mM, respectively). Mutation of the lysine and aspartate residues in TMDs II and IV, respectively, can decrease the fraction of sites having high affinity. In three-dimensional structures of related transporters, these TMDs lie next to each other and distantly from TMDs VIII and X, which probably contain the binding sites for ACh and the allosteric inhibitor vesamicol. Importantly, mutation of the aspartate in TMD XI can create extra-high affinities for ACh (K-d approximate to 4 mM) and vesamicol (K-d approximate to 2 nM compared to approximate to 20 nM). Effects of different external pH values on transport indicate a site that must be protonated (apparent pK(a) approximate to 7.6) likely is the aspartate in TMD XI. The observations suggest a model in which the known ion pair between lysine in TMD 11 and aspartate in TMD XI controls the conformation or relative position of TMD XI, which in turn controls additional TMDs in the C-terminal half of VAChT. The pH effects also indicate that sites that must be unprotonated for transport (apparent pK(a) approximate to 6.4) and vesamicol binding (apparent pK(a) approximate to 6.3) remain unidentified.