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 transmembrane domains (TMDs) of vesicular acetylcholine transporter (VAChT). Rat VAChT in wild-type 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 approximately 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) approximately 4 mM) and vesamicol (K(d) approximately 2 nM compared to approximately 20 nM). Effects of different external pH values on transport indicate a site that must be protonated (apparent pK(a) approximately 7.6) likely is the aspartate in TMD XI. The observations suggest a model in which the known ion pair between lysine in TMD II 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) approximately 6.4) and vesamicol binding (apparent pK(a) approximately 6.3) remain unidentified.
Bravo
Dawn T
DT
Department of Chemistry and Biochemistry, University of California, Santa Barbara, California 93106, USA.
Kolmakova
Natalia G
NG
Parsons
Stanley M
SM
D000109:Acetylcholine / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D001224:Aspartic Acid / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D001692:Biological Transport / Q000235:genetics; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism; D019085:Fluorescent Antibody Technique, Direct; D006639:Histidine / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008239:Lysine / Q000235:genetics; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D018613:Microscopy, Confocal; D016297:Mutagenesis, Site-Directed; D010880:Piperidines / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000235:genetics; D051381:Rats; D055672:Static Electricity; D050494:Vesicular Acetylcholine Transport Proteins
D026901:Membrane Transport Proteins; D010880:Piperidines; C493461:Slc18a3 protein, rat; D050494:Vesicular Acetylcholine Transport Proteins; D001224:Aspartic Acid; C006189:vesamicol; D006639:Histidine; D008239:Lysine; D000109:Acetylcholine