Mutational analysis of aspartate residues in the transmembrane regions and cytoplasmic loops of rat vesicular acetylcholine transporter.
The vesicular acetylcholine transporter (VAChT) is responsible for the transport of the neurotransmitter acetylcholine (ACh) into synaptic vesicles using an electrochemical gradient to drive transport. Rat VAChT has a number of aspartate residues within its predicted transmembrane domains (TM) and cytoplasmic loops, which may play important structural or functional roles in acetylcholine transport. In order to identify functional charged residues, site-directed mutagenesis of rVAChT was undertaken. No effect on ACh transport was observed when any of the five aspartate residues in the cytoplasmic loop were converted to asparagine. Similarly, changing Asp-46 (D46N) in TM1 or Asp-255 (D255N) in TM6 had no effect on ACh transport or vesamicol binding. However, replacement of Asp-398 in TM10 with Asn completely eliminated both ACh transport and vesamicol binding. The conservative mutant D398E retained transport activity, but not vesamicol binding, suggesting this residue is critical for transport. Mutation of Asp-193 in TM4 did not affect ACh transport activity; however, vesamicol binding was dramatically reduced. With mutant D425N of TM11 transport activity for ACh was completely blocked, without an effect on vesamicol binding. Activity was not restored in the conservative mutant D425E, suggesting the side chain as well as the negative charge of Asp-425 is important for substrate binding. These mutants, as well as mutant D193N, clearly dissociated ACh binding and transport from vesamicol binding. These data suggest that Asp-398 in TM10 and Asp-425 in TM11 are important for ACh binding and transport, while Asp-193 and Asp-398 in TM4 and TM10, respectively, are involved in vesamicol binding.
Kim
M H
MH
Department of Biochemistry, University of Kentucky, Lexington, Kentucky 40536, USA.
Lu
M
M
Lim
E J
EJ
Chai
Y G
YG
Hersh
L B
LB
D000109:Acetylcholine / Q000378:metabolism*; D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D006801:Humans; D026901:Membrane Transport Proteins; D016297:Mutagenesis, Site-Directed; D016716:PC12 Cells; D010880:Piperidines / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D050494:Vesicular Acetylcholine Transport Proteins; D033921:Vesicular Transport Proteins
D002352:Carrier Proteins; D026901:Membrane Transport Proteins; D010880:Piperidines; C493459:SLC18A3 protein, human; C493461:Slc18a3 protein, rat; D050494:Vesicular Acetylcholine Transport Proteins; D033921:Vesicular Transport Proteins; D001224:Aspartic Acid; C006189:vesamicol; D000109:Acetylcholine