Mutational analysis of basic residues in the rat vesicular acetylcholine transporter. Identification of a transmembrane ion pair and evidence that histidine is not involved in proton translocation.
The function of positively charged residues and the interaction of positively and negatively charged residues of the rat vesicular acetylcholine transporter (rVAChT) were studied. Changing Lys-131 in transmembrane domain helix 2 (TM2) to Ala or Leu eliminated transport activity, with no effect on vesamicol binding. However, replacement by His or Arg retained transport activity, suggesting a positive charge in this position is critical. Mutation of His-444 in TM12 or His-413 in the cytoplasmic loop between TM10 and TM11 was without effect on ACh transport, but vesamicol binding was reduced with His-413 mutants. Changing His-338 in TM8 to Ala or Lys did not effect ACh transport, however replacement with Cys or Arg abolished activity. Mutation of both of the transmembrane histidines or all three of the luminal loop histidines showed no change in acetylcholine transport. The mutant H338A/D398N between oppositely charged residues in transmembrane domains showed no vesamicol binding, however the charge reversal mutant H338D/D398H restored binding. This suggests that His-338 forms an ion pair with Asp-398. The charge neutralizing mutant K131A/D425N or the charge exchanged mutant K131D/D425K did not restore ACh transport. Taken together these results provide new insights into the tertiary structure in VAChT.
Kim
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MH
Department of Biochemistry, University of Kentucky, Lexington, Kentucky 40536-0298, USA.
Lu
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Kelly
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Hersh
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LB
D000109:Acetylcholine / Q000378:metabolism; D000818:Animals; D001692:Biological Transport / Q000235:genetics; D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics*; D002499:Centrifugation, Density Gradient; D006031:Glycosylation; D006639:Histidine / Q000235:genetics; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics; D026901:Membrane Transport Proteins; D009154:Mutation; D010880:Piperidines / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary; D011522:Protons; D051381:Rats; D017386:Sequence Homology, Amino Acid; D055672:Static Electricity; D050494:Vesicular Acetylcholine Transport Proteins; D033921:Vesicular Transport Proteins
D002352:Carrier Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D010880:Piperidines; D011522:Protons; C493461:Slc18a3 protein, rat; D050494:Vesicular Acetylcholine Transport Proteins; D033921:Vesicular Transport Proteins; C006189:vesamicol; D006639:Histidine; D000109:Acetylcholine