Active transport of acetylcholine by the human vesicular acetylcholine transporter.
The characteristics of ATP-dependent transport of acetylcholine (ACh) in homogenates of pheochromocytoma (PC-12) cells stably transfected with the human vesicular acetylcholine transporter (VAChT) cDNA are described. The human VAChT protein was abundantly expressed in this line and appeared as a diffuse band with a molecular mass of approximately 75 kDa on Western blots. Vesicular [3H]ACh accumulation increased approximately 20 times over levels attained by the endogenous rat VAChT, expressed at low levels in control PC-12 cells. The transport of [3H]ACh by human VAChT was dependent upon the addition of exogenous ATP at 37 degrees C. Uptake was abolished by low temperature (4 degrees C), the proton ionophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (2.5 microM) and bafilomycin A1 (1 microM), a specific inhibitor of the vesicular H+-ATPase. The kinetics of [3H]ACh uptake by human VAChT were saturable, exhibiting an apparent Km of 0.97 +/- 0.1 mM and Vmax of 0.58 +/- 0.04 nmol/min/mg. Maximal steady-state levels of vesicular [3H]ACh accumulation were directly proportional to the concentration of substrate present in the medium with saturation occurring at approximately 4 mM. Uptake was stereospecifically inhibited by L-vesamicol with an IC50 of 14.7 +/- 1.5 nM. The apparent affinity (Kd) of [3H]vesamicol for human VAChT was 4.1 +/- 0.5 nM, and the Bmax was 8.9 +/- 0.6 pmol/mg. The turnover (Vmax/Bmax) of the human VAChT was approximately 65/min. This expression system should prove useful for the structure/function analysis of VAChT.
Section on Molecular Neuroscience, Laboratory of Cell Biology, National Institute of Mental Health, Bethesda, Maryland 20892, USA. disbrow@codon.nih.gov
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D000109:Acetylcholine / Q000378:metabolism*; D000310:Adrenal Gland Neoplasms; D000818:Animals; D001693:Biological Transport, Active / Q000187:drug effects; D002352:Carrier Proteins / Q000096:biosynthesis / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D026901:Membrane Transport Proteins; D009467:Neuromuscular Depolarizing Agents / Q000378:metabolism / Q000494:pharmacology; D016716:PC12 Cells; D010673:Pheochromocytoma; D010880:Piperidines / Q000378:metabolism / Q000494:pharmacology; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D013379:Substrate Specificity; D013747:Tetrabenazine / Q000378:metabolism / Q000494:pharmacology; D014162:Transfection; D050494:Vesicular Acetylcholine Transport Proteins; D033921:Vesicular Transport Proteins
D002352:Carrier Proteins; D026901:Membrane Transport Proteins; D009467:Neuromuscular Depolarizing Agents; D010880:Piperidines; D011994:Recombinant Proteins; C493459:SLC18A3 protein, human; C493461:Slc18a3 protein, rat; D050494:Vesicular Acetylcholine Transport Proteins; D033921:Vesicular Transport Proteins; C006189:vesamicol; D000109:Acetylcholine; D013747:Tetrabenazine