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 similar to 75 kDa on Western blots, Vesicular [H-3]ACh accumulation increased similar to 20 times over levels attained by the endogenous rat VAChT, expressed at low levels in control PC-12 cells, The transport of [H-3]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 mu M) and bafilomycin A(1) (1 mu M), a specific inhibitor of the vesicular H+-ATPase, The kinetics of [H-3]ACh uptake by human VAChT were saturable, exhibiting an apparent K-m of 0.97 +/- 0.1 mM and V-max of 0.58 +/- 0.04 nmol/min/mg, Maximal steady-state levels of vesicular [H-3]ACh accumulation were directly proportional to the concentration of substrate present in the medium with saturation occurring at similar to 4 mM. Uptake was stereospecifically inhibited by L-vesamicol with an IC50 of 14.7 +/- 1.5 nM. The apparent affinity (K-d) of [H-3] vesamicol for human VAChT was 4.1 +/- 0.5 nM, and the B-max was 8.9 +/- 0.6 pmol/mg. The turnover (VmaxBmax) of the human VAChT was similar to 65/min. This expression system should prove useful for the structure/function analysis of VAChT.