Publications

• Choline is transported by vesicular acetylcholine transporter.

  Bravo D.T., Kolmakova N.G., Parsons S.M.

  Previously published results appeared to show that vesicular acetylcholine transporter (VAChT) does not transport choline (Ch). Because it is uniquely suited to detect transport of weakly bound substrates, a recently developed assay that detects transmembrane reorientation of the substrate binding ... >> More

  Previously published results appeared to show that vesicular acetylcholine transporter (VAChT) does not transport choline (Ch). Because it is uniquely suited to detect transport of weakly bound substrates, a recently developed assay that detects transmembrane reorientation of the substrate binding site was used to re-examine transport selectivity. Rat VAChT was expressed in PC12(A1237) cells, postnuclear supernatant-containing microvesicles was prepared, and the reorientation assay was conducted with unlabeled Ch and tetramethylammonium (TMA). Also, [(14)C]Ch and [(3)H]acetylcholine (ACh) were used in an optimized accumulation assay. The results demonstrate that Ch is transported at least as well as ACh is, but with sevenfold lower affinity. Even TMA is transported, but with 26-fold lower affinity. Ch transport by VAChT is of interest in view of the possibilities that Ch (i) occurs at higher concentration than ACh does in terminal cytoplasm under some conditions, and (ii) is an agonist for alpha 7 nicotinic receptors. << Less

  J. Neurochem. 91:766-768(2004) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Kinetic parameters for the vesicular acetylcholine transporter: two protons are exchanged for one acetylcholine.

  Nguyen M.L., Cox G.D., Parsons S.M.

  The vesicular acetylcholine transporter (VAChT) mediates ACh storage in synaptic vesicles by exchanging cytoplasmic ACh with vesicular protons. This study sought to determine the stoichiometry of exchange by analysis of ligand binding and transport kinetics. The effects of different pH values insi ... >> More

  The vesicular acetylcholine transporter (VAChT) mediates ACh storage in synaptic vesicles by exchanging cytoplasmic ACh with vesicular protons. This study sought to determine the stoichiometry of exchange by analysis of ligand binding and transport kinetics. The effects of different pH values inside and outside, external ACh concentrations, and electrical potential gradients on ACh transport by vesicles isolated from the electric organ of Torpedo were determined using a pH-jump protocol. The equilibrium binding of a high-affinity analogue of ACh is inhibited by protonation with a pKa of 7.4 +/-0.3. A two-proton model fits the transport data much better than a one-proton model does, and uptake increases at more positive internal electrical potential, as expected for the two-proton model. Thus, the results support the two-proton model. The transport cycle begins with binding of external ACh to outwardly oriented site 2 (KACho = 20 mM) and protonation of inwardly oriented site 1 (pKa1 = 4.73 +/-0.05). Loaded VAChT reorients quickly (73 000 min-1) and releases ACh to the inside (KAChi = 44 000 mM) and the proton to the outside. Unloaded, internally oriented site 2 binds a proton (pKa2 = 7.0), after which VAChT reorients (150 +/-20 min-1) in the rate-limiting step and releases the proton to the outside to complete the cycle. Rate constants for the reverse direction also were estimated. Two protons provide a thermodynamic driving force beyond that utilized in vivo, which suggests that vesicular filling is regulated. Other phenomena related to VAChT, namely the time required to fill synaptic vesicles, the fractional orientation of the ACh binding site toward cytoplasm, orientational lifetimes, and the rate of nonquantal release of ACh from cholinergic nerve terminals, were computer-simulated, and the results are compared with physiological observations. << Less

  Biochemistry 37:13400-13410(1998) [PubMed] [EuropePMC]

• Functional identification of a vesicular acetylcholine transporter and its expression from a 'cholinergic' gene locus.

  Erickson J.D., Varoqui H., Schafer M.K.-H., Modi W., Diebler M.-F., Weihe E., Rand J.B., Eiden L.E., Bonner T.I., Usdin T.B.

  The vesicular acetylcholine transporter (VAChT) has been identified and characterized based on the acquisition of high affinity vesamicol binding and proton-dependent, vesamicol-sensitive acetylcholine accumulation by a fibroblast cell line transfected with a clone from a rat pheochromocytoma cDNA ... >> More

  The vesicular acetylcholine transporter (VAChT) has been identified and characterized based on the acquisition of high affinity vesamicol binding and proton-dependent, vesamicol-sensitive acetylcholine accumulation by a fibroblast cell line transfected with a clone from a rat pheochromocytoma cDNA library encoding this protein. The distribution of VAChT mRNA coincides with that reported for choline acetyltransferase (ChAT), the enzyme required for acetylcholine biosynthesis, in the peripheral and central cholinergic nervous systems. A human VAChT cDNA was used to localize the VAChT gene to chromosome 10q11.2, which is also the location of the ChAT gene. The entire sequence of the human VAChT cDNA is contained uninterrupted within the first intron of the ChAT gene locus. Transcription of VAChT and ChAT mRNA from the same or contiguous promoters within a single regulatory locus provides a previously undescribed genetic mechanism for coordinate regulation of two proteins whose expression is required to establish a mammalian neuronal phenotype. << Less

  J. Biol. Chem. 269:21929-21932(1994) [PubMed] [EuropePMC]

• Active transport of acetylcholine by the human vesicular acetylcholine transporter.

  Varoqui H., Erickson J.D.

  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 d ... >> More

  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. << Less

  J. Biol. Chem. 271:27229-27232(1996) [PubMed] [EuropePMC]

• Identification of a mammalian vesicular polyamine transporter.

  Hiasa M., Miyaji T., Haruna Y., Takeuchi T., Harada Y., Moriyama S., Yamamoto A., Omote H., Moriyama Y.

  Spermine and spermidine act as neuromodulators upon binding to the extracellular site(s) of various ionotropic receptors, such as N-methyl-d-aspartate receptors. To gain access to the receptors, polyamines synthesized in neurons and astrocytes are stored in secretory vesicles and released upon dep ... >> More

  Spermine and spermidine act as neuromodulators upon binding to the extracellular site(s) of various ionotropic receptors, such as N-methyl-d-aspartate receptors. To gain access to the receptors, polyamines synthesized in neurons and astrocytes are stored in secretory vesicles and released upon depolarization. Although vesicular storage is mediated in an ATP-dependent, reserpine-sensitive fashion, the transporter responsible for this process remains unknown. SLC18B1 is the fourth member of the SLC18 transporter family, which includes vesicular monoamine transporters and vesicular acetylcholine transporter. Proteoliposomes containing purified human SLC18B1 protein actively transport spermine and spermidine by exchange of H(+). SLC18B1 protein is predominantly expressed in the hippocampus and is associated with vesicles in astrocytes. SLC18B1 gene knockdown decreased both SLC18B1 protein and spermine/spermidine contents in astrocytes. These results indicated that SLC18B1 encodes a vesicular polyamine transporter (VPAT). << Less

  Sci. Rep. 4:6836-6836(2014) [PubMed] [EuropePMC]

  This publication is cited by 4 other entries.