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Cited by 66Cellular transport and secretionIon Transport and Channel RegulationNeuroscience and Neuropharmacology Research

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

Nguyen, Marie L., Cox, Gregory D., Parsons, Stanley M.·1998·Biochemistry
DOI 10.1021/bi9802263
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Abstract

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.

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Crossref · 10.1021/bi9802263

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  • vMAT1/2 and VAChT transport positively charged neurotransmitters in exchange for two protons and depend more on the pH gradient than on membrane potential.

    “Vesicular transporters for monoamines (vMAT1/2) and Ach (VAChT) transport positively charged neurotransmitters in exchange for 2H+ and are more dependent on DpH than DJ.18,19”

    From Synaptic and circuit functions of multitransmitter neurons in the mammalian brain (2023) · cites this paper as [19]

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Authors
Nguyen, M. L., Cox, G. D., & Parsons, S. M.
Affiliations
University of California, Santa Barbara (US)
Corresponding author
Parsons, Stanley M.
ORCID
Parsons, Stanley M.
Venue
Biochemistry
Publisher
American Chemical Society (ACS)
Published
1998-09-01
Volume
37
Issue
38
Pages
13400-13410
ISSN
  • 0006-2960 (ISSN-L)
  • 1520-4995
DOI
10.1021/bi9802263
OpenAlex
W1985977357
Cited by
66
References
13
Citations by year
2026: 2 · 2025: 3 · 2024: 1 · 2023: 3 · 2021: 4 · 2018: 2 · 2017: 4 · 2016: 3 · 2015: 3 · 2014: 3 · 2013: 5 · 2012: 1
FWCI
1.32
Type
journal article
Language
en
Open access
closed
Locations
  • Biochemistry (publishedVersion)
  • PubMed (publishedVersion)
Funders
National Institute of Neurological Disorders and Stroke (R01NS015047, NS 15047)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Cellular transport and secretion
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Ion Transport and Channel Regulation
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Neuroscience and Neuropharmacology Research
Keywords
vesicular acetylcholine transporter, acetylcholine transport, proton exchange, stoichiometry, protonation, ligand binding, transport cycle, protonation kinetics, cholinergic nerve terminals
MeSH terms
Acetylcholine, Animals, Biological Transport, Carrier Proteins, Cell Fractionation, Electric Organ, Hydrogen-Ion Concentration, Kinetics, Membrane Potentials, Osmolar Concentration, Protons, Regression Analysis, Synaptic Vesicles, Time Factors, Torpedo, Tritium, Membrane Transport Proteins, Vesicular Transport Proteins, Vesicular Acetylcholine Transport Proteins

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