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Written by | November
Vacuolar-type adenosine triphosphatases is a proton pump associated with the structure of F-type ATP synthetase, which uses the energy released during ATP hydrolysis to pump protons across the cell membrane, which is necessary for intracellular acidification and acid secretion 【1-3】
。 V-ATPases were first discovered in the vacuole membrane of yeast cells, hence the name
.
In neurons, each synaptic vesicle has approximately one V-ATPases, which provides energy for all neurotransmitters to enter the synaptic vesicle [4].
However, so far, the single-molecule model of V-ATPases is unknown
.
On November 23, 2022, the Dimitrios Stamou research group at the University of Copenhagen in Denmark published an article in Nature Regulation of the mammalian-brain V-ATPase through ultraslow mode-switching.
It was found that V-ATPases did not transport protons continuously, but had three different modes of action, namely proton-pumping, inactive mode and proton leakage mode (Proton-leaky), which unveils the working principle
of V-ATPase from the single-molecule level.
The function of V-ATPases is important in many different cellular, physiological, and pathological processes, including membrane trafficking, signaling, and cancer metastasis
.
In neuronal synaptic vesicles, V-ATPases transfer protons into the
synaptic vesicle cavity.
To understand V-ATPases at the single-molecule level, the authors measured the function of V-ATPases enzymes and revealed
their mechanism of action.
To maintain proton pump activity for the V-ATPases enzyme in mammalian brains, the authors isolated endogenous V-ATPases directly from intact synaptic vesicles, and can characterize proton transport
using genetically encoded pH sensors to measure changes in pH in synaptic vesicles.
To observe the activity of V-ATPases in a single synaptic vesicle for up to 3 hours, the authors added 16-300 photostable synthetic pH indicator DOPE-pHrodo to the synaptic vesicle (Figure 1).
To activate V-ATPases, the authors added ATP, a pH plateau phase reached approximately 15 minutes later, reflecting the dynamic equilibrium
achieved between proton active pumping into the cavity and proton passive penetration down a concentration gradient down the membrane.
By blocking the action of the proton pump with the addition of V-ATPases-specific inhibitors, the proton gradient undergoes irreversible collapse
.
Fig.
1 Observation of V-ATPases single-molecule level mode of action In order to quantify the transition of
different modes of V-ATPases, the authors developed a random Bayesian test model, which revealed the mode of action of V-ATPases by analyzing the residence time
。 The authors found that activated V-ATPases could exit proton pump mode and switch to deactivation or proton leakage mode
.
Further, the authors measured the proton gradient of the in-situ single-molecule V-ATPases proton pump and found that the gradient change in pH modulates the transition between different modes of the V-ATPases proton pump
.
The availability of ATP, a catalytic substrate, is also essential
for the activity of V-ATPases proton pumps.
In this work, the authors tested the effect of ATP on proton pumps at the single-molecule level for the first time, and found that a wide range of physiologically relevant ATP concentrations were also involved in mode switching
that regulated proton pumps.
Figure 2 Working modelIn
general, the authors used V-ATPases proton pumps directly purified in mouse brains to unveil their different change patterns at the single-molecule level for the first time V-ATPases proton pumps in mammals can switch from active proton pump mode to inactive or proton leakage mode under pH gradient and ATP substrate adjustment (Figure 2), emphasizing the mechanism and biological importance
of neurotransmitter delivery and proton pump mode switching.
Original link:
https://doi.
org/10.
1038/s41586-022-05472-9
Platemaker: Eleven
References
1.
Vasanthakumar, T.
& Rubinstein, J.
L.
Structure and roles of V-type ATPases.
Trends Biochem.
Sci.
45, 295–307 (2020).
2.
Ueno, H.
, Suzuki, K.
& Murata, T.
Structure and dynamics of rotary V1 motor.
Cell.
Mol.
Life Sci.
75, 1789–1802 (2018).
3.
Forgac, M.
Vacuolar ATPases: rotary proton pumps in physiology and pathophysiology.
Nat.
Rev.
Mol.
Cell Biol.
8, 917–929 (2007)
4.
Takamori, S.
et al.
Molecular anatomy of a trafficking organelle.
Cell 127, 831–846 (2006).
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