Article

Presynaptic regulation of quantal size: K+/H+ exchange stimulates vesicular glutamate transport

Department of Physiology, Graduate Program in Neuroscience, University of California, San Francisco, San Francisco, California, USA.
Nature Neuroscience (Impact Factor: 14.98). 08/2011; 14(10):1285-92. DOI: 10.1038/nn.2898
Source: PubMed

ABSTRACT The amount of neurotransmitter stored in a single synaptic vesicle can determine the size of the postsynaptic response, but the factors that regulate vesicle filling are poorly understood. A proton electrochemical gradient (Δμ(H+)) generated by the vacuolar H(+)-ATPase drives the accumulation of classical transmitters into synaptic vesicles. The chemical component of Δμ(H+) (ΔpH) has received particular attention for its role in the vesicular transport of cationic transmitters as well as in protein sorting and degradation. Thus, considerable work has addressed the factors that promote ΔpH. However, synaptic vesicle uptake of the principal excitatory transmitter glutamate depends on the electrical component of Δμ(H+) (Δψ). We found that rat brain synaptic vesicles express monovalent cation/H(+) exchange activity that converts ΔpH into Δψ, and that this promotes synaptic vesicle filling with glutamate. Manipulating presynaptic K(+) at a glutamatergic synapse influenced quantal size, indicating that synaptic vesicle K(+)/H(+) exchange regulates glutamate release and synaptic transmission.

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    • "ΔpH was determined by measuring the fluorescence quench - ing of acridine orange ( 1 . 5 μM ) in a Spectra Max M5 microplate reader spectrophotometer as previously described using an exci - tation wavelength of 492 nm and an emission wavelength of 537 nm ( Goh et al . , 2011 ) . The reaction was initiated by the addition of 1 mM MgCl2 in the presence of 1 mM ATP to a reaction medium contain - ing 30 μg of synaptic vesicle protein , 140 mM KCl , and 10 mM Mops – Tris ( pH 7 . 4 ) . The reaction was terminated by the addition of Triton X - 100 to a final concentration of 10 μM . JM - 20 effects were evalu - a"
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    ABSTRACT: Acidification of synaptic vesicles relies on the vacuolar-type ATPase (V-ATPase) and provides the electrochemical driving force for neurotransmitter exchange. The regulatory mechanisms that ensure assembly of the V-ATPase holoenzyme on synaptic vesicles are unknown. Rabconnectin3α (Rbc3α) is a potential candidate for regulation of V-ATPase activity because of its association with synaptic vesicles and its requirement for acidification of intracellular compartments. Here, we provide the first evidence for a role of Rbc3α in synaptic vesicle acidification and neurotransmission. In this study, we characterized mutant alleles of rbc3α isolated from a large-scale screen for zebrafish with auditory/vestibular defects. We show that Rbc3α is localized to basal regions of hair cells in which synaptic vesicles are present. To determine whether Rbc3α regulates V-ATPase activity, we examined the acidification of synaptic vesicles and localization of the V-ATPase in hair cells. In contrast to wild-type hair cells, we observed that synaptic vesicles had elevated pH, and a cytosolic subunit of the V-ATPase was no longer enriched in synaptic regions of mutant hair cells. As a consequence of defective acidification of synaptic vesicles, afferent neurons in rbc3α mutants had reduced firing rates and reduced accuracy of phase-locked action potentials in response to mechanical stimulation of hair cells. Collectively, our data suggest that Rbc3α modulates synaptic transmission in hair cells by promoting V-ATPase activity in synaptic vesicles.
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Hai Huang