|dc.description.abstracteng||During neuronal activity, synapses sustain neurotransmission by a high fidelity multi-step process called synaptic vesicle (SV) recycling. This process involves endocytosis, neurotransmitter loading and exocytosis of SVs within a timeframe lasting from a few seconds to tens of seconds at different synapses. Despite extensive studies on endo/exocytosis, there is a scarcity of details regarding vesicle loading and its regulation.
Vesicle filling requires two distinct but dependent processes. First, the vacuolar H+-ATPase (V-ATPase) builds a concentration gradient (∆pH) as well as an electrical potential (∆ψ) across the membrane of SVs by pumping of protons into the lumen of the vesicle at the expense of ATP. Vesicular transporters then use this combined electrochemical gradient (∆µH+) to drive the loading of transmitters into the SVs.
Neurotransmitter molecules are differently charged at neutral pH, and although ∆µH+ is required for their transport, the contribution of ∆pH and ∆ψ to their transport is different and depends on their charge. For positively charged monoamines and acetylcholine, ∆pH provides the main driving force. In case of negatively charged glutamate, ∆ψ is more important and for neutral GABA, both components of ∆µH+ play equal roles. Therefore, accumulation of massive amounts of either of these transmitters within the short lifetime of a recycling SV would demand additional compensating mechanisms to maintain the right balance between ∆pH and ∆ψ during each cycle of neurotransmitter uptake.
Existing models so far have proposed that uptake of these transmitters are probably associated with the compensating ion fluxes which are either mediated by the transporter itself or provided by other ion exchangers present on SVs. However, there are still major disagreements. Moreover, whether these compensating mechanisms are different in distinct vesicles, and if this is the case, the molecular mechanisms underlying these differences are still enigmatic, particularly when considering that SVs share the majority of their molecular composition.
In the current study, a novel single vesicle assay was developed to first explore the basic questions about the extent and kinetics of the two components of ∆µH+ at the single vesicle level, and second, to unravel how the balance between ∆pH and ∆ψ is regulated in glutamatergic and GABAergic SVs, which have different bioenergetics requirements. In this assay, SVs purified from transgenic mice expressing super-ecliptic pHluorin in the vesicular lumen (spH-SVs) were imaged using TIRF (total-internal reflection fluorescence) microscopy to accurately measure luminal pH changes above pH 6. In addition, SVs were labeled with voltage sensitive dye VF2.1.Cl to quantitatively measure changes in membrane potential across the lipid bilayer of single SVs for the first time. After measuring ∆pH or ∆ψ, antibody labeling against VGAT or VGLUT1 allowed for unequivocally distinguishing GABAergic from glutamatergic SVs.
This study provides evidence that SVs can acidify with sub-second kinetics and their biophysical characteristics such as buffering capacity and proton permeability fall within the range of reported values for other trafficking organelles. Moreover, a detailed comparison of ∆pH and ∆ψ in glutamatergic and GABAergic SVs at the single vesicle level revealed that regulatory mechanisms do exist to optimize the balance of the electrochemical gradient, and that the vesicular transporter itself bears responsibility. It was demonstrated that VGAT transports GABA with a GABA/H+ anitport mechanism. This transport mechanism enables VGAT to keep the balance between the two components of ∆µH+ during neurotransmitter loading. In addition, it was shown that VGLUT can transport Cl- and also functions as a K+/H+ exchanger, both of which assist the transporter to tailor the balance to greater ∆ψ which is the main driving force for glutamate uptake. Together, these findings introduce vesicular transporters as novel regulators of the electrochemical gradient, which would grant additional significance to their role in synaptic transmission regulation.||de