Neurotransmitter uptake of synaptic vesicles studied by X-ray diffraction

The size, polydispersity, and electron density profile of synaptic vesicles (SVs) can be studied by small-angle X-ray scattering (SAXS), i.e. by X-ray diffraction from purified SV suspensions in solution. Here we show that size and shape transformations, as they appear in the functional context of these important synaptic organelles, can also be monitored by SAXS. In particular, we have investigated the active uptake of neurotransmitters, and find a mean vesicle radius increase of about 12% after the uptake of glutamate, which indicates an unusually large extensibility of the vesicle surface, likely to be accompanied by conformational changes of membrane proteins and rearrangements of the bilayer. Changes in the electron density profile (EDP) give first indications for such a rearrangement. Details of the protein structure are screened, however, by SVs polydispersity. To overcome the limitations of large ensemble averages and heterogeneous structures, we therefore propose serial X-ray diffraction by single free electron laser pulses. Using simulated data for realistic parameters, we show that this is in principle feasible, and that even spatial distances between vesicle proteins could be assessed by this approach

In vitro fusion of single synaptic and dense core vesicles reproduces key physiological properties.

Regulated exocytosis of synaptic vesicles is substantially faster than of endocrine dense core vesicles despite similar molecular machineries. The reasons for this difference are unknown and could be due to different regulatory proteins, different spatial arrangements, different vesicle sizes, or other factors. To address these questions, we take a reconstitution approach and compare regulated SNARE-mediated fusion of purified synaptic and dense core chromaffin and insulin vesicles using a single vesicle-supported membrane fusion assay. In all cases, Munc18 and complexin are required to restrict fusion in the absence of calcium. Calcium triggers fusion of all docked vesicles. Munc13 (C1C2MUN domain) is required for synaptic and enhanced insulin vesicle fusion, but not for chromaffin vesicles, correlating inversely with the presence of CAPS protein on purified vesicles. Striking disparities in calcium-triggered fusion rates are observed, increasing with curvature with time constants 0.23 s (synaptic vesicles), 3.3 s (chromaffin vesicles), and 9.1 s (insulin vesicles) and correlating with rate differences in cells

Elevated synaptic vesicle release probability in synaptophysin/gyrin family quadruple knockouts

Synaptophysins 1 and 2 and synaptogyrins 1 and 3 constitute a major family of synaptic vesicle membrane proteins. Unlike other widely expressed synaptic vesicle proteins such as vSNAREs and synaptotagmins, the primary function has not been resolved. Here, we report robust elevation in the probability of release of readily releasable vesicles with both high and low release probabilities at a variety of synapse types from knockout mice missing all four family members. Neither the number of readily releasable vesicles, nor the timing of recruitment to the readily releasable pool was affected. The results suggest that family members serve as negative regulators of neurotransmission, acting directly at the level of exocytosis to dampen connection strength selectively when presynaptic action potentials fire at low frequency. The widespread expression suggests that chemical synapses may play a frequency filtering role in biological computation that is more elemental than presently envisioned

Colocalization of different neurotransmitter transporters on synaptic vesicles is sparse except for VGLUT1 and ZnT3

Vesicular transporters (VTs) define the type of neurotransmitter that synaptic vesicles (SVs) store and release. While certain mammalian neurons release multiple transmitters, it is not clear whether the release occurs from the same or distinct vesicle pools at the synapse. Using quantitative single-vesicle imaging, we show that a vast majority of SVs in the rodent brain contain only one type of VT, indicating specificity for a single neurotransmitter. Interestingly, SVs containing dual transporters are highly diverse (27 types) but small in proportion (2% of all SVs), excluding the largest pool that carries VGLUT1 and ZnT3 (34%). Using VGLUT1-ZnT3 SVs, we demonstrate that the transporter colocalization influences the SV content and synaptic quantal size. Thus, the presence of diverse transporters on the same vesicle is bona fide, and depending on the VT types, this may act to regulate neurotransmitter type, content, and release in space and time

Vesicular glutamate transporters use flexible anion and cation binding sites for efficient accumulation of neurotransmitter.

Vesicular glutamate transporters (VGLUTs) accumulate the neurotransmitter glutamate in synaptic vesicles. Transport depends on a V-ATPase-dependent electrochemical proton gradient (Delta mu H+) and requires chloride ions, but how chloride acts and how ionic and charge balance is maintained during transport is controversial. Using a reconstitution approach, we used an exogenous proton pump to drive VGLUT-mediated transport either in liposomes containing purified VGLUT1 or in synaptic vesicles fused with proton-pump-containing liposomes. Our data show that chloride stimulation can be induced at both sides of the membrane. Moreover, chloride competes with glutamate at high concentrations. In addition, VGLUT1 possesses a cation binding site capable of binding H+ or K+ ions, allowing for proton antiport or K+/H+ exchange. We conclude that VGLUTs contain two anion binding sites and one cation binding site, allowing the transporter to adjust to the changing ionic conditions during vesicle filling without being dependent on other transporters or channels

Dual and direction-selective mechanisms of phosphate transport by the vesicular glutamate transporter.

Vesicular glutamate transporters (VGLUTs) fill synaptic vesicles with glutamate and are thus essential for glutamatergic neurotransmission. However, VGLUTs were originally discovered as members of a transporter subfamily specific for inorganic phosphate (Pi). It is still unclear how VGLUTs accommodate glutamate transport coupled to an electrochemical proton gradient ΔμH+ with inversely directed Pi transport coupled to the Na+ gradient and the membrane potential. Using both functional reconstitution and heterologous expression, we show that VGLUT transports glutamate and Pi using a single substrate binding site but different coupling to cation gradients. When facing the cytoplasm, both ions are transported into synaptic vesicles in a ΔμH+-dependent fashion, with glutamate preferred over Pi. When facing the extracellular space, Pi is transported in a Na+-coupled manner, with glutamate competing for binding but at lower affinity. We conclude that VGLUTs have dual functions in both vesicle transmitter loading and Pi homeostasis within glutamatergic neurons

Elevated synaptic vesicle release probability in synaptophysin/gyrin family quadruple knockouts.

Synaptophysins 1 and 2 and synaptogyrins 1 and 3 constitute a major family of synaptic vesicle membrane proteins. Unlike other widely expressed synaptic vesicle proteins such as vSNAREs and synaptotagmins, the primary function has not been resolved. Here, we report robust elevation in the probability of release of readily releasable vesicles with both high and low release probabilities at a variety of synapse types from knockout mice missing all four family members. Neither the number of readily releasable vesicles, nor the timing of recruitment to the readily releasable pool was affected. The results suggest that family members serve as negative regulators of neurotransmission, acting directly at the level of exocytosis to dampen connection strength selectively when presynaptic action potentials fire at low frequency. The widespread expression suggests that chemical synapses may play a frequency filtering role in biological computation that is more elemental than presently envisioned

Single-vesicle imaging reveals different transport mechanisms between glutamatergic and GABAergic vesicles.

Synaptic transmission is mediated by the release of neurotransmitters, which involves exo-endocytotic cycling of synaptic vesicles. To maintain synaptic function, synaptic vesicles are refilled with thousands of neurotransmitter molecules within seconds after endocytosis, using the energy provided by an electrochemical proton gradient. However, it is unclear how transmitter molecules carrying different net charges can be efficiently sequestered while maintaining charge neutrality and osmotic balance. We used single-vesicle imaging to monitor pH and electrical gradients and directly showed different uptake mechanisms for glutamate and {gamma}-aminobutyric acid (GABA) operating in parallel. In contrast to glutamate, GABA was exchanged for protons, with no other ions participating in the transport cycle. Thus, only a few components are needed to guarantee reliable vesicle filling with different neurotransmitters

alpha-SNAP interferes with the zippering of the SNARE protein membrane fusion machinery.

Neuronal exocytosis is mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. Before fusion, SNARE proteins form complexes bridging the membrane followed by assembly toward the C-terminal membrane anchors, thus initiating membrane fusion. After fusion, the SNARE complex is disassembled by the AAA-ATPase N-ethylmaleimide-sensitive factor that requires the cofactor alpha-SNAP to first bind to the assembled SNARE complex. Using chromaffin granules and liposomes we now show that alpha-SNAP on its own interferes with the zippering of membrane-anchored SNARE complexes midway through the zippering reaction, arresting SNAREs in a partially assembled trans-complex and preventing fusion. Intriguingly, the interference does not result in an inhibitory effect on synaptic vesicles, suggesting that membrane properties also influence the final outcome of alpha-SNAP interference with SNARE zippering. We suggest that binding of alpha-SNAP to the SNARE complex affects the ability of the SNARE complex to harness energy or transmit force to the membrane