Molecular and functional characterization of vesicular glutamate and GABA transporters.
Final Report Abstract
Communication between two neurons is mediated at synapses where one neuron “talks” (sender neuron) and the second “listens” (receiving neuron). When an electrical signal arrives in the sender neuron, it releases signal molecules, the neurotransmitters that then are recognized by the receiving neuron. The neurotransmitters are stored in small membrane organelles, the synaptic vesicles, within the cytoplasm of the presynaptic nerve ending. Incoming electrical signals lead to an influx of calcium ions that then trigger the fusion of the vesicle with the surrounding plasma membrane, causing the discharge of the neurotransmitter content. Neurotransmitters are synthesized in the cytoplasm of neurons. For synaptic transmission they first need to be actively concentrated inside synaptic vesicles, which is mediated by vesicular transporters specific for each neurotransmitter. In recent years, the transporters for all major classes of neurotransmitters such as glutamate, GABA, glycine, acetylcholine, dopamine, noradrenaline, serotonin, and ATP, have been identified. They are all fueled by an ion pump of the V-ATPase type, which electrogenically pumps protons in the vesicle interior, but the transport mechanisms are different and in many cases not well understood. In the project, we have mainly worked on the transport of the major excitatory neurotransmitter glutamate, which is mediated by the vesicular glutamate transporters (VGluTs, 3 variants). For our transport studies we used three different experimental systems: (i) vesicles highly purified from brain or from neuroendocrine cell lines that contain endogenous or exogenously expressed VGluT, (ii) purified vesicles that were fused with artificial vesicles containing a defined ionic composition and an exogenous proton pump in order to manipulate not only the external but also the internal solution, and (iii) artificial vesicles containing purified VGluT and an exogenous proton pump. Our results show that the transport mechanism of VGluT is complex, with the transporter possessing at least three ion binding sites: one for the substrate glutamate that, however, also binds chloride but with lower affinity, (ii) one for chloride that is probably not involved in transport but serves to increase the activity of the transporter, and (iii) a cation binding site that functions as a potassium/proton exchanger under physiological conditions, thus lowering the acid load of the vesicle during continuous transport and proton pumping. The results explain how the transporter can fill an “empty” vesicle with up to 100-200 mM glutamate without being dependent on any other compensatory ion channel or transporter, solely relying on the energy provided by the proton pump. In addition, we have also compared the transport mechanisms of VGluTs with those of the vesicular GABA (VGAT) and monoamine transporters (VMATs). Our results confirm the differences in energy dependence (i.e. VGAT and VMATs are proton antiporters whereas VGluT uses the voltage difference as energy source). Furthermore, they suggest, contrary to reports in the literature, that chloride ions are not directly involved in the transport mechanisms of VGAT and VMATs.
Publications
- (2010): Quantitative comparison of glutamatergic and GABAergic synaptic vesicles unveils selectivity for few proteins including MAL2, a novel synaptic vesicle protein. J Neurosci 30: 2-12
Grønborg M, Pavlos NJ, Brunk I, Chua JJE, Münster-Wandowski A, Riedel D, Ahnert-Hilger G, Urlaub H, Jahn R
(See online at https://doi.org/10.1523/jneurosci.4074-09.2010) - (2011): ClC-3 spices up GABAergic synaptic vesicles. Nat. Neurosci. 14: 405-407
Ahnert-Hilger G, Jahn R
(See online at https://doi.org/10.1038/nn.2786) - (2014) Vesicular glutamate transporters (VGLUTs) use flexible anion and cation binding sites for efficient accumulation of neurotransmitter. Neuron 84, 1287-1301
Preobraschenski J, Zander J-F, Suzuki T, Ahnert-Hilger G, Jahn, R
(See online at https://doi.org/10.1016/j.neuron.2014.11.008)
