Results & Discussion
Chemical signal transmission
Chemical signal transmission is a key process for intracellular communication (2). In neuron, neurotransmitters are accumulated in the synaptic vesicles and are released upon stimulation. Released neurotransmitters stimulate target cells by binding to the specific receptors.
The Vesicular Glutamate Transporter
Among the various neurotransmitters used in the biological system, glutamate is a most important neurotransmitter for excitatory signal transmission in neuron and plays key role in higher order brain functions including memory and learning. The vesicular glutamate transporter (VGLUT) is a protein molecule located on the membrane of synaptic vesicles and responsible for vesicular accumulation of glutamate in the vesicles (2). Thus, VGLUT is an essential component of excitatory signal transmission in brain.
Purification & Reconstitution: a novel and classical approach to analyze transporter
Although importance of VGLUT in chemical transmission, molecular nature of VGLUT was not well understood. We analyzed kinetic features of VGLUT through newly developed assay system (3). In this system, VGLUT is over-expressed in the insect cells and purified by Ni-NTA column chromatography after solubilization of membrane by detergent. The purified VGLUT is reconstituted in liposomes (artificial synaptic vesicles) and used for glutamate transport analysis.
Chloride is a regulator of VGLUT
One of the interesting feature of VGLUT found in our kinetic studies was the Cl- switch (1). VGLUT requires low concentration of Cl- for glutamate transport activity. Interestingly, such Cl- stimulation shows strong cooperativity. With this strong cooperativity, Cl- can tightly control VGLUT as a molecular switch.
Vesicular Glutamate Transporter & Epilepsy
We also found that ketone bodies (acetoacetate) inhibits VGLUT by reducing affinity to Cl-. Ketone body is the metabolic intermediates of fatty acid and produced during starvation. Our study showed that ketone body inhibits neuronal activity and seizures evoked by epilepsy. By inhibiting VGLUT, glutamate can not be accumulated in the synaptic vesicles and hence chemical transmission is reduced. It is noteworthy that ketogenic diet has been used for treatment of epilepsy. However, molecular mechanism of this treatment was not known. Our study revealed that the metabolic control of vesicular glutamate transporter is the key process to control epilepsy. Our results also indicate that chemical signaling in brain is affected by the metabolic state of the animals.
In addition to the postsynaptic receptors site(s), loading of neurotransmitters into vesicles is novel site for controlling chemical transmission. This means that vesicular neurotransmitter transporters are a new set of potential pharmacological targets for drugs that control brain function.
- Juge N, Gray JA, Omote H, Miyaji T, Inoue T, Hara C, Uneyama H, Edwards RH, Nicoll RA, Moriyama Y. Metabolic control of vesicular glutamate transport and release. Neuron 68: 99-112, 2010.
- Omote H, Moriyama Y. Vesicular neurotransmitter transporters: an approach for studying transporters with purified proteins. Physiology (Bethesda) 28:39-50, 2013
- Juge N, Yoshida Y, Yatsushiro S, Omote H, Moriyama Y. Vesicular glutamate transporter contains two independent transport machineries. J Biol Chem 281: 39499-39506, 2006.
- Hartman, A.L., Gasior, M., Vining, E.P., and Rogawski, M.A. The neuropharmacology of the ketogenic diet. Pediatr. Neurol. 36: 281-292, 2007.