The glutamate transport system in the synaptic vesicle: Mechanism and regulation.

Abstract

Transport of glutamate, the major excitatory neurotransmitter in the mammalian central nervous system (CNS), into synaptic vesicles may represent the first critical step in the neurotransmitter pathway for glutamate. This thesis describes biochemical and bioenergetic analyses of vesicular glutamate transport. In the first section of the thesis, steady-state transport experiments demonstrated that under physiological conditions, $\Delta\Psi$ and $\Delta$pH both contribute to the driving force of glutamate transport and that $\Delta\mu$-driven glutamate uptake relies upon counter-transport of H$\sp+$ and Cl$\sp-$. Kinetic studies demonstrated that in the absence of $\Delta\mu$, the neutral form of glutamate is capable of being transported with a K$\sb{\rm m}$ of 5.47 mM and a V$\sb{\rm max}$ of 2.76 nmol/min/mg. The presence of $\Delta\Psi$ led to an 8.2-fold decrease in K$\sb{\rm m}$ for anionic glutamate with a 2.3-fold decrease in V$\sb{\rm max}$. Conversely, the imposition of $\Delta$pH (in the presence of $\Delta\Psi$) increased V$\sb{\rm max}$ up to 2.9-fold with little change in K$\sb{\rm m}$. These studies suggest that anionic glutamate uptake requires not only a transporter with the proper affinity, which is set by $\Delta\Psi,$ but also a translocation process, whose maximal rate depends on a proton gradient. In the second section, studies demonstrated the partial purification (230-fold enrichment) of a proteinaceous factor that modulates vesicular glutamate levels. This factor has an approximate molecular weight of 180 kDa and is temperature and protease sensitive. In addition, the factor itself appears to require activation that involves the presence of synaptic vesicles and ATP hydrolysis. The factor is postulated to be a component of a regulatory system that modulates presynaptic glutamate levels. In the third section, the vesicular glutamate uptake system in epileptic (EL) mice, an animal model of temporal lobe epilepsy, was investigated. The total and specific activities of vesicular glutamate uptake were significantly higher in the hippocampus of stimulated EL mice than of stimulated B6 mice (704% and 207%, respectively). This enhancement was not only specific to one brain region but also dependent on age and seizure history. The results suggest that elevated vesicular glutamate levels in the hippocampus may be involved in seizure manifestation in these animals.