Glutamate plays an important metabolic role in virtually every vertebrate cell. In particular, glutamate is the most common excitatory neurotransmitter in the vertebrate central nervous system. As such, the mechanism by which glutamate is diverted from its normal metabolic activities toward its role as a neurotransmitter has, in recent years, been systematically investigated. In glutamatergic nerve endings, synaptic vesicles accumulate and store a proportion of the cellular glutamate pool and, in response to appropriate signals, release glutamate into the synaptic cleft by exocytosis. Glutamate accumulation is accomplished by virtue of a glutamate uptake system present in the synaptic vesicle membrane. The uptake system consists of a transport protein, remarkably specific for glutamate, and a vacuolar-type H+-ATPase, which provides the coupling between ATP hydrolysis and glutamate transport. The precise manner in which the glutamate transporter and H+-ATPase operate is currently the subject of debate. Recent data relevant to this debate are reviewed in this article. Additionally, pharmacological agents thought to specifically interact with the vesicular glutamate transporter are discussed. Finally, a newly discovered, endogenous inhibitor of vesicular uptake, inhibitory protein factor (IPF), is discussed with some speculations as to its potential role as a presynaptic modulator of neurotransmission.

Glutamate, the major excitatory neurotransmitter in the mammalian central nervous system, is transported into and stored in synaptic vesicles. We have purified to apparent homogeneity a protein from brain cytosol that inhibits glutamate and ␥-aminobutyric acid uptake into synaptic vesicles and have termed this protein ''inhibitory protein factor'' (IPF). IPF refers to three distinct proteins with relative molecular weights of 138,000 (IPF ␣), 135,000 (IPF ␤), and 132,000 (IPF ␥), respectively. Gel filtration and sedimentation data suggest that all three proteins share an elongated structure, identical Stokes radius (60 Å), and identical sedimentation coefficient (4.3 S). Using these values and a partial specific volume of 0.716 ml͞g, we determined the native molecular weight for IPF ␣ to be 103,000. Partial sequence analysis shows that IPF ␣ is derived from ␣ fodrin, a protein implicated in several diverse cellular activities. IPF ␣ inhibits ATP-dependent glutamate uptake into purified synaptic vesicles with an IC 50 of Ϸ26 nM, while showing no ability to inhibit ATP-independent uptake at concentrations up to 100 nM. Moreover, IPF ␣ inhibited neither norepinephrine uptake into chromaffin vesicles nor Na ؉ -dependent glutamate uptake into synaptosomes. However, IPF ␣ inhibited uptake of ␥-aminobutyric acid into synaptic vesicles derived from spinal cord, suggesting that inhibition may not be limited to glutamatergic systems. We propose that IPF could be a novel component of a presynaptic regulatory system. Such a system might modulate neurotransmitter accumulation into synaptic vesicles and thus regulate the overall efficacy of neurotransmission.

Synaptic vesicles in the nerve terminal play a pivotal role in neurotransmission. Neurotransmitter accumulation into synaptic vesicles is catalyzed by distinct vesicular transporters, harnessing an electrochemical proton gradient generated by V-type proton-pump ATPase. However, little is known about regulation of the transmitter pool size, particularly in regard to amino acid neurotransmitters. We previously provided evidence for the existence of a potent endogenous inhibitory protein factor (IPF), which causes reduction of glutamate and GABA accumulation into isolated, puri®ed synaptic vesicles. In this study we demonstrate that IPF is concentrated most in the synaptosomal cytosol fraction and that, when introduced into the synaptosome, it leads to a decrease in calcium-dependent exocytotic (but not calcium-independent) release of glutamate in a concentration-dependent manner. In contrast, a-fodrin (nonerythroid spectrin), which is structurally related to IPF and thought to serve as the precursor for IPF, is devoid of such inhibitory activity. Intrasynaptosomal IPF also caused reduction in exocytotic release of GABA and the monoamine neurotransmitter serotonin. Whether IPF affects vesicular storage of multiple neurotransmitters in vivo would depend upon the localization of IPF. These results raise the possibility that IPF may modulate synaptic transmission by acting as a quantal size regulator of one or more neurotransmitters.

Reserpine, a competitive inhibitor of catecholamine transport into adrenal medullary chromaffin vesicles, consists of a trimethoxybenzoyl group esterified to an alkaloid ring system. Reserpine inhibits norepinephrine transport with a Ki of approximately 1 nM and binds to chromaffin-vesicle membranes with a KD of about the same value. Methyl reserpate and reserpinediol, derivatives that incorporate the alkaloid ring system, also competitively inhibit norepinephrine transport into chromaffin vesicles with Ki values of 38 +/- 10 nM and 440 +/- 240 nM, respectively. Similar concentrations inhibit [3H]reserpine binding to chromaffin-vesicle membranes. 3,4,5-Trimethoxybenzyl alcohol and 3,4,5-trimethoxybenzoic acid, derivatives of the other part of the reserpine molecule, do not inhibit either norepinephrine transport or [3H]reserpine binding at concentrations up to 100 microM. Moreover, trimethoxybenzyl alcohol does not potentiate the inhibitory action of methyl reserpate. Therefore, the amine binding site of the catecholamine transporter appears to bind the alkaloid ring system of reserpine rather than the trimethoxybenzoyl moiety. The more potent inhibitors are more hydrophobic compounds, suggesting that the reserpine binding site is hydrophobic.

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