Synaptic Vesicle-bound Pyruvate Kinase can Support Vesicular Glutamate Uptake

Abstract

Glucose metabolism is essential for normal brain function and plays a vital role in synaptic transmission. Recent evidence suggests that ATP synthesized locally by glycolysis, particularly via glyceraldehyde 3-phosphate dehydrogenase/3-phosphoglycerate kinase, is critical for synaptic transmission. We present evidence that ATP generated by synaptic vesicle-associated pyruvate kinase is harnessed to transport glutamate into synaptic vesicles. Isolated synaptic vesicles incorporated [ 3 H]glutamate in the presence of phosphoenolpyruvate (PEP) and ADP. Pyruvate kinase activators and inhibitors stimulated and reduced PEP/ADP-dependent glutamate uptake, respectively. Membrane potential was also formed in the presence of pyruvate kinase activators. "ATP-trapping" experiments using hexokinase and glucose suggest that ATP produced by vesicleassociated pyruvate kinase is more readily used than exogenously added ATP. Other neurotransmitters such as GABA, dopamine, and serotonin were also taken up into crude synaptic vesicles in a PEP/ADP-dependent manner. The possibility that ATP locally generated by glycolysis supports vesicular accumulation of neurotransmitters is discussed.

Direct evidence for activity-dependent glucose phosphorylation in neurons with implications for the astrocyte-to-neuron lactate shuttle

“…The finding of a large fraction of activity-dependent glucose uptake in neurons requires an alternative explanation of the 1:1 relationship. Two alternate mechanisms have been postulated, one involving the coupling of neuronal glycolysis (or glycolytic ATP) to vesicular loading of glutamate (37,38) and another involving the coupling of neuronal glutamate formed from glutamine to redox movements into mitochondria via the malate aspartate shuttle (39,40). Glutamate accumulation into synaptic vesicles is driven by a H + electrochemical gradient produced by a vacuolar (H + )-ATPase, the energetic cost of which was estimated to be ∼0.33 ATP/glutamate (26).…”

Glutamate Release

“…Evidence suggests that a sub-population of synaptic vesicles bear pyruvate kinase, which is also capable of supporting vesicular glutamate uptake [111]. These observations together support the notion that locally synthesized glycolytic ATP is the major energy source harnessed for efficiently refilling of synaptic vesicles with glutamate [112].…”

Enhanced Glutamate Uptake into Synaptic Vesicles Fueled by Vesicle-generated ATP from Phosphoenolpyruvate and ADP

“…Ishida et al [32] reported evidence suggesting that ATP generated endogenously by synaptic vesicles in the presence of PEP and ADP is preferentially used compared to exogenous ATP; exogenous hexokinase has less access to the former ATP than to the latter. This supports the notion that ATP locally produced at the synaptic vesicle site is important in efficient fueling glutamate loading into synaptic vesicles.…”

“…A pyruvate kinase-rich synaptic vesicle fraction was prepared from frozen rat cerebrum, and glutamate uptake into these synaptic vesicles measured by a filtration-based assay using Whatman GF/C filters, as described previously [32]. The vesicle fraction (30 or 15 μg) was incubated at 30° C in a solution (final volume, 0.1 ml), which contained 50 μM [ 3 H]glutamate (280 mCi/mmol), 20 mM Hepes-Tris (pH 7.4), 4 mM MgSO 4 , 4 mM KCl, 2 mM aspartate, 100 mM potassium gluconate, and 50 mM sucrose, in the absence or presence of 0.8 mM ADP alone, a combination of 0.8 mM ADP and 1 mM PEP, or 1 mM ATP alone (Fig.…”

“…Compatible with this notion, Ishida et al [32] have recently shown that ATP produced by synaptic-vesicle bound pyruvate kinase, the other glycolytic ATP-generating enzyme, at the expense of the glycolytic intermediate PEP, can also support vesicular glutamate uptake. Thus, since glycolytic ATP production (which occurs considerably prior to mitochondrial ATP synthesis) can occur at the synaptic vesicle site, local glycolytic ATP synthesis could represent an efficient mechanism for the rapid energy supply needed to sustain normal synaptic transmission.…”