Vacuolar H+-ATPase subunits Voa1 and Voa2 cooperatively regulate secretory vesicle acidification, transmitter uptake, and storage

The Vo sector of the vacuolar H+-ATPase is a multisubunit complex that forms a proteolipid pore. Among the four isoforms (a1–a4) of subunit Voa, the isoform(s) critical for secretory vesicle acidification have yet to be identified. An independent function of Voa1 in exocytosis has been suggested. Here we investigate the function of Voa isoforms in secretory vesicle acidification and exocytosis by using neurosecretory PC12 cells. Fluorescence-tagged and endogenous Voa1 are primarily localized on secretory vesicles, whereas fluorescence-tagged Voa2 and Voa3 are enriched on the Golgi and early endosomes, respectively. To elucidate the functional roles of Voa1 and Voa2, we engineered PC12 cells in which Voa1, Voa2, or both are stably down-regulated. Our results reveal significant reductions in the acidification and transmitter uptake/storage of dense-core vesicles by knockdown of Voa1 and more dramatically of Voa1/Voa2 but not of Voa2. Overexpressing knockdown-resistant Voa1 suppresses the acidification defect caused by the Voa1/Voa2 knockdown. Unexpectedly, Ca2+-dependent peptide secretion is largely unaffected in Voa1 or Voa1/Voa2 knockdown cells. Our data demonstrate that Voa1 and Voa2 cooperatively regulate the acidification and transmitter uptake/storage of dense-core vesicles, whereas they might not be as critical for exocytosis as recently proposed.

Molecules of the mind: integrating synaptic biochemistry to understand brain function

The focused meeting entitled ‘Molecular Determinants of Synaptic Function: Molecules and Models’ brought together several molecules and experimental models that are furthering our understanding of the biochemical basis of integrative brain function. Invited speakers and short communications from more junior scientists highlighted how individual molecules or protein networks underlie defined subcellular functions (e.g. transmitter release, receptor activation and transmitter uptake) can be used to unravel integrative function at cellular, systems and behavioural levels.

The uptake inhibitor L-trans-PDC enhances responses to glutamate but fails to alter the kinetics of excitatory synaptic currents in the hippocampus

1. We have used patch-clamp recording techniques to study the physiological properties of a recently described glutamate uptake blocker, L-trans-pyrrolidine-2,4-dicarboxylic acid (L-trans-PDC), in the CA1 region of the guinea pig hippocampus. 2. L-trans-PDC markedly potentiated the action of exogenously applied glutamate and raised the ambient extracellular levels of glutamate in hippocampal slices. Despite these actions, L-trans-PDC did not affect the time course of either the N-methyl-D-aspartate (NMDA) or non-NMDA receptor-mediated synaptic currents evoked by the stimulation of a large number of neighboring synapses. 3. These findings are consistent with models of fast synaptic transmission in which transmitter is rapidly cleared from the synaptic cleft by diffusion. However, in marked contrast to fast gamma-aminobutyric acid A (GABAA) synapses in the hippocampus, uptake does not appear to play a role in regulating the "spill-over" of transmitter from neighboring, co-activated glutamatergic synapses. Therefore, either diffusion alone can effectively limit the temporal and spatial domain of synaptically released glutamate, or alternatively, L-trans-PDC like other currently available blockers is not sufficiently potent to reveal a role for transmitter uptake at glutamatergic synapses.