The V0 membrane domain of the V-ATPase reversibly dissociates from V1 at acidic intragranular pH and is necessary for normal exocytosis and synaptic transmission.

Myasthenia gravis is an autoimmune disease associated with thymic pathologies, including hyperplasia. In this study, we investigated the processes that may lead to thymic overexpression of the triggering Ag, the acetylcholine receptor (AChR). Using microarray technology, we found that IFN-regulated genes are more highly expressed in these pathological thymic tissues compared with age- and sex-matched normal thymus controls. Therefore, we investigated whether proinflammatory cytokines could locally modify AChR expression in myoid and thymic epithelial cells. We found that AChR transcripts are up-regulated by IFN-γ, and even more so by IFN-γ and TNF-α, as assessed by real-time RT-PCR, with the α-AChR subunit being the most sensitive to this regulation. The expression of AChR protein was increased at the cytoplasmic level in thymic epithelial cells and at the membrane in myoid cells. To examine whether IFN-γ could influence AChR expression in vivo, we analyzed AChR transcripts in IFN-γ gene knock-out mice, and found a significant decrease in AChR transcript levels in the thymus but not in the muscle, compared with wild-type mice. However, up-regulation of AChR protein expression was found in the muscles of animals with myasthenic symptoms treated with TNF-α. Altogether, these results indicate that proinflammatory cytokines influence the expression of AChR in vitro and in vivo. Because proinflammatory cytokine activity is evidenced in the thymus of myasthenia gravis patients, it could influence AChR expression and thereby contribute to the initiation of the autoimmune anti-AChR response.

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Vacuolar proton ATPase accumulates protons inside various intracellular organelles such as synaptic vesicles; its membrane domain V0 could also be involved in membrane fusion. These different functions could require vacuolar proton ATPases possessing different V0 subunit a isoforms. In vertebrates, four genes encode isoforms a1-a4, and a1 variants are also generated by alternative splicing. We identified a novel a1 splice variant a1-IV and showed that the two a1 variants containing exon C are specifically expressed in neurons. Single neurons coexpress a2, a1-I, and a1-IV, and these subunit a isoforms are targeted to different membrane compartments. Recombinant a2 was accumulated in the trans-Golgi network, and a1-I was concentrated in axonal varicosities, whereas a1-IV was sorted to both distal dendrites and axons. Our results indicate that alternative splicing of exon N controls differential sorting of a1 variants to nerve terminals or distal dendrites, whereas exon C regulates their neuronal expression.

pH is an important parameter that affects many functions of live cells, from protein structure or function to several crucial steps of their metabolism. Genetically encoded pH sensors based on pH-sensitive fluorescent proteins have been developed and used to monitor the pH of intracellular compartments. The quantitative analysis of pH variations can be performed either by ratiometric or fluorescence lifetime detection. However, most available genetically encoded pH sensors are based on green and yellow fluorescent proteins and are not compatible with multicolor approaches. Taking advantage of the strong pH sensitivity of enhanced cyan fluorescent protein (ECFP), we demonstrate here its suitability as a sensitive pH sensor using fluorescence lifetime imaging. The intracellular ECFP lifetime undergoes large changes (32 %) in the pH 5 to pH 7 range, which allows accurate pH measurements to better than 0.2 pH units. By fusion of ECFP with the granular chromogranin A, we successfully measured the pH in secretory granules of PC12 cells, and we performed a kinetic analysis of intragranular pH variations in living cells exposed to ammonium chloride.

V-ATPases are multimeric enzymes made of two sectors, a V1 catalytic domain and a V0 membrane domain. They accumulate protons in various intracellular organelles. Acidification of synaptic vesicles by V-ATPase energizes the accumulation of neurotransmitters in these storage organelles and is therefore required for efficient synaptic transmission. In addition to this well-accepted role, functional studies have unraveled additional hidden roles of V0 in neurotransmitter exocytosis that are independent of the transport of protons. V0 interacts with SNAREs and calmodulin, and perturbing these interactions affects neurotransmitter release. Here, we discuss these data in relation with previous results obtained in reconstituted membranes and on yeast vacuole fusion. We propose that V0 could be a sensor of intra-vesicular pH that controls the exocytotic machinery, probably regulating SNARE complex assembly during the synaptic vesicle priming step, and that, during the membrane fusion step, V0 might favor lipid mixing and fusion pore stability.

Vacuolar H(+) -ATPases (v-ATPases) are multimeric proton pumps which acidify various intra-cellular organelles and may participate in pHe and pHi regulation in cancer cell lines. The ATP6V0A4 gene encodes the a4 subunit which is expressed in kidney and epididymis. Because we found a4 mRNA highly expressed in C6Bu1 glioma cell line, we measured it in 205 glioma biopsies and 11 brain biopsies from epileptic patients. a4 was absent in epileptic brain biopsies, but was expressed by 34% (11/32) of grade III oligodendrogliomas, independently of the chromosome 1p19q codeletion. a4 expression in grade III oligodendrogliomas and oligoastrocytomas without the 1p19q codeletion tended to be associated with a shorter overall survival of patients. We also observed a4 expression in biopsies of pilocytic astrocytomas (68%; 19/28) and gangliogliomas (37%; 6/16). In pilocytic astrocytomas a4 expression was associated with a tandem duplication of the 7q34 chromosome region, distant 0.5 Mb to the ATP6V0A4 gene locus. In conclusion, a4 expression identifies subtypes of oligodendrogliomas, pilocytic astrocytomas and gangliogliomas and may contribute to refine characterization of these tumors. © 2012 Wiley Periodicals, Inc.