José Márcio Silva Barbosa

Trafficking of the vesicular acetylcholine transporter in SN56 cells: a dynamin-sensitive step and interaction with the AP-2 adaptor complex.

The pathways by which synaptic vesicle proteins reach their destination are not completely defined. Here we investigated the traffic of a green fluorescent protein (GFP)‐tagged version of the vesicular acetylcholine transporter (VAChT) in cholinergic SN56 cells, a model system for neuronal processing of this cargo. GFP‐VAChT accumulates in small vesicular compartments in varicosities, but perturbation of endocytosis with a dominant negative mutant of dynamin I‐K44A impaired GFP‐VAChT trafficking to these processes. The protein in this condition accumulated in the cell body plasma membrane and in large vesicular patches therein. A VAChT endocytic mutant (L485A/L486A) was also located at the plasma membrane, however, the protein was not sorted to dynamin I‐K44A generated vesicles. A fusion protein containing the VAChT C‐terminal tail precipitated the AP‐2 adaptor protein complex from rat brain, suggesting that VAChT directly interacts with the endocytic complex. In addition, yeast two hybrid experiments indicated that the C‐terminal tail of VAChT interacts with the µ subunit of AP‐2 in a di‐leucine (L485A/L486A) dependent fashion. These observations suggest that the di‐leucine motif regulates sorting of VAChT from the soma plasma membrane through a clathrin dependent mechanism prior to the targeting of the transporter to varicosities.

Effect of Protein Kinase C Activation on the Release of [3H]Acetylcholine in the Presence of Vesamicol

Abstract: The present work tested whether pharmacological activation of protein kinase C (PKC) influences the release of [3H]‐acetylcholine ([3H]ACh) synthesized in the presence of vesamicol, an inhibitor of the vesicular acetylcholine transporter (VAChT). Newly synthesized [3H]ACh was released from hippocampal slices by field stimulation (15 Hz) in the absence of vesamicol, but as expected [3H]ACh synthesized during exposure to vesamicol was not released significantly by stimulation. Treatment of slices with the PKC activator phorbol myristate acetate (PMA) decreased the inhibitory effect of vesamicol on [3H]ACh release. The effect of PMA was dose‐dependent, was sensitive to calphostin C, a PKC‐selective inhibitor, and could not be mimicked by α‐PMA, an inactive phorbol ester. PMA did not alter the release of [3H]ACh in the absence of vesamicol, suggesting that the site of PKC action could be related to the VAChT. In agreement with this observation, immunoprecipitation of VAChT from 32P‐labeled synaptosomes showed that phosphorylation occurs and that incorporation of 32P in the VAChT protein increases in the presence of PMA. We suggest that PKC alters the output of [3H]ACh formed in the presence of vesamicol and also provide circumstantial evidence for a role of phosphorylation of VAChT in this process.

Trafficking of green fluorescent protein tagged-vesicular acetylcholine transporter to varicosities in a cholinergic cell line: VAChT traffic in living cells

Synaptic vesicle proteins are suggested to travel from the trans‐Golgi network to active zones via tubulovesicular organelles, but the participation of different populations of endosomes in trafficking remains a matter of debate. Therefore, we generated a green fluorescent protein (GFP)‐tagged version of the vesicular acetylcholine transporter (VAChT) and studied the localization of VAChT in organelles in the cell body and varicosities of living cholinergic cells. GFP–VAChT is distributed to both early and recycling endosomes in the cell body and is also observed to accumulate in endocytic organelles within varicosities of SN56 cells. GFP–VAChT positive organelles in varicosities are localized close to plasma membrane and are labeled with FM4‐64 and GFP–Rab5, markers of endocytic vesicles and early endosomes, respectively. A GFP–VAChT mutant lacking a dileucine endocytosis motif (leucine residues 485 and 486 changed to alanine residues) accumulated at the plasma membrane in SN56 cells. This endocytosis‐defective GFP–VAChT mutant is localized primarily at the somal plasma membrane and exhibits reduced neuritic targeting. Furthermore, the VAChT mutant did not accumulate in varicosities, as did VAChT. Our data suggest that clathrin‐mediated internalization of VAChT to endosomes at the cell body might be involved in proper sorting and trafficking of VAChT to varicosities. We conclude that genesis of competent cholinergic secretory vesicles depends on multiple interactions of VAChT with endocytic proteins.

Structural requirements for steady-state localization of the vesicular acetylcholine transporter

The vesicular acetylcholine transporter (VAChT) regulates the amount of acetylcholine stored in synaptic vesicles. However, the mechanisms that control the targeting of VAChT and other synaptic vesicle proteins are still poorly comprehended. These processes are likely to depend, at least partially, on structural determinants present in the primary sequence of the protein. Here, we use site‐directed mutagenesis to evaluate the contribution of the C‐terminal tail of VAChT to the targeting of this transporter to synaptic‐like microvesicles in cholinergic SN56 cells. We found that residues 481–490 contain the trafficking information necessary for VAChT localization and that within this region L485 and L486 are strictly necessary. Deletion and alanine‐scanning mutants lacking most of the carboxyl tail of VAChT, but containing residues 481–490, were still targeted to microvesicles. Moreover, we found that clathrin‐mediated endocytosis of VAChT is required for targeting to microvesicles in SN56 and PC12 cells. The data provide novel information on the mechanisms and structural determinants necessary for VAChT localization to synaptic vesicles.

Halothane-induced intracellular calcium release in cholinergic cells

Low concentrations of halothane and isoflurane can release acetylcholine in an extracellular Ca(2+)-independent manner. In the present study, a cholinergic cell line (SN56) was used to examine whether release of calcium from intracellular stores occurs in the presence of halothane. Changes in intracellular calcium concentration ([Ca(2+)](i)) were measured using fluo-3, a fluorescent calcium-sensitive dye and laser scanning confocal microscopy. Halothane, at sub-anesthetic concentrations (14, 28, 40 and 56 microM), increased [Ca(2+)](i) in SN56 cells. This effect remained even when the cells were perfused with medium lacking extracellular calcium, suggesting the involvement of intracellular Ca(2+) sources. SN56 cells responded to ryanodine by increasing [Ca(2+)](i) and this effect was blocked by dantrolene, an inhibitor of Ca(2+)-release from ryanodine-sensitive stores. The effect of halothane was attenuated after the increase in [Ca(2+)](i) induced by ryanodine and it was suppressed by dantrolene, suggesting the participation of ryanodine-sensitive stores. Using cyclopiazonic acid, a Ca(2+)-ATPase inhibitor, we investigated whether the depletion of intracellular Ca(2+) stores interfered with the effect of halothane. Cyclopiazonic acid significantly decreased the increase in [Ca(2+)](i) induced by the volatile anesthetic. It is suggested that sub-anesthetic concentrations of halothane may increase [Ca(2+)](i) by releasing Ca(2+) from intracellular stores in cholinergic cells.

Visualization and trafficking of the vesicular acetylcholine transporter in living cholinergic cells

Abstract: The present experiments investigated the trafficking of the vesicular acetylcholine transporter (VAChT) tagged with the enhanced green fluorescent protein (EGFP) in living cholinergic cells (SN56). The EGFP‐VAChT chimera was located in endosomal‐like compartments in the soma of SN56 cells, and it was also targeted to varicosities of neurites. In contrast, EGFP alone in cells was soluble in the cytoplasm. The C‐terminal cytoplasmic tail of VAChT has been implicated in targeting of VAChT to synaptic vesicles; thus, we have examined the role of the C‐terminal region in the trafficking to varicosities. A C‐terminal fragment tagged with EGFP appeared to be selectively accumulated in varicosities when expressed in SN56 cells. Interestingly, the protein was not freely soluble in the cytosol, and it presented a punctate pattern of expression. However, EGFP‐C terminus did not present this peculiar pattern of expression in a nonneuronal cell line (HEK 293). Moreover, the C‐terminal region of VAChT did not seem to be essential for VAChT trafficking, as a construct that lacks the C‐terminal tail was, similar to EGFP‐VAChT, partially targeted to endocytic organelles in the soma and sorted to varicosities. These experiments visualize VAChT for the first time in living cells and suggest that there might be multiple signals that participate in trafficking of VAChT to sites of synaptic vesicle accumulation.

Translocation of protein kinase C by halothane in cholinergic cells

Protein kinase C (PKC) is a signal transducing enzyme that is an important regulator of multiple physiologic processes and a potential molecular target for volatile anaesthetic actions. However, the effects of these agents on PKC activity are not yet fully understood. Volatile anaesthetics increase intracellular calcium concentration ([Ca(2+)](i)) in a variety of cells, thus their effects on PKC activity may be indirect due to [Ca(2+)](i) increase. Alternatively, the anaesthetics could directly stimulate PKC activity. In order to distinguish these two possibilities in intact cells, we used a fully functional green fluorescent protein conjugated PKCbetaII (GFP-PKCbetaII) and confocal microscopy to evaluate the dynamic redistribution of PKC in living SN56 cells, a cholinergic cell line, in response to halothane. Halothane induced PKC translocation in SN56 cells transfected with GFP-PKCbetaII. This effect was not suppressed by dantrolene, a drug that blocks halothane-induced Ca(2+) release from intracellular stores in these cells. These findings indicate that halothane induces PKC translocation in SN56 cells independently of its ability to release calcium from internal stores.

Visualization of the Vesicular Acetylcholine Transporter in Living Cholinergic Cells

We regret that we must retract the article Visualization of the Vesicular Acetylcholine Transporter in Living Cholinergic Cells by M. S. Santos, J. Barbosa Jr., C. Kushmerick, M. V. Gomez, V. F. Prado, and M. A. M. Prado (J. Neurochem. 74, 2425‐2435, 2000). We have made an unintentional mistake in the construction of the enhanced green fluorescent protein (EGFP) vectors, and consequently the vesicular acetylcholine transporter (VAChT) and its truncated forms are incorrectly expressed. We have repeated the key experiments with proper constructs and have found that the expression pattern is clearly different from that reported in the article. The truncated form of VAChT, without the C‐terminal tail, presents a distinct pattern of expression when compared to VAChT, and we have found no evidence that the C‐terminal tail of VAChT is able to drive EGFP to varicosity sites. As a consequence of this problem, our earlier conclusions were incorrect. We apologize for this mistake and for any problems that we may have caused.