scholarly journals Thy-1 Is a Component Common to Multiple Populations of Synaptic Vesicles

1998 ◽  
Vol 140 (3) ◽  
pp. 685-698 ◽  
Author(s):  
Chung-Jiuan Jeng ◽  
Steven A. McCarroll ◽  
Thomas F. J. Martin ◽  
Erik Floor ◽  
James Adams ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Plasma Membrane ◽  
Membrane Protein ◽  
Pc12 Cells ◽  
Synaptic Vesicles ◽  
Integral Membrane Protein ◽  
Immunoglobulin Superfamily ◽  
Secretory Vesicles ◽  
Vesicular Release

Thy-1, a glycosylphosphatidylinositol-linked integral membrane protein of the immunoglobulin superfamily, is a component of both large dense-core and small clear vesicles in PC12 cells. A majority of this protein, formerly recognized only on the plasma membrane of neurons, is localized to regulated secretory vesicles. Thy-1 is also present in synaptic vesicles in rat central nervous system. Experiments on permeabilized PC12 cells demonstrate that antibodies against Thy-1 inhibit the regulated release of neurotransmitter; this inhibition appears to be independent of any effect on the Ca2+ channel. These findings suggest Thy-1 is an integral component of many types of regulated secretory vesicles, and plays an important role in the regulated vesicular release of neurotransmitter at the synapse.

scholarly journals Protein p38: an integral membrane protein specific for small vesicles of neurons and neuroendocrine cells.

1986 ◽  
Vol 103 (6) ◽  
pp. 2511-2527 ◽  
Author(s):  
F Navone ◽  
R Jahn ◽  
G Di Gioia ◽  
H Stukenbrok ◽  
P Greengard ◽  
...  
Keyword(s):  
Nervous System ◽  
Membrane Protein ◽  
Synaptic Vesicles ◽  
Secretory Granules ◽  
Light And Electron Microscopy ◽  
Immunoblot Analysis ◽  
Integral Membrane Protein ◽  
Neuroendocrine Cells ◽  
Synapsin I ◽  
Large Dense Core Vesicles

An intrinsic membrane protein of brain synaptic vesicles with Mr 38,000 (p38, synaptophysin) has recently been partially characterized (Jahn, R., W. Schiebler, C. Ouimet, and P. Greengard, 1985, Proc. Natl. Acad. Sci. USA, 83:4137-4141; Wiedenmann, B., and W. W. Franke, 1985, Cell, 41:1017-1028). We have now studied the presence of p38 in a variety of tissues by light and electron microscopy immunocytochemistry and by immunochemistry. Our results indicate that, within the nervous system, p38, like the neuron-specific phosphoprotein synapsin I, is present in virtually all nerve terminals and is selectively associated with small synaptic vesicles (SSVs). No p38 was detectable on large dense-core vesicles (LDCVs). p38 and synapsin I were found to be present in similar concentrations throughout the brain. Outside the nervous system, p38 was found in a variety of neuroendocrine cells, but not in any other cell type. In neuroendocrine cells p38 was localized on a pleiomorphic population of small, smooth-surfaced vesicles, which were interspersed among secretory granules and concentrated in the Golgi area, but not on the secretory granules themselves. Immunoblot analysis of endocrine tissues and cell lines revealed a band with a mobility slightly different from that of neuronal p38. This difference was attributable to a difference in glycosylation. The finding that p38, like synapsin I, is a component of SSVs of virtually all neurons, but not of LDCVs, supports the idea that SSVs and LDCVs are organelles of two distinct pathways for regulated neuronal secretion. In addition, our results indicate the presence in a variety of neuroendocrine cells of an endomembrane system, which is related to SSVs of neurons but is distinct from secretory granules.

scholarly journals Inhibition of regulated catecholamine secretion from PC12 cells by the Ca2+/calmodulin kinase II inhibitor KN-62

1995 ◽  
Vol 108 (7) ◽  
pp. 2619-2628 ◽  
Author(s):  
E.S. Schweitzer ◽  
M.J. Sanderson ◽  
C.G. Wasterlain
Keyword(s):  
Plasma Membrane ◽  
Pc12 Cells ◽  
Synaptic Vesicles ◽  
Large Fraction ◽  
Dense Core ◽  
Secretory Vesicles ◽  
Dependent Manner ◽  
Regulated Secretion ◽  
Kinase C ◽  
Calmodulin Kinase

When stimulated by the cholinergic agonist carbachol, PC12 cells rapidly secrete a large fraction of the intracellular catecholamines by exocytotic release from the large dense-core secretory vesicles in a Ca(2+)-dependent manner. To investigate whether Ca2+/calmodulin kinase II plays a role in the regulated secretion of catecholamines, we examined the effect of the specific Ca2+/calmodulin kinase II inhibitor KN-62 on the carbachol-induced release of norepinephrine from PC12 cells. Approximately 50% of the regulated release of norepinephrine, stimulated either by carbachol or direct depolarization, was inhibited by pretreatment with KN-62, while the remaining 50% was resistant to KN-62 and therefore independent of Ca2+/calmodulin kinase II. In contrast, H7, an inhibitor of protein kinase C, had no effect on any of the stimulated release. FURA 2 imaging experiments demonstrated that KN-62 does not act by blocking the stimulation-induced increase in intracellular [Ca2+]. The most likely model consistent with these data is that all the dense-core vesicles fuse with the plasma membrane in a Ca(2+)-dependent process, but that approximately 50% of the vesicles require an additional step that is dependent on the action of Ca2+/calmodulin kinase II. This step occurs between the influx of Ca2+ and the fusion of vesicle membranes with the plasma membrane, and may be analogous to the Ca2+/calmodulin kinase II phosphorylation of synapsin which mobilizes small, clear synaptic vesicles for exocytosis at the synapse.

scholarly journals Synaptophysin binds to physophilin, a putative synaptic plasma membrane protein.

1990 ◽  
Vol 111 (5) ◽  
pp. 2041-2052 ◽  
Author(s):  
L Thomas ◽  
H Betz
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Synaptic Vesicle ◽  
Synaptic Vesicles ◽  
Binding Protein ◽  
Primary Cultures ◽  
Specific Interaction ◽  
Integral Membrane Protein ◽  
Synaptic Plasma Membrane ◽  
Plasma Membrane Protein

We have developed procedures for detecting synaptic vesicle-binding proteins by using glutaraldehyde-fixed or native vesicle fractions as absorbent matrices. Both adsorbents identify a prominent synaptic vesicle-binding protein of 36 kD in rat brain synaptosomes and mouse brain primary cultures. The binding of this protein to synaptic vesicles is competed by synaptophysin, a major integral membrane protein of synaptic vesicles, with half-maximal inhibition seen between 10(-8) and 10(-7) M synaptophysin. Because of its affinity for synaptophysin, we named the 36-kD synaptic vesicle-binding protein physophilin (psi nu sigma alpha, greek = bubble, vesicle; psi iota lambda os, greek = friend). Physophilin exhibits an isoelectric point of approximately 7.8, a Stokes radius of 6.6 nm, and an apparent sedimentation coefficient of 5.6 S, pointing to an oligomeric structure of this protein. It is present in synaptic plasma membranes prepared from synaptosomes but not in synaptic vesicles. In solubilization experiments, physophilin behaves as an integral membrane protein. Thus, a putative synaptic plasma membrane protein exhibits a specific interaction with one of the major membrane proteins of synaptic vesicles. This interaction may play a role in docking and/or fusion of synaptic vesicles to the presynaptic plasma membrane.

Substance P: localization in synaptic vesicles in rat central nervous system

1977 ◽  
Vol 29 (4) ◽  
pp. 747-751 ◽  
Author(s):  
A. C. CUELLO ◽  
T. M. JESSELL ◽  
I. KANAZAWA ◽  
L. L. IVERSEN
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Substance P ◽  
Synaptic Vesicles

Lipocortin 1 (annexin 1) in patches associated with the membrane of a lung adenocarcinoma cell line and in the cell cytoplasm

1998 ◽  
Vol 111 (10) ◽  
pp. 1405-1418 ◽  
Author(s):  
V. Traverso ◽  
J.F. Morris ◽  
R.J. Flower ◽  
J. Buckingham
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Lung Adenocarcinoma ◽  
Western Blotting ◽  
Signal Sequence ◽  
Membrane Surface ◽  
Integral Membrane Protein ◽  
Subcellular Fractionation ◽  
Cell Fractionation ◽  
Electron Microscopic

Lipocortin 1 (annexin I) is a calcium- and phospholipid-binding annexin protein which can be externalised from cells despite the lack of a signal sequence. To determine its cellular distribution lipocortin 1 in A549 human lung adenocarcinoma cells was localised by light- and electron-microscopic immunocytochemistry and by cell fractionation and western blotting. Lipocortin 1 immunoreactivity is concentrated in prominent patches associated with the plasma membrane. The intensity of these patches varied with the confluence and duration of the culture and was not detectably diminished by an EDTA wash before fixation. Tubulin and cytokeratin 8 were colocalized with lipocortin 1 in the patches. Within the cells lipocortin 1 was distributed throughout the cytoplasm. Electron microscopy revealed prominent immunoreactivity along the plasma membrane with occasional large clusters of gold particles in contact with the membrane surface of the cells; within the cytoplasm the membrane of some vesicle/vacuole structures and some small electron-dense bodies was immunoreactive, but no immunogold particles were associated with the multilamellar bodies. Subcellular fractionation, extraction and western blotting showed that lipocortin 1 in the membrane pellet was present as two distinct fractions; one, intimately associated with the lipid bilayer, which behaved like an integral membrane protein and one loosely attached which behaved like a peripheral membrane protein. The results show that a substantial amounts of lipocortin 1 is concentrated in focal structures associated with and immediately beneath the plasma membrane. These might form part of the mechanism by which lipocortin 1 is released from the cells.

scholarly journals Interaction Profiles of Central Nervous System Active Drugs at Human Organic Cation Transporters 1–3 and Human Plasma Membrane Monoamine Transporter

2021 ◽  
Vol 22 (23) ◽  
pp. 12995
Author(s):  
Thomas J. F. Angenoorth ◽  
Stevan Stankovic ◽  
Marco Niello ◽  
Marion Holy ◽  
Simon D. Brandt ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Plasma Membrane ◽  
Human Plasma ◽  
Organic Cation ◽  
Hek293 Cells ◽  
Monoamine Transporters ◽  
Organic Cation Transporters ◽  
Monoamine Transporter ◽  
Cation Transporters

Many psychoactive compounds have been shown to primarily interact with high-affinity and low-capacity solute carrier 6 (SLC6) monoamine transporters for norepinephrine (NET; norepinephrine transporter), dopamine (DAT; dopamine transporter) and serotonin (SERT; serotonin transporter). Previous studies indicate an overlap between the inhibitory capacities of substances at SLC6 and SLC22 human organic cation transporters (SLC22A1–3; hOCT1–3) and the human plasma membrane monoamine transporter (SLC29A4; hPMAT), which can be classified as high-capacity, low-affinity monoamine transporters. However, interactions between central nervous system active substances, the OCTs, and the functionally-related PMAT have largely been understudied. Herein, we report data from 17 psychoactive substances interacting with the SLC6 monoamine transporters, concerning their potential to interact with the human OCT isoforms and hPMAT by utilizing radiotracer-based in vitro uptake inhibition assays at stably expressing human embryonic kidney 293 cells (HEK293) cells. Many compounds inhibit substrate uptake by hOCT1 and hOCT2 in the low micromolar range, whereas only a few substances interact with hOCT3 and hPMAT. Interestingly, methylphenidate and ketamine selectively interact with hOCT1 or hOCT2, respectively. Additionally, 3,4-methylenedioxymethamphetamine (MDMA) is a potent inhibitor of hOCT1 and 2 and hPMAT. Enantiospecific differences of R- and S-α-pyrrolidinovalerophenone (R- and S-α-PVP) and R- and S-citalopram and the effects of aromatic substituents are explored. Our results highlight the significance of investigating drug interactions with hOCTs and hPMAT, due to their role in regulating monoamine concentrations and xenobiotic clearance.

scholarly journals Analysis of the Antimalarial Drug Resistance Protein Pfcrt Expressed in Yeast

2002 ◽  
Vol 277 (51) ◽  
pp. 49767-49775 ◽  
Author(s):  
Hanbang Zhang ◽  
Ellen M. Howard ◽  
Paul D. Roepe
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Membrane Vesicle ◽  
Membrane Vesicles ◽  
Integral Membrane Protein ◽  
Chloroquine Resistance ◽  
Malarial Parasite ◽  
Plasma Membrane Vesicle ◽  
Wild Type ◽  
Stem Loop

Mutations in the novel membrane protein Pfcrt were recently found to be essential for chloroquine resistance (CQR) inPlasmodium falciparum, the parasite responsible for most lethal human malaria (Fidock, D. A., Nomura, T., Talley, A. K., Cooper, R. A., Dzekunov, S. M., Ferdig, M. T., Ursos, L. M., Sidhu, A. B., Naude, B., Deitsch, K. W., Su, X. Z., Wootton, J. C., Roepe, P. D., and Wellems, T. E. (2000)Mol. Cell6, 861–871). Pfcrt is localized to the digestive vacuolar membrane of the intraerythrocytic parasite and may function as a transporter. Study of this putative transport function would be greatly assisted by overexpression in yeast followed by characterization of membrane vesicles. Unfortunately, the very high AT content of malarial genes precludes efficient heterologous expression. Thus, we back-translated Pfcrt to design idealized genes with preferred yeast codons, no long poly(A) sequences, and minimal stem-loop structure. We synthesized a designed gene with a two-step PCR method, fused this to N- and C-terminal sequences to aid membrane insertion and purification, and now report efficient expression of wild type and mutant Pfcrt proteins in the plasma membrane ofSaccharomyces cerevisiaeandPichia pastorisyeast. To our knowledge, this is the first successful expression of a full-length malarial parasite integral membrane protein in yeast. Purified membranes and inside-out plasma membrane vesicle preparations were used to analyze wild typeversusCQR-conferring mutant Pfcrt function, which may include effects on H+transport (Dzekunov, S., Ursos, L. M. B., and Roepe, P. D. (2000)Mol. Biochem. Parasitol.110, 107–124), and to perfect a rapid purification of biotinylated Pfcrt. These data expand on the role of Pfcrt in conferring CQR and define a productive route for analysis of importantP. falciparumtransport proteins and membrane associated vaccine candidates.

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