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1.
Mol Cell ; 71(6): 1092-1104.e5, 2018 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-30174291

RESUMO

Activation of class I phosphatidylinositol 3-kinase (PI3K) leads to formation of phosphatidylinositol-3,4,5-trisphophate (PIP3) and phosphatidylinositol-3,4-bisphophate (PI34P2), which spatiotemporally coordinate and regulate a myriad of cellular processes. By simultaneous quantitative imaging of PIP3 and PI34P2 in live cells, we here show that they have a distinctively different spatiotemporal distribution and history in response to growth factor stimulation, which allows them to selectively induce the membrane recruitment and activation of Akt isoforms. PI34P2 selectively activates Akt2 at both the plasma membrane and early endosomes, whereas PIP3 selectively stimulates Akt1 and Akt3 exclusively at the plasma membrane. These spatiotemporally distinct activation patterns of Akt isoforms provide a mechanism for their differential regulation of downstream signaling molecules. Collectively, our studies show that different spatiotemporal dynamics of PIP3 and PI34P2 and their ability to selectively activate key signaling proteins allow them to mediate class I PI3K signaling pathways in a spatiotemporally specific manner.


Assuntos
Imagem Óptica/métodos , Fosfatos de Fosfatidilinositol/fisiologia , Imagem Individual de Molécula/métodos , Animais , Linhagem Celular , Membrana Celular , Humanos , Fosfatos de Inositol , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositol 3-Quinases/fisiologia , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilinositóis , Isoformas de Proteínas , Transporte Proteico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais
2.
J Neurosci ; 43(21): 3807-3824, 2023 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-37185099

RESUMO

Sphingosine-1-phosphate (S1P), a bioactive sphingolipid concentrated in the brain, is essential for normal brain functions, such as learning and memory and feeding behaviors. Sphingosine kinase 1 (SphK1), the primary kinase responsible for S1P production in the brain, is abundant within presynaptic terminals, indicating a potential role of the SphK1/S1P axis in presynaptic physiology. Altered S1P levels have been highlighted in many neurologic diseases with endocytic malfunctions. However, it remains unknown whether the SphK1/S1P axis may regulate synaptic vesicle endocytosis in neurons. The present study evaluates potential functions of the SphK1/S1P axis in synaptic vesicle endocytosis by determining effects of a dominant negative catalytically inactive SphK1. Our data for the first time identify a critical role of the SphK1/S1P axis in endocytosis in both neuroendocrine chromaffin cells and neurons from mice of both sexes. Furthermore, our Ca2+ imaging data indicate that the SphK1/S1P axis may be important for presynaptic Ca2+ increases during prolonged stimulations by regulating the Ca2+ permeable TRPC5 channels, which per se regulate synaptic vesicle endocytosis. Collectively, our data point out a critical role of the regulation of TRPC5 by the SphK1/S1P axis in synaptic vesicle endocytosis.SIGNIFICANCE STATEMENT Sphingosine kinase 1 (SphK1), the primary kinase responsible for brain sphingosine-1-phosphate (S1P) production, is abundant within presynaptic terminals. Altered SphK1/S1P metabolisms has been highlighted in many neurologic disorders with defective synaptic vesicle endocytosis. However, whether the SphK1/S1P axis may regulate synaptic vesicle endocytosis is unknown. Here, we identify that the SphK1/S1P axis regulates the kinetics of synaptic vesicle endocytosis in neurons, in addition to controlling fission-pore duration during single vesicle endocytosis in neuroendocrine chromaffin cells. The regulation of the SphK1/S1P axis in synaptic vesicle endocytosis is specific since it has a distinguished signaling pathway, which involves regulation of Ca2+ influx via TRPC5 channels. This discovery may provide novel mechanistic implications for the SphK1/S1P axis in brain functions under physiological and pathologic conditions.


Assuntos
Fosfotransferases (Aceptor do Grupo Álcool) , Vesículas Sinápticas , Masculino , Feminino , Camundongos , Animais , Vesículas Sinápticas/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Esfingosina/metabolismo , Endocitose , Lisofosfolipídeos/metabolismo , Canais de Cátion TRPC
3.
J Proteome Res ; 23(8): 3174-3187, 2024 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-38686625

RESUMO

NPC intracellular cholesterol transporter 1 (NPC1) is a multipass, transmembrane glycoprotein mostly recognized for its key role in facilitating cholesterol efflux. Mutations in the NPC1 gene result in Niemann-Pick disease, type C (NPC), a fatal, lysosomal storage disease. Due to the progressively expanding implications of NPC1-related disorders, we investigated endogenous NPC1 protein-protein interactions in the mouse cortex and human-derived iPSCs neuronal models of the disease through coimmunoprecipitation-coupled with LC-MS based proteomics. The current study investigated protein-protein interactions specific to the wild-type and the most prevalent NPC1 mutation (NPC1I1061T) while filtering out any protein interactor identified in the Npc1-/- mouse model. Additionally, the results were matched across the two species to map the parallel interactome of wild-type and mutant NPC1I1061T. Most of the identified wild-type NPC1 interactors were related to cytoskeleton organization, synaptic vesicle activity, and translation. We found many putative NPC1 interactors not previously reported, including two SCAR/WAVE complex proteins that regulate ARP 2/3 complex actin nucleation and multiple membrane proteins important for neuronal activity at synapse. Moreover, we identified proteins important in trafficking specific to wild-type and mutant NPC1I1061T. Together, the findings are essential for a comprehensive understanding of NPC1 biological functions in addition to its classical role in sterol efflux.


Assuntos
Córtex Cerebral , Proteína C1 de Niemann-Pick , Mapas de Interação de Proteínas , Animais , Córtex Cerebral/metabolismo , Camundongos , Humanos , Proteômica/métodos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Doença de Niemann-Pick Tipo C/metabolismo , Doença de Niemann-Pick Tipo C/genética , Mutação , Camundongos Knockout , Colesterol/metabolismo , Neurônios/metabolismo
4.
J Neurochem ; 152(1): 48-60, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31587282

RESUMO

Phosphatidylserine (PS), a negatively charged phospholipid present predominantly at the inner leaflet of the plasma membrane, has been widely implicated in many cellular processes including membrane trafficking. Along this line, PS has been demonstrated to be important for endocytosis, however, the involved mechanisms remain uncertain. By monitoring clathrin-mediated endocytosis (CME) of single vesicles in mouse chromaffin cells using cell-attached capacitance measurements that offer millisecond time resolution, we demonstrate in the present study that the fission-pore duration is reduced by PS addition, indicating a stimulatory role of PS in regulating the dynamics of vesicle fission during CME. Furthermore, our results show that the PS-mediated effect on the fission-pore duration is Ca2+ -dependent and abolished in the absence of synaptotagmin 1 (Syt1), implying that Syt1 is necessary for the stimulatory role of PS in vesicle fission during CME. Consistently, a Syt1 mutant with a defective PS-Syt1 interaction increases the fission-pore duration. Taken together, our study suggests that PS-Syt1 interaction may be critical in regulating fission dynamics during CME.


Assuntos
Células Cromafins/fisiologia , Vesículas Revestidas por Clatrina/fisiologia , Clatrina/fisiologia , Fosfatidilserinas/fisiologia , Animais , Células Cultivadas , Endocitose/fisiologia , Exocitose/fisiologia , Feminino , Técnicas de Inativação de Genes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Sinaptotagmina I/genética , Sinaptotagmina I/fisiologia
5.
J Neurochem ; 149(6): 729-746, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30963576

RESUMO

Sphingosine-1-phosphate (S1P) is an essential bioactive sphingosine lipid involved in many neurological disorders. Sphingosine kinase 1 (SphK1), a key enzyme for S1P production, is concentrated in presynaptic terminals. However, the role of S1P/SphK1 signaling in exocytosis remains elusive. By detecting catecholamine release from single vesicles in chromaffin cells, we show that a dominant negative SphK1 (SphK1DN ) reduces the number of amperometric spikes and increases the duration of foot, which reflects release through a fusion pore, implying critical roles for S1P in regulating the rate of exocytosis and fusion pore expansion. Similar phenotypes were observed in chromaffin cells obtained from SphK1 knockout mice compared to those from wild-type mice. In addition, extracellular S1P treatment increased the number of amperometric spikes, and this increase, in turn, was inhibited by a selective S1P3 receptor blocker, suggesting extracellular S1P may regulate the rate of exocytosis via activation of S1P3. Furthermore, intracellular S1P application induced a decrease in foot duration of amperometric spikes in control cells, indicating intracellular S1P may regulate fusion pore expansion during exocytosis. Taken together, our study represents the first demonstration that S1P regulates exocytosis through distinct mechanisms: extracellular S1P may modulate the rate of exocytosis via activation of S1P receptors while intracellular S1P may directly control fusion pore expansion during exocytosis. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.


Assuntos
Células Cromafins/metabolismo , Exocitose/fisiologia , Lisofosfolipídeos/metabolismo , Esfingosina/análogos & derivados , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Esfingosina/metabolismo
6.
J Cell Sci ; 128(12): 2229-35, 2015 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-25964652

RESUMO

Synaptotagmin 1 (Syt1) is a synaptic vesicle protein that is important for the kinetics of both exocytosis and endocytosis, and is thus a candidate molecule to link these two processes. Although the tandem Ca(2+)-binding C2 domains of Syt1 have important roles in exocytosis and endocytosis, the function of the conserved juxtamembrane (jxm) linker region has yet to be determined. We now demonstrate that the jxm region of Syt1 interacts directly with the pleckstrin homology (PH) domain of the endocytic protein dynamin 1. By using cell-attached capacitance recordings with millisecond time resolution to monitor clathrin-mediated endocytosis of single vesicles in neuroendocrine chromaffin cells, we find that loss of this interaction prolongs the lifetime of the fission pore leading to defects in the dynamics of vesicle fission. These results indicate a previously undescribed interaction between two major regulatory proteins in the secretory vesicle cycle and that this interaction regulates endocytosis.


Assuntos
Encéfalo/metabolismo , Células Cromafins/metabolismo , Dinamina I/metabolismo , Vesículas Sinápticas/fisiologia , Sinaptotagmina I/fisiologia , Sequência de Aminoácidos , Animais , Western Blotting , Encéfalo/citologia , Cálcio/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Células Cromafins/citologia , Clatrina/metabolismo , Endocitose/fisiologia , Exocitose/fisiologia , Feminino , Humanos , Imunoprecipitação , Masculino , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Domínios e Motivos de Interação entre Proteínas , Ratos , Homologia de Sequência de Aminoácidos , Sinapses/fisiologia
7.
J Neurosci ; 33(40): 15793-8, 2013 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-24089486

RESUMO

Actin polymerization is important for vesicle fission during clathrin-mediated endocytosis (CME), and it has been proposed that actin polymerization may promote vesicle fission during CME by providing direct mechanical forces. However, there is no direct evidence in support of this hypothesis. In the present study, the role of actin polymerization in vesicle fission was tested by analyzing the kinetics of the endocytic tubular membrane neck (the fission-pore) with cell-attached capacitance measurements to detect CME of single vesicles in a millisecond time resolution in mouse chromaffin cells. Inhibition in dynamin GTPase activity increased the fission-pore conductance (Gp), supporting the mechanical role of dynamin GTPase in vesicle fission. However, disruptions in actin polymerization did not alter the fission-pore conductance Gp, thus arguing against the force-generating role of actin polymerization in vesicle fission during CME. Similar to disruptions of actin polymerization, cholesterol depletion results in an increase in the fission-pore duration, indicating a role for cholesterol-dependent membrane reorganization in vesicle fission. Further experiments suggested that actin polymerization and cholesterol might function in vesicle fission during CME in the same pathway. Our results thus support a model in which actin polymerization promotes vesicle fission during CME by inducing cholesterol-dependent membrane reorganization.


Assuntos
Actinas/metabolismo , Clatrina/metabolismo , Endocitose/fisiologia , Endossomos/metabolismo , Animais , Membrana Celular/metabolismo , Células Cromafins/metabolismo , Dinaminas/metabolismo , Camundongos , Polimerização
8.
Nat Cell Biol ; 9(8): 915-22, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17643118

RESUMO

Release of charged neurotransmitter molecules through a narrow fusion pore requires charge compensation by other ions. It has been proposed that this may occur by ion flow from the cytosol through channels in the vesicle membrane, which would generate a net outward current. This hypothesis was tested in chromaffin cells using cell-attached patch amperometry that simultaneously measured catecholamine release from single vesicles and ionic current across the patch membrane. No detectable current was associated with catecholamine release indicating that <2% of cations, if any, enter the vesicle through its membrane. Instead, we show that flux of catecholamines through the fusion pore, measured as an amperometric foot signal, decreases when the extracellular cation concentration is reduced. The results reveal that the rate of transmitter release through the fusion pore is coupled to net Na+ influx through the fusion pore, as predicted by electrodiffusion theory applied to fusion-pore permeation, and suggest a prefusion rather than postfusion role for vesicular cation channels.


Assuntos
Catecolaminas/metabolismo , Cátions/metabolismo , Exocitose/fisiologia , Canais Iônicos/metabolismo , Fusão de Membrana/fisiologia , Vesículas Secretórias/metabolismo , Sódio/metabolismo , Animais , Transporte Biológico/fisiologia , Bovinos , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular , Células Cultivadas , Células Cromafins/citologia , Células Cromafins/metabolismo , Capacitância Elétrica , Técnicas de Patch-Clamp
9.
J Neurosci ; 32(11): 3778-85, 2012 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-22423098

RESUMO

The role of Ca²âº in synaptic vesicle endocytosis remains uncertain due to the diversity in various preparations where several forms of endocytosis may contribute variably in different conditions. Although recent studies have demonstrated that Ca²âº is important for clathrin-mediated endocytosis (CME), the mechanistic role of Ca²âº in CME remains to be elucidated. By monitoring CME of single vesicles in mouse chromaffin cells with cell-attached capacitance measurements that offer millisecond time resolution, we demonstrate that the dynamics of vesicle fission during CME is Ca²âº dependent but becomes Ca²âº independent in synaptotagmin 1 (Syt1) knock-out cells. Our results thus suggest that Syt1 is necessary for the Ca²âº dependence of CME.


Assuntos
Cálcio/metabolismo , Clatrina/fisiologia , Endocitose/fisiologia , Sinaptotagmina I/deficiência , Animais , Células Cultivadas , Células Cromafins/metabolismo , Feminino , Masculino , Camundongos , Camundongos Knockout
10.
J Biol Chem ; 285(37): 28708-14, 2010 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-20622009

RESUMO

The predominant pathway for phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P(2)) synthesis is thought to be phosphorylation of phosphatidylinositol 4-phosphate at the 5 position of the inositol ring by type I phosphatidylinositol phosphate kinases (PIPK): PIPKIalpha, PIPKIbeta, and PIPKIgamma. PIPKIgamma has been shown to play a role in PI(4,5)P(2) synthesis in brain, and the absence of PIPKIgamma is incompatible with postnatal life. Conversely, mice lacking PIPKIalpha or PIPKIbeta (isoforms are referred to according to the nomenclature of human PIPKIs) live to adulthood, although functional effects in specific cell types are observed. To determine the contribution of PIPKIalpha and PIPKIbeta to PI(4,5)P(2) synthesis in brain, we investigated the impact of disrupting multiple PIPKI genes. Our results show that a single allele of PIPKIgamma, in the absence of both PIPKIalpha and PIPKIbeta, can support life to adulthood. In addition, PIPKIalpha alone, but not PIPKIbeta alone, can support prenatal development, indicating an essential and partially overlapping function of PIPKIalpha and PIPKIgamma during embryogenesis. This is consistent with early embryonic expression of PIPKIalpha and PIPKIgamma but not of PIPKIbeta. PIPKIbeta expression in brain correlates with neuronal differentiation. The absence of PIPKIbeta does not impact embryonic development in the PIPKIgamma knock-out (KO) background but worsens the early postnatal phenotype of the PIPKIgamma KO (death occurs within minutes rather than hours). Analysis of PIP(2) in brain reveals that only the absence of PIPKIgamma significantly impacts its levels. Collectively, our results provide new evidence for the dominant importance of PIPKIgamma in mammals and imply that PIPKIalpha and PIPKIbeta function in the generation of specific PI(4,5)P(2) pools that, at least in brain, do not have a major impact on overall PI(4,5)P(2) levels.


Assuntos
Encéfalo/enzimologia , Diferenciação Celular , Embrião de Mamíferos/enzimologia , Neurônios/enzimologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositol 4,5-Difosfato/biossíntese , Animais , Encéfalo/embriologia , Química Encefálica/genética , Embrião de Mamíferos/embriologia , Desenvolvimento Embrionário/genética , Humanos , Camundongos , Camundongos Knockout , Neurônios/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 4,5-Difosfato/genética
11.
Proc Natl Acad Sci U S A ; 105(45): 17561-6, 2008 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-18987319

RESUMO

Endocytosis of postsynaptic AMPA receptors is a mechanism through which efficiency of neurotransmission is regulated. We have genetically tested the hypothesis that synaptojanin 1, a phosphoinositide phosphatase implicated in the endocytosis of synaptic vesicles presynaptically, may also function in the endocytosis of AMPA receptors postsynaptically. Electrophysiological recordings of cultured hippocampal neurons showed that miniature excitatory postsynaptic current amplitudes were larger in synaptojanin 1 knockout (KO) neurons because of an increase of surface-exposed AMPA receptors. This change did not represent an adaptive response to decreased presynaptic release in KO cultures and was rescued by the expression of wild type, but not catalytically inactive synaptojanin 1, in the postsynaptic neuron. NMDA-induced internalization of pHluorin-tagged AMPA receptors (GluR2) was impaired in KO neurons. These results reveal a function of synaptojanin 1 in constitutive and triggered internalization of AMPA receptors and thus indicate a role for phosphatidylinositol(4,5)-bisphosphate metabolism in the regulation of postsynaptic AMPA responses.


Assuntos
Endocitose/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Receptores de AMPA/metabolismo , Transmissão Sináptica/fisiologia , Análise de Variância , Animais , Eletrofisiologia , Proteínas de Fluorescência Verde , Hipocampo/metabolismo , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Fosfatidilinositol 4,5-Difosfato/metabolismo , Monoéster Fosfórico Hidrolases/genética
12.
Proc Natl Acad Sci U S A ; 105(40): 15388-92, 2008 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-18829435

RESUMO

Formation of a fusion pore between a vesicle and its target membrane is thought to involve the so-called SNARE protein complex. However, there is no mechanistic model explaining how the fusion pore is opened by conformational changes in the SNARE complex. It has been suggested that C-terminal zipping triggers fusion pore opening. A SNAP-25 mutant named SNAP-25Delta9 (lacking the last nine C-terminal residues) should lead to a less-tight C-terminal zipping. Single exocytotic events in chromaffin cells expressing this mutant were characterized by carbon fiber amperometry and cell-attached patch capacitance measurements. Cells expressing SNAP-25Delta9 displayed smaller amperometric "foot-current" currents, reduced fusion pore conductances, and lower fusion pore expansion rates. We propose that SNARE/lipid complexes form proteolipid fusion pores. Fusion pores involving the SNAP-25Delta9 mutant will be less tightly zipped and may lead to a longer fusion pore structure, consistent with the observed decrease of fusion pore conductance.


Assuntos
Fusão de Membrana , Proteína 25 Associada a Sinaptossoma/química , Proteína 25 Associada a Sinaptossoma/metabolismo , Animais , Sinalização do Cálcio , Bovinos , Células Cromafins/citologia , Células Cromafins/metabolismo , Exocitose , Cinética
13.
Elife ; 102021 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-34569930

RESUMO

Transient receptor potential melastatin 7 (TRPM7) contributes to a variety of physiological and pathological processes in many tissues and cells. With a widespread distribution in the nervous system, TRPM7 is involved in animal behaviors and neuronal death induced by ischemia. However, the physiological role of TRPM7 in central nervous system (CNS) neuron remains unclear. Here, we identify endocytic defects in neuroendocrine cells and neurons from TRPM7 knockout (KO) mice, indicating a role of TRPM7 in synaptic vesicle endocytosis. Our experiments further pinpoint the importance of TRPM7 as an ion channel in synaptic vesicle endocytosis. Ca2+ imaging detects a defect in presynaptic Ca2+ dynamics in TRPM7 KO neuron, suggesting an importance of Ca2+ influx via TRPM7 in synaptic vesicle endocytosis. Moreover, the short-term depression is enhanced in both excitatory and inhibitory synaptic transmissions from TRPM7 KO mice. Taken together, our data suggests that Ca2+ influx via TRPM7 may be critical for short-term plasticity of synaptic strength by regulating synaptic vesicle endocytosis in neurons.


Assuntos
Endocitose , Inibição Neural , Plasticidade Neuronal , Neurônios/metabolismo , Transmissão Sináptica , Vesículas Sinápticas/metabolismo , Canais de Cátion TRPM/metabolismo , Animais , Cálcio/metabolismo , Sinalização do Cálcio , Células Cromafins/metabolismo , Potenciais Pós-Sinápticos Excitadores , Feminino , Células HEK293 , Humanos , Potenciais Pós-Sinápticos Inibidores , Cinética , Masculino , Camundongos Knockout , Vesículas Sinápticas/genética , Canais de Cátion TRPM/genética
14.
J Am Soc Mass Spectrom ; 30(8): 1359-1367, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31140077

RESUMO

The study of membrane proteins, and in particular ion channels, is crucial to understanding cellular function. Mass spectrometry-based approaches including bottom-up strategies to study membrane proteins have been successful yet still can remain challenging. In this study, we sought to evaluate the phosphorylation patterns of the ion channel TRPM7 which is involved in a range of critical physiological functions. To overcome extraction obstacles associated with analyzing membrane proteins, we incorporated the use of 5% SDS solubilization coupled with SCAD and S-Trap digestion methods to eliminate detergent interference in downstream LC-MS/MS analysis. We found that the SCAD method was more efficient, yielding 84% of the overall identified proteins; however, the variability was greater than the S-Trap method. Using both methods together with TiO2 and Fe-NTA phospho-enrichment protocols, we successfully observed the phosphorylation pattern of TRPM7 in a transfected cell line. An average of 22 ± 6% of the TRPM7 amino acid sequence was observed. In addition to several previously reported phosphorylation sites, we identified six new phosphosites (S5, S233, S554, S824, T1265, and S1401), providing new targets for further functional analyses of TRPM7.


Assuntos
Fosfopeptídeos/análise , Proteínas Serina-Treonina Quinases/química , Canais de Cátion TRPM/química , Sequência de Aminoácidos , Cromatografia Líquida , Células HEK293 , Humanos , Fosforilação , Espectrometria de Massas em Tandem
15.
J Neurosci ; 23(13): 5835-45, 2003 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-12843288

RESUMO

Alterations in the cytosolic pool directly affect neurotransmitter synthesis and release and are suggested to be key factors in various neurodegenerative disorders. Although this cytosolic pool is the most metabolically active, it is miniscule compared with the amount of vesicular transmitter and has never been quantified separately. Here, we introduce intracellular patch electrochemistry (IPE), a technique that for the first time provides direct measurements of cytosolic oxidizable molecules in single mammalian cells. In amperometric mode, IPE detects total catechols, whereas in cyclic voltammetric mode, it preferentially measures catecholamines. In cultured chromaffin cells, the total cytosolic catechol concentration was 50-500 microm, of which approximately 10% were catecholamines. Reserpine, a vesicular monoamine transporter inhibitor, had no effect on the catecholamine pool but increased total catechols by fourfold to fivefold. Combined with pargyline, a monoamine oxidase inhibitor, reserpine increased catecholamine levels in the cytosol by approximately sixfold. Amphetamine induced a transient approximately fivefold accumulation of cytosolic catecholamines and a slow increase of total catechols. In cells incubated with 3,4-dihydroxy-L-phenylalanine (L-DOPA), catecholamines increased by approximately 2.5-fold and total catechols increased by approximately fourfold. Cytosolic catecholamines returned to control levels

Assuntos
Catecolaminas/metabolismo , Células Cromafins/metabolismo , Citosol/metabolismo , Eletroquímica/métodos , Inibidores da Captação Adrenérgica/farmacologia , Anfetamina/farmacologia , Animais , Calibragem , Catecóis/metabolismo , Bovinos , Células Cultivadas , Estimulantes do Sistema Nervoso Central/farmacologia , Células Cromafins/citologia , Células Cromafins/efeitos dos fármacos , Dopaminérgicos/farmacologia , Eletroquímica/instrumentação , Homeostase/efeitos dos fármacos , Homeostase/fisiologia , Levodopa/farmacologia , Microeletrodos , Inibidores da Monoaminoxidase/farmacologia , Pargilina/farmacologia , Técnicas de Patch-Clamp/métodos , Ratos , Reserpina/farmacologia
16.
J Neurosci ; 23(21): 7917-21, 2003 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-12944522

RESUMO

The number of transmitter molecules released in a quantal event can be regulated, and recent studies suggest that the modulation of quantal size is associated with corresponding changes in vesicle volume (Colliver et al., 2000; Pothos et al., 2002). If so, this could occur either by distension of the vesicle membrane or by incorporation and removal of vesicle membrane. We performed simultaneous measurements of vesicle membrane area and catecholamine release in individual quantal events from chromaffin cells using cell-attached patch amperometry. Cells were treated with reserpine, a vesicular monoamine transport blocker that decreases quantal size, or l-dopa, a catecholamine precursor that increases quantal size. We show that decrease and increase in quantal size are associated with a respective decrease and increase in vesicle membrane area. These results point to a novel mechanism of vesicle membrane dynamics by which vesicles physically change their membrane area in response to changes in transmitter content such that the intravesicular concentration of transmitter is maintained.


Assuntos
Células Cromafins/ultraestrutura , Grânulos Cromafim/química , Grânulos Cromafim/ultraestrutura , Vesículas Secretórias/química , Vesículas Secretórias/ultraestrutura , Animais , Catecolaminas/análise , Bovinos , Células Cultivadas , Células Cromafins/química , Células Cromafins/fisiologia , Grânulos Cromafim/efeitos dos fármacos , Exocitose , Membranas Intracelulares/ultraestrutura , Levodopa/farmacologia , Neurotransmissores/análise , Técnicas de Patch-Clamp , Reserpina/farmacologia , Vesículas Secretórias/efeitos dos fármacos
17.
Neurosci Lett ; 332(2): 141-5, 2002 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-12384230

RESUMO

Using inside-out configuration of patch clamp techniques, actin cytoskeleton modulation of large conductance calcium-activated potassium (BK(Ca)) channels was studied in hippocampal CA1 pyramidal neurons from adult rat. Disruption of actin filament after brief treatment with cytochalasin D (CD) markedly decreased activity of BK(Ca) channels and this inhibitory action persisted even after CD washout. This CD-produced decrease in BK(Ca) channel activity was characterized by a shortened open lifetime and reduced opening frequency. Disruption of actin filament with another toxin cytochalasin B also exerted similar effects as CD. Phalloidin, the actin filament stabilizer, had no significant effect on BK(Ca) channel activity, while pre-treatment with phalloidin could prevent the CD-induced inhibitory effect on channel activity. The present study demonstrates that disruption of actin cytoskeleton can inhibit BK(Ca) channel activity, suggesting that BK(Ca) channels in rat hippocampal CA1 pyramidal neurons may be directly modulated by dynamic assembly and disassembly of submembranous actin cytoskeleton.


Assuntos
Actinas/fisiologia , Citoesqueleto/fisiologia , Hipocampo/metabolismo , Neurônios/metabolismo , Canais de Potássio Cálcio-Ativados/fisiologia , Células Piramidais/metabolismo , Animais , Hipocampo/citologia , Cinética , Masculino , Potássio/metabolismo , Ratos , Ratos Wistar
18.
J Vis Exp ; (92): e52024, 2014 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-25408421

RESUMO

Neuronal transmission is an integral part of cellular communication within the brain. Depolarization of the presynaptic membrane leads to vesicle fusion known as exocytosis that mediates synaptic transmission. Subsequent retrieval of synaptic vesicles is necessary to generate new neurotransmitter-filled vesicles in a process identified as endocytosis. During exocytosis, fusing vesicle membranes will result in an increase in surface area and subsequent endocytosis results in a decrease in the surface area. Here, our lab demonstrates a basic introduction to cell-attached capacitance recordings of single endocytic events in the mouse adrenal chromaffin cell. This type of electrical recording is useful for high-resolution recordings of exocytosis and endocytosis at the single vesicle level. While this technique can detect both vesicle exocytosis and endocytosis, the focus of our lab is vesicle endocytosis. Moreover, this technique allows us to analyze the kinetics of single endocytic events. Here the methods for mouse adrenal gland tissue dissection, chromaffin cell culture, basic cell-attached techniques, and subsequent examples of individual traces measuring singular endocytic event are described.


Assuntos
Glândulas Suprarrenais/fisiologia , Técnicas de Cultura de Células/métodos , Células Cromafins/fisiologia , Técnicas de Patch-Clamp/métodos , Glândulas Suprarrenais/citologia , Glândulas Suprarrenais/cirurgia , Animais , Células Cromafins/citologia , Dissecação , Capacitância Elétrica , Camundongos
19.
Eur J Pharmacol ; 714(1-3): 249-53, 2013 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-23819912

RESUMO

Cordycepin (3'-deoxyadenosine) is the major bioactive component of Cordyceps militaris that has been widely used in oriental countries as a Traditional Chinese Medicine and healthy food for preventing early aging, improving physical performance and increasing lifespan. Cordyceps militaris extracts other than cordycepin have been reported to improve cognitive function. Although cordycepin is one of the most utilized Cordyceps militaris components, it remains unknown whether cordycepin could improve learning and memory. Here we investigated effects of cordycepin on learning and memory in healthy and ischemic mice using Y-maze test. We found that oral cordycepin administration at dose of 10 mg/kg significantly improved Y-maze learning performance both in healthy and ischemic mice. However, cordycepin at dose of 5 mg/kg enhanced Y-maze learning only in ischemic mice but not healthy mice. In this study, simultaneously, we found that orally administrated cordycepin significantly decreased the neuronal loss induced by ischemia in hippocampal CA1 and CA3 regions. Collectively, our results can provide valuable evidence that cordycepin may act as a nootropic product or potential clinical application in improving cognitive function of patients with ischemic stroke in the future.


Assuntos
Desoxiadenosinas/farmacologia , Aprendizagem em Labirinto/efeitos dos fármacos , Nootrópicos/farmacologia , Animais , Isquemia Encefálica/patologia , Isquemia Encefálica/fisiopatologia , Cognição/efeitos dos fármacos , Relação Dose-Resposta a Droga , Hipocampo/patologia , Masculino , Camundongos , Neurônios/efeitos dos fármacos , Neurônios/patologia
20.
Behav Brain Res ; 246: 111-5, 2013 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-23499702

RESUMO

Polygala tenuifolia root has been used to improve memory and cognitive function in Traditional Chinese Medicine for more than 2000 years. Since tenuigenin (TEN) is one of the most utilized P. tenuifolia root extracts, it is surprising there is no evidence for the effects of TEN on learning and memory so far. In the present study, we investigated the effects of TEN on learning and memory with Y-maze test in mice. We found that oral administration of 4mg/kg TEN significantly improved learning and memory in Y-maze task. Treatment with 4mg/kg TEN markedly reduced the acetylcholinesterase (AChE) activity and malondialdehyde (MDA) level, and increased superoxide dismutase (SOD) activity in hippocampus. In the electrophysiological test of hippocampal brain slice, 2µg/ml TEN perfusion substantially enhanced field excitatory postsynaptic potential (fEPSP) amplitude both in basic synaptic transmission and after high frequency stimulation (HFS) in Schaffer to CA1 pathway (Scha-CA1). These results indicate that TEN enhancing learning and memory may result from inhibiting AChE activity, improving antioxidation and enhancing synaptic plasticity in mice. Therefore, TEN shows promise as a potential nootropic product in improving learning and memory.


Assuntos
Medicamentos de Ervas Chinesas/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Aprendizagem em Labirinto/efeitos dos fármacos , Memória/efeitos dos fármacos , Nootrópicos/farmacologia , Acetilcolinesterase/metabolismo , Animais , Aprendizagem por Discriminação/efeitos dos fármacos , Relação Dose-Resposta a Droga , Estimulação Elétrica , Hipocampo/efeitos dos fármacos , Técnicas In Vitro , Masculino , Malondialdeído/metabolismo , Camundongos , Camundongos Endogâmicos , Vias Neurais/efeitos dos fármacos , Vias Neurais/fisiologia , Especificidade da Espécie , Superóxido Dismutase/metabolismo , Fatores de Tempo
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