whole-cell-recording-calcium-release-activated-calcium-crac-currents

Whole-Cell Recording of Calcium Release-Activated Calcium (CRAC) Currents in Human T Lymphocytes

In T lymphocytes, depletion of Ca2+ from the intracellular Ca2+ store leads to activation of plasmalemmal Ca2+ channels, called Calcium Release-Activated ...

The central nervous system is one of the main target organs in cyanide toxicity. In this study, primary cultures of chick embryonic neurons were used to characterize sodium cyanide (NaCN)-induced cell death and to investigate the mechanism of NaCN-mediated preconditioning. After treatment of the cells with 1mM NaCN for 1h followed by a NaCN-free incubation period of 23 h, we observed features of apoptosis such as a reduction in nuclear size, chromatin condensation and nuclear fragmentation as evaluated by nuclear staining with Hoechst 33258 and electron microscopy. In addition, NaCN-induced neurotoxicity was reduced by the protein synthesis inhibitor cycloheximide (CHX) suggesting an active type of cell death. Most of the neurons with condensed chromatin and a shrunken nuclei also showed membrane damage at a late stage. Mitochondrial membrane potential as well as the protein levels of Bcl-2 and Bcl-x(L) decreased 15-60 min and 1-3 h after the exposure to NaCN (1mM, 1h), respectively. Preconditioning caused by incubating chick neurons with 100 microM NaCN for 30 min followed by a NaCN-free interval of 24h significantly protected the neurons against subsequent NaCN (1mM, 1h)-induced damage. Preconditioning prevented NaCN-induced decrease in the mitochondrial membrane potential as well as in the protein levels of Bcl-2 and Bcl-x(L) suggesting that preconditioning-induced neuroprotection is mediated by preserving mitochondrial function.

Preconditioning-induced protection against cyanide-induced neurotoxicity is mediated by preserving mitochondrial function.

Vorkonditionierung-induzierten Schutz gegen Zyanid-induzierten Neurotoxizit&#xE4;t wird durch die Erhaltung der mitochondrialen Funktion vermittelt.

Use-dependent activation of protein kinase A (PKA) modulates transmitter release, contributing to synaptic plasticity. Snapin, a PKA substrate in neurons, associates with the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, and its phosphorylation leads to increased binding of synaptotagmin to the SNARE complex. We investigated the role of PKA-dependent phosphorylation of Snapin in hippocampal neurons. Overexpression of Snapin S50D, a mutant mimicking the phosphorylated state, resulted in a decreased number of readily releasable vesicles. In addition, both the release probability of individual vesicles and the depression rate during high-frequency stimulation were increased. Overexpression of Snapin S50A, a mutant that cannot be phosphorylated, did not alter the size of the pool or the probability of release. Furthermore, dialysis of Sp-cAMPS, a nonhydrolyzable analog of cAMP that will promote phosphorylation by PKA, also led to increased synaptic depression in cells overexpressing wild-type Snapin. These results establish Snapin as an important target of PKA in CNS synapses and indicate a role for Snapin in the plasticity of transmitter release.

Effects of PKA-mediated phosphorylation of Snapin on synaptic transmission in cultured hippocampal neurons.

Effekte von PKA-vermittelte Phosphorylierung von Snapin auf die synaptische &#xDC;bertragung in kultivierten hippocampalen Neuronen.

SV2 proteins are abundant synaptic vesicle proteins expressed in two major (SV2A and SV2B) and one minor (SV2C) isoform. SV2A and SV2B have been shown to be involved in the regulation of synaptic vesicle exocytosis. Previous studies found that SV2A, but not SV2B, can interact with the cytoplasmic domain of synaptotagmin 1, a Ca2+ sensor for synaptic vesicle exocytosis. To determine whether SV2B can interact with full-length synaptotagmin 1, we performed immunoprecipitations from brain protein extracts and found that SV2B interacts strongly with synaptotagmin 1 in a detergent-resistant, Ca2+ -independent manner. In contrast, an interaction between native SV2A and synaptotagmin 1 was not detectable under these conditions. The SV2B-synaptotagmin 1 complex also contained the synaptic t-SNARE proteins, syntaxin 1 and SNAP-25, suggesting that SV2B may participate in exocytosis by modulating the interaction of synaptotagmin 1 with t-SNARE proteins. Analysis of retinae in SV2B knock-out mice revealed a strong reduction in the level of synaptotagmin 1 in rod photoreceptor synapses, which are unique in that they express only the SV2B isoform. In contrast, other synaptic vesicle proteins were not affected by SV2B knock out, indicating a specific role for SV2B in the regulation of synaptotagmin 1 levels at certain synapses. These experiments suggest that the SV2B-synaptotagmin 1 complex is involved in the regulation of synaptotagmin 1 stability and/or trafficking. This study has demonstrated a new role of SV2B as a regulator of synaptotagmin 1 that is likely mediated by direct interaction of these two synaptic proteins.

SV2B regulates synaptotagmin 1 by direct interaction.

SV2B regelt Synaptotagmin 1 durch eine direkte Interaktion.

The mouse is an important model system for understanding the molecular basis of neuronal signaling and diseases of synaptic communication. However, the best-characterized retinal ribbon-style synapses are those of nonmammalian vertebrates. To remedy this situation, we asked whether it would be feasible to track synaptic vesicle dynamics in the isolated mouse rod bipolar cell using time-resolved capacitance measurements. The results demonstrate that membrane depolarization triggered an increase in membrane capacitance that was Ca(2+) dependent and restricted to the synaptic compartment, consistent with exocytosis. The amplitude of the capacitance response recorded from the easily accessible soma of an intact mouse rod bipolar cell was identical to that recorded directly from the small synaptic terminal, suggesting that in the carefully selected cohort of cells presented here, axonal resistance was not a significant barrier to current flow. This supposition was supported by the analysis of passive membrane properties and a comparison of membrane capacitance measurements in cells with and without synaptic terminals and reinforced by the lack of an effect of sine-wave frequency (200-1,600 Hz) on the measured capacitance increase. The magnitude of the capacitance response increased with Ca(2+) entry until a plateau was reached at a spatially averaged intraterminal calcium of about 600 nM. We interpret this plateau, nominally 30 fF, as corresponding to a releasable pool of synaptic vesicles. The robustness of this measure suggests that capacitance measurements may be used in the mouse rod bipolar cell to compare pool size across treatment conditions.

Capacitance measurements in the mouse rod bipolar cell identify a pool of releasable synaptic vesicles.

Die Maus ist ein wichtiges Modellsystem f&uuml;r das Verst&auml;ndnis der molekularen Basis neuronaler signalisieren und Krankheiten der synaptische Kommunikation. Die best-characterized Netzhaut Ribbon-Style-Synapsen sind jedoch diejenigen der Nonmammalian Wirbeltiere. Um hier Abhilfe zu schaffen, haben wir gefragt, ob es machbar w&auml;re, synaptische Vesikel Dynamik in der isolierten Maus Stab bipolare Zelle mit Kapazit&auml;tsmessungen Zeit aufgel&ouml;st zu verfolgen. Die Ergebnisse zeigen, dass die Membran-Depolarisierung eine Zunahme der Membran Kapazitiv ausgel&ouml;st, die Ca(2+) angewiesen und auf die synaptische Fach, mit Exozytose beschr&auml;nkt war. Die Amplitude der Kapazitiv-Antwort aus den leicht zug&auml;nglichen Soma einer intakten Maus Stab bipolare Zelle aufgenommen war identisch mit dem direkt aus dem kleinen synaptische Terminal, was darauf hindeutet, dass in der sorgf&auml;ltig ausgew&auml;hlten Kohorte von Zellen, die hier vorgestellten, axonale Widerstand kein ernsthaftes Hindernis f&uuml;r Stromfluss war aufgezeichnet. Diese Vermutung wurde unterst&uuml;tzt durch die Analyse der passiven Membran-Eigenschaften und einen Vergleich der Membran Kapazit&auml;tsmessungen in Zellen mit und ohne synaptische Klemmen und verst&auml;rkt durch das Fehlen eines Effekts der Sinus-Wellen-Frequenz (200-1.600 Hz) gemessene Kapazit&auml;t zunimmt. Das Ausma&szlig; der Kapazitiv Antwort mit Ca(2+) Eintrag erh&ouml;ht, bis ein Plateau, bei einer r&auml;umlich gemittelte intraterminal Kalzium von etwa 600 erreicht wurde nM. Wir interpretieren diese Hochebene, die nominell 30 fF, dass eine l&ouml;sbare Pool von synaptische Vesikel entsprechen. Die Robustheit dieser Ma&szlig;nahme schl&auml;gt Kapazit&auml;tsmessungen in der Maus Stab bipolare Zelle verwendet werden k&ouml;nnen, um die Gr&ouml;&szlig;e des Pufferpools Behandlung Bedingungen vergleichen.

Kapazit&auml;tsmessungen in der Maus Stab Bipolare Zellen identifizieren einen Pool von l&ouml;sbare synaptische Vesikel.

Mast cell degranulation is a highly regulated, calcium-dependent process, which is important for the acute release of inflammatory mediators during the course of many pathological conditions. We previously found that Synaptotagmin-2, a calcium sensor in neuronal exocytosis, was expressed in a mast cell line. We postulated that this protein may be involved in the control of mast cell-regulated exocytosis, and we generated Synaptotagmin-2 knock-out mice to test our hypothesis. Mast cells from this mutant animal conferred an abnormally decreased passive cutaneous anaphylaxis reaction on mast cell-deficient mice that correlated with a specific defect in mast cell-regulated exocytosis, leaving constitutive exocytosis and nonexocytic mast cell effector responses intact. This defect was not secondary to abnormalities in the development, maturation, migration, morphology, synthesis, and storage of inflammatory mediators, or intracellular calcium transients of the mast cells. Unlike neurons, the lack of Synaptotagmin-2 in mast cells was not associated with increased spontaneous exocytosis.

Synaptotagmin-2 controls regulated exocytosis but not other secretory responses of mast cells.

Mastzelle Degranulation ist ein stark regulierten, Kalzium-abh&auml;ngigen Prozess, der im Laufe der vielen pathologischen Bedingungen f&uuml;r die akute Freisetzung von entz&uuml;ndlichen Mediatoren wichtig ist. Vorher finden wir, dass Synaptotagmin-2, ein Kalzium-Sensor im neuronalen Exozytose in eine Mastzell-Linie ausgedr&uuml;ckt wurde. Wir postuliert, dass dieses Protein die Kontrolle der Mastzell-regulierten Exozytose beteiligt sein kann, und wir Synaptotagmin-2 Knock-out-M&auml;use generiert um unsere Hypothese zu testen. Mastzellen von dieser mutant Tier &uuml;bertragen eine abnorm verringerte passive kutane Anaphylaxie-Reaktion auf Mastzell-defizienten M&auml;usen, die mit bestimmten Mastzell-regulierten Exozytose, intakt konstitutive Exozytose und Nonexocytic Mastzelle Effektor Antworten korreliert. Dieser Fehler war nicht zu Abweichungen in der Entwicklung, Reifung, Migration, Morphologie, Synthese und Lagerung von entz&uuml;ndlichen Mediatoren oder intrazellul&auml;re Kalzium Transienten von den Mastzellen zweitrangig. Im Gegensatz zu Neuronen war das Fehlen von Synaptotagmin-2 in Mastzellen nicht erh&ouml;hte spontane Exozytose zugeordnet.

Synaptotagmin-2 Steuerelemente regulierten Exozytose aber nicht andere sekretorischen Antworten von Mastzellen.

Synaptic vesicle 2 (SV2) proteins, critical for proper nervous system function, are implicated in human epilepsy, yet little is known about their function. We demonstrate, using direct approaches, that loss of the major SV2 isoform in a central nervous system nerve terminal is associated with an elevation in both resting and evoked presynaptic Ca(2+) signals. This increase is essential for the expression of the SV2B(-/-) secretory phenotype, characterized by changes in synaptic vesicle dynamics, synaptic plasticity, and synaptic strength. Short-term reproduction of the Ca(2+) phenotype in wild-type nerve terminals reproduces almost all aspects of the SV2B(-/-) secretory phenotype, while rescue of the Ca(2+) phenotype in SV2B(-/-) neurons relieves every facet of the SV2B(-/-) secretory phenotype. Thus, SV2 controls key aspects of synaptic functionality via its ability to regulate presynaptic Ca(2+), suggesting a potential new target for therapeutic intervention in the treatment of epilepsy.

SV2 acts via presynaptic calcium to regulate neurotransmitter release.

Synaptische Vesikel 2 (SV2) Proteine, kritische f&uuml;r ordnungsgem&auml;&szlig;e Nervensystem Funktion in menschlichen Epilepsie verwickelt sind, doch ist wenig bekannt &uuml;ber ihre Funktion. Wir demonstrieren, direkte Ans&auml;tze, diesen Verlust der wichtigen SV2-Isoform in einem zentralen Nervensystems Nerve Terminal mit einer H&ouml;he in beiden ruhelosigkeit zugeordnet ist und pr&auml;synaptischen Ca(2+) Signale hervorgerufen. Dieser Anstieg ist wesentlich f&uuml;r den Ausdruck des SV2B(-/-) sekretorischen Ph&auml;notyps, zeichnet sich durch &Auml;nderungen in synaptische Vesikel Dynamik, synaptische Plastizit&auml;t und synaptische St&auml;rke. Kurzfristige Reproduktion des Ph&auml;notyps in Wildtyp Nervenenden Ca(2+) reproduziert fast alle Aspekte des SV2B(-/-) sekretorischen Ph&auml;notyps, w&auml;hrend Rettung des Ph&auml;notyps Ca(2+) in SV2B(-/-) Neuronen jede Facette des SV2B(-/-) sekretorischen Ph&auml;notyps entlastet. So steuert SV2 Schl&uuml;sselaspekte der synaptischen Funktionen &uuml;ber seine F&auml;higkeit zur Regulierung der pr&auml;synaptischen Ca(2+), was auf ein potenzielles neues Ziel f&uuml;r therapeutische Intervention bei der Behandlung von Epilepsie.

SV2 agiert &uuml;ber pr&auml;synaptische Kalzium, Neurotransmitter-Freisetzung zu regulieren.

CRAC channel-mediated Ca ( 2+) Â entry plays a crucial role in T lymphocyte activation. Activated T cells display enhanced Ca ( 2+) Â signaling compared with resting T cells; this is partially attributed to activation-induced upregulation of CRAC channel expression.Â OraiÂ andÂ StimÂ family genes encode CRAC channel structural elements and regulatory proteins, respectively, but studies of their expression in T cells have led to controversial results. We re-examined Orai and Stim gene expression in resting, activated, and Jurkat T cells. Levels ofÂ Orai1Â transcripts, encoding the human T cell CRAC channel subunit, were not significantly different between resting T and activated T cells. The total amount of allÂ OraiÂ transcripts was 2-fold higher in activated T cells than in resting T cells.Â Orai1Â and totalÂ OraiÂ transcript levels were significantly higher in Jurkat T cells than those in resting T cells.Â StimÂ expression did not vary significantly among cell types. Maximal whole-cell CRAC current amplitudes were 1.4-fold and 2.3-fold higher in activated and Jurkat T cells, respectively, than in resting T cells. Due to the small size of resting T cells, the surface CRAC channel density was 2.5-fold and 1.6-fold higher in resting T cells than in activated and Jurkat T cells, respectively. Predicted the rates of cytosolic Ca2+ elevation calculated using the average values of CRAC channel currents and cell volumes showed that < 2-fold increase in the functional CRAC channel expression level cannot account for the enhanced rate of store-operated Ca ( 2+) Â entry in activated T cells compared with resting T cells.

Density of functional Ca ( 2+) release-activated Ca ( 2+) (CRAC) channels declines after T-cell activation.

CRAC Kanal-vermittelte Ca (2 +) Eintrag spielt eine entscheidende Rolle in T-Lymphozyten-Aktivierung. Aktivierte T-Zellen anzeigen verbesserte Ca (2 +) signalisieren gegen&uuml;ber ruhen T-Zellen; Dies wird teilweise Aktivierung-induzierte Hochregulation von CRAC Kanal Ausdruck zugeschrieben.&nbsp;Orai und Stim Familie Gene kodieren CRAC Kanal Strukturelemente und regulatorischen Proteine, bzw., aber Studien &uuml;ber ihren Ausdruck in den T-Zellen zu umstrittenen Ergebnissen gef&uuml;hrt haben. Wir &uuml;berpr&uuml;ft Orai und Stim-Genexpression in ruhen, aktiviert, und Jurkat T Zellen. Ebenen der Orai1 Abschriften, Codierung der menschlichen T Zelle CRAC Kanal Untereinheit waren nicht signifikant verschieden zwischen ruhenden T und aktivierten T-Zellen. Der Gesamtbetrag der alle Orai Abschriften wurde 2-fach h&ouml;her aktivierten T-Zellen als in Ruhe T-Zellen.&nbsp;Orai1 und total Orai Abschrift Ebenen waren signifikant h&ouml;her in Jurkat T-Zellen als in Ruhe T-Zellen.&nbsp;STIM Ausdruck unter Zelltypen nicht erheblich variieren. Maximale gesamte-Zelle CRAC aktuelle Amplituden waren 1.4-fold und 2.3-fold in h&ouml;her aktiviert und Jurkat T-Zellen, bzw. als in Ruhe T-Zellen. Aufgrund der geringen Gr&ouml;&szlig;e der ruhelosigkeit T-Zellen die Oberfl&auml;chendichte des CRAC-Kanal war 2.5-fold und 1.6-fold h&ouml;her in Ruhe T-Zellen als in aktiviert und Jurkat T-Zellen, beziehungsweise. Prognostiziert die Preise der cytosolic Ca2 + H&ouml;he berechnet unter Verwendung der durchschnittlichen Werte CRAC Kanal Str&ouml;me und Zelle-B&auml;nde, die zeigten, dass &lt; 2-fold Zunahme der Funktionsebene des CRAC-Kanal-Ausdruck kann nicht die erh&ouml;hte Rate von Speicher-betrieben von Ca (2 +)-Eintrag im aktivierten T-Zellen, die ruhelosigkeit T-Zellen gegen&uuml;ber entfallen.

Article	Year
Preconditioning-induced protection against cyanide-induced neurotoxicity is mediated by preserving mitochondrial function. Neurochemistry international\| PubMed ID: 11792457	2002
Effects of PKA-mediated phosphorylation of Snapin on synaptic transmission in cultured hippocampal neurons. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 15269257	2004
SV2B regulates synaptotagmin 1 by direct interaction. The Journal of biological chemistry\| PubMed ID: 15466855	2004
Capacitance measurements in the mouse rod bipolar cell identify a pool of releasable synaptic vesicles. Journal of neurophysiology\| PubMed ID: 16914610	2006
Synaptotagmin-2 controls regulated exocytosis but not other secretory responses of mast cells. The Journal of biological chemistry\| PubMed ID: 19473977	2009
SV2 acts via presynaptic calcium to regulate neurotransmitter release. Neuron\| PubMed ID: 20620874	2010
Density of functional Ca ( 2+) release-activated Ca ( 2+) (CRAC) channels declines after T-cell activation. Channels (Austin, Tex.)\| PubMed ID: 22172731	2011

Pratima Thakur

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