Synaptotagmin oligomerization is essential for calcium control of regulated exocytosis

Regulated exocytosis, which underlies many intercellular signaling events, is a tightly controlled process often triggered by calcium ion(s) (Ca2+). Despite considerable insight into the central components involved, namely, the core fusion machinery [soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)] and the principal Ca2+ sensor [C2-domain proteins like synaptotagmin (Syt)], the molecular mechanism of Ca2+-dependent release has been unclear. Here, we report that the Ca2+-sensitive oligomers of Syt1, a conserved structural feature among several C2-domain proteins, play a critical role in orchestrating Ca2+-coupled vesicular release. This follows from pHluorin-based imaging of single-vesicle exocytosis in pheochromocytoma (PC12) cells showing that selective disruption of Syt1 oligomerization using a structure-directed mutation (F349A) dramatically increases the normally low levels of constitutive exocytosis to effectively occlude Ca2+-stimulated release. We propose a parsimonious model whereby Ca2+-sensitive oligomers of Syt (or a similar C2-domain protein) assembled at the site of docking physically block spontaneous fusion until disrupted by Ca2+ Our data further suggest Ca2+-coupled vesicular release is triggered by removal of the inhibition, rather than by direct activation of the fusion machinery

Loss of Sugar Detection by GLUT2 Affects Glucose Homeostasis in Mice

International audienceBACKGROUND: Mammals must sense the amount of sugar available to them and respond appropriately. For many years attention has focused on intracellular glucose sensing derived from glucose metabolism. Here, we studied the detection of extracellular glucose concentrations in vivo by invalidating the transduction pathway downstream from the transporter-detector GLUT2 and measured the physiological impact of this pathway. METHODOLOGY/PRINCIPAL FINDINGS: We produced mice that ubiquitously express the largest cytoplasmic loop of GLUT2, blocking glucose-mediated gene expression in vitro without affecting glucose metabolism. Impairment of GLUT2-mediated sugar detection transiently protected transgenic mice against starvation and streptozotocin-induced diabetes, suggesting that both low- and high-glucose concentrations were not detected. Transgenic mice favored lipid oxidation, and oral glucose was slowly cleared from blood due to low insulin production, despite massive urinary glucose excretion. Kidney adaptation was characterized by a lower rate of glucose reabsorption, whereas pancreatic adaptation was associated with a larger number of small islets. CONCLUSIONS/SIGNIFICANCE: Molecular invalidation of sugar sensing in GLUT2-loop transgenic mice changed multiple aspects of glucose homeostasis, highlighting by a top-down approach, the role of membrane glucose receptors as potential therapeutic targets

Rate-dependent Ca2+ signalling underlying the force-frequency response in rat ventricular myocytes: A coupled electromechanical modeling study

Rate-dependent effects on the Ca2+ sub-system in a rat ventricular myocyte are investigated. Here, we employ a deterministic mathematical model describing various Ca2+ signalling pathways under voltage clamp (VC) conditions, to better understand the important role of calmodulin (CaM) in modulating the key control variables Ca2+/calmodulin-dependent protein kinase-II (CaMKII), calcineurin (CaN), and cyclic adenosine monophosphate (cAMP) as they affect various intracellular targets. In particular, we study the frequency dependence of the peak force generated by the myofilaments, the force-frequency response (FFR). Our cell model incorporates frequency-dependent CaM-mediated spatially heterogenous interaction of CaMKII and CaN with their principal targets (dihydropyridine (DHPR) and ryanodine (RyR) receptors and the SERCA pump). It also accounts for the rate-dependent effects of phospholamban (PLB) on the SERCA pump; the rate-dependent role of cAMP in up-regulation of the L-type Ca2+ channel (ICa;L); and the enhancement in SERCA pump activity via phosphorylation of PLB.Our model reproduces positive peak FFR observed in rat ventricular myocytes during voltage-clamp studies both in the presence/absence of cAMP mediated -adrenergic stimulation. This study provides quantitative insight into the rate-dependence of Ca2+-induced Ca2+-release (CICR) by investigating the frequency-dependence of the trigger current (ICa;L) and RyR-release. It also highlights the relative role of the sodium-calcium exchanger (NCX) and the SERCA pump at higher frequencies, as well as the rate-dependence of sarcoplasmic reticulum (SR) Ca2+ content. A rigorous Ca2+ balance imposed on our investigation of these Ca2+ signalling pathways clarifies their individual roles. Here, we present a coupled electromechanical study emphasizing the rate-dependence of isometric force developed and also investigate the temperature-dependence of FFR. Our model provides mechanistic biophysically based explanations for the rate-dependence of CICR, generating useful and testable hypotheses. Although rat ventricular myocytes exhibit a positive peak FFR in the presence/absence of beta-adrenergic stimulation, they show a characteristic increase in the positive slope in FFR due to the presence of Norepinephrine or Isoproterenol. Our study identifies cAMP-mediated stimulation, and rate-dependent CaMKII-mediated up-regulation of ICa;L as the key mechanisms underlying the aforementioned positive FFR

Synaptotagmin oligomerization is essential for calcium control of regulated exocytosis

Regulated exocytosis, which underlies many intercellular signaling events, is a tightly controlled process often triggered by calcium ion(s) (Ca2+). Despite considerable insight into the central components involved, namely, the core fusion machinery [soluble N-ethylmaleimide?sensitive factor attachment protein receptor (SNARE)] and the principal Ca2+ sensor [C2-domain proteins like synaptotagmin (Syt)], the molecular mechanism of Ca2+-dependent release has been unclear. Here, we report that the Ca2+-sensitive oligomers of Syt1, a conserved structural feature among several C2-domain proteins, play a critical role in orchestrating Ca2+-coupled vesicular release. This follows from pHluorin-based imaging of single-vesicle exocytosis in pheochromocytoma (PC12) cells showing that selective disruption of Syt1 oligomerization using a structure-directed mutation (F349A) dramatically increases the normally low levels of constitutive exocytosis to effectively occlude Ca2+-stimulated release. We propose a parsimonious model whereby Ca2+-sensitive oligomers of Syt (or a similar C2-domain protein) assembled at the site of docking physically block spontaneous fusion until disrupted by Ca2+. Our data further suggest Ca2+-coupled vesicular release is triggered by removal of the inhibition, rather than by direct activation of the fusion machinery.Fil: Bello, Oscar Daniel. University of Yale. School of Medicine; Estados Unidos. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Mendoza. Instituto de Histologia y Embriologia de Mendoza Dr. Mario H. Burgos. Grupo Vinculado de Investigacion y Desarrollo Biotecnologico Aplicado Al Diagnostico Al Ihem | Universidad Nacional de Cuyo. Facultad de Ciencias Medicas. Instituto de Histologia y Embriologia de Mendoza Dr. Mario H. Burgos. Grupo Vinculado de Investigacion y Desarrollo Biotecnologico Aplicado Al Diagnostico Al Ihem.; Argentina. University College London; Estados UnidosFil: Jouannot, Ouardane. University of Yale. School of Medicine; Estados UnidosFil: Chaudhuri, Arunima. University of Yale. School of Medicine; Estados UnidosFil: Stroeva, Ekaterina. University of Yale. School of Medicine; Estados UnidosFil: Coleman, Jeff. University of Yale. School of Medicine; Estados UnidosFil: Volynski, Kirill E.. University College London; Reino UnidoFil: Rothman, James E.. University of Yale. School of Medicine; Estados Unidos. University College London; Estados UnidosFil: Krishnakumar, Shyam S.. University of Yale. School of Medicine; Estados Unidos. University College London; Estados Unido

PRRT2 Regulates Synaptic Fusion by Directly Modulating SNARE Complex Assembly

Mutations in proline-rich transmembrane protein 2 (PRRT2) are associated with a range of paroxysmal neurological disorders. PRRT2 predominantly localizes to the pre-synaptic terminals and is believed to regulate neurotransmitter release. However, the mechanism of action is unclear. Here, we use reconstituted single vesicle and bulk fusion assays, combined with live cell imaging of single exocytotic events in PC12 cells and biophysical analysis, to delineate the physiological role of PRRT2. We report that PRRT2 selectively blocks the trans SNARE complex assembly and thus negatively regulates synaptic vesicle priming. This inhibition is actualized via weak interactions of the N-terminal proline-rich domain with the synaptic SNARE proteins. Furthermore, we demonstrate that paroxysmal dyskinesia-associated mutations in PRRT2 disrupt this SNARE-modulatory function and with efficiencies corresponding to the severity of the disease phenotype. Our findings provide insights into the molecular mechanisms through which loss-of-function mutations in PRRT2 result in paroxysmal neurological disorders