Modular architecture of Munc13/calmodulin complexes: dual regulation by Ca2+ and possible function in short‐term synaptic plasticity

Ca²⁺ signalling in neurons through calmodulin (CaM) has a prominent function in regulating synaptic vesicle trafficking, transport, and fusion. Importantly, Ca²⁺–CaM binds a conserved region in the priming proteins Munc13‐1 and ubMunc13‐2 and thus regulates synaptic neurotransmitter release in neurons in response to residual Ca²⁺ signals. We solved the structure of Ca²⁺₄–CaM in complex with the CaM‐binding domain of Munc13‐1, which features a novel 1‐5‐8‐26 CaM‐binding motif with two separated mobile structural modules, each involving a CaM domain. Photoaffinity labelling data reveal the same modular architecture in the complex with the ubMunc13‐2 isoform. The N‐module can be dissociated with EGTA to form the half‐loaded Munc13/Ca²⁺₂–CaM complex. The Ca²⁺ regulation of these Munc13 isoforms can therefore be explained by the modular nature of the Munc13/Ca²⁺–CaM interactions, where the C‐module provides a high‐affinity interaction activated at nanomolar [Ca²⁺]_i, whereas the N‐module acts as a sensor at micromolar [Ca²⁺]_i. This Ca²⁺/CaM‐binding mode of Munc13 likely constitutes a key molecular correlate of the characteristic Ca²⁺‐dependent modulation of short‐term synaptic plasticity.     

Involvement of Rab3A in vesicle priming during exocytosis: interaction with Munc13-1 and Munc18-1

Rab3A is a small G-protein of the Rab family that is involved in the late steps of exocytosis. Here, we studied the role of Rab3A and its relationship with Munc13-1 and Munc18-1 during vesicle priming. Phorbol 12-myristate 13-acetate (PMA) is known to enhance the percentage of fusion-competent vesicles and this is mediated by protein kinase C (PKC)-independent Munc13-1 activation and PKC-dependent dissociation of Munc18-1 from syntaxin 1a. Our results show that the effects of PMA varied in cells overexpressing Rab3A or mutants of Rab3A and in cells with Rab3A knockdown. When Munc13-1 was overexpressed in Rab3A knockdown cells, secretion was completely inhibited. In cells overexpressing a Rab-interacting molecule (RIM)-binding deficient Munc13-1 mutant, 128-Munc13-1, the effects of Rab3A on PMA-induced secretion was abolished. The effect of PMA, which disappeared in cells overexpressing GTP-Rab3A (Q81L), could be reversed by co-expressing Munc18-1 but not its mutant R39C, which is unable to bind to syntaxin 1a. In cells overexpressing Munc18-1, manipulation of Rab3A activity had no effect on secretion. Finally, Munc18-1 enhanced the dissociation of Rab3A, and such enhancement correlated with exocytosis. In summary, our results support the hypothesis that the Rab3A cycle is coupled with the activation of Munc13-1 via RIM, which accounts for the regulation of secretion by Rab3A. Munc18-1 acts downstream of Munc13-1/RIM/Rab3A and interacts with syntaxin 1a allowing vesicle priming. Furthermore, Munc18-1 promotes Rab3A dissociation from vesicles, which then results in fusion.     

S100A1 codistributes with synapsin I in discrete brain areas and inhibits the F-actin-bundling activity of synapsin I

The Ca2+-sensor protein S100A1 was recently shown to bind in vitro to synapsins, a family of synaptic vesicle phosphoproteins involved in the regulation of neurotransmitter release. In this paper, we analyzed the distribution of S100A1 and synapsin I in the CNS and investigated the effects of the S100A1/synapsin binding on the synapsin functional properties. Subcellular fractionation of rat brain homogenate revealed that S100A1 is present in the soluble fraction of isolated nerve endings. Confocal laser scanning microscopy and immunogold immunocytochemistry demonstrated that S100A1 and synapsin codistribute in a subpopulation (5-20%) of nerve terminals in the mouse cerebral and cerebellar cortices. By forming heterocomplexes with either dephosphorylated or phosphorylated synapsin I, S100A1 caused a dose- and Ca2+-dependent inhibition of synapsin-induced F-actin bundling and abolished synapsin dimerization, without affecting the binding of synapsin to F-actin, G-actin or synaptic vesicles. These data indicate that: (i) synapsins and S100A1 can interact in the nerve terminals where they are coexpresssed; (ii) S100A1 is unable to bind to SV-associated synapsin I and may function as a cytoplasmic store of monomeric synapsin I; and (iii) synapsin dimerization and interaction with S100A1 are mutually exclusive, suggesting an involvement of S100A1 in the Ca2+-dependent regulation of synaptic vesicle trafficking.     

A Ca2+‐binding protein with numerous roles and uses: parvalbumin in molecular biology and physiology

Parvalbumins (PVs) are acidic, intracellular Ca²⁺‐binding proteins of low molecular weight. They are associated with several Ca²⁺‐mediated cellular activities and physiological processes. It has been suggested that PV might function as a “Ca²⁺ shuttle” transporting Ca²⁺ from troponin‐C (TnC) to the sarcoplasmic reticulum (SR) Ca²⁺ pump during muscle relaxation. Thus, PV may contribute to the performance of rapid, phasic movements by accelerating the contraction–relaxation cycle of fast‐twitch muscle fibers. Interestingly, PVs promote the generation of power stroke in fish by speeding up the rate of relaxation and thus provide impetus to attain maximal sustainable speeds. However, immunological monitoring of diverse tissues demonstrated that PVs are also present in non‐muscle cells. The axoplasmic transport and various intracellular secretory mechanisms including the endocrine secretions seem to be controlled by the Ca²⁺ regulation machinery. Any defect in the Ca²⁺ handling apparatus may cause several clinical problems; for instance, PV deficiency alters the neuronal activity, a key mechanism leading to epileptic seizures. Moreover, atypical relaxation of the heart results in diastolic dysfunction, which is a major cause of heart failure predominantly among the aged people. PV may offer a unique potential to correct defective relaxation in energetically compromised failing hearts through PV gene transfer. Consequently, PV gene transfer may present a new therapeutic approach to correct cellular disturbances in Ca²⁺ signaling pathways of diseased organs. Hence, PVs appear to be amazingly useful candidate proteins regulating a variety of cellular functions through action on Ca²⁺ flux management.     

Neuroprotective effects of donepezil against Aβ42‐induced neuronal toxicity are mediated through not only enhancing PP2A activity but also regulating GSK‐3β and nAChRs activity

The main purpose of this study was to evaluate whether donepezil, acetylcholinesterase inhibitor, shown to play a protective role through inhibiting glycogen synthesis kinase‐3β (GSK‐3β) activity, could also exert neuroprotective effects by stimulating protein phosphatase 2A (PP2A) activity in the amyloid‐beta (Aβ)42‐induced neuronal toxicity model of Alzheimer's disease. In Aβ42‐induced toxic conditions, each PP2A and GSK‐3β activity measured at different times showed time‐dependent reverse pattern toward the direction of accelerating neuronal deaths with the passage of time. In addition, donepezil pre‐treatment showed dose‐dependent stepwise increase of neuronal viability and stimulation of PP2A activity. However, such effects on them were significantly reduced through the depletion of PP2A activity with either okadaic acid or PP2Ac siRNA. In spite of blocked PP2A activity in this Aβ42 insult, however, donepezil pretreatment showed additional significant recovering effect on neuronal viability when compared to the value without donepezil. Moreover, donepezil partially recovered its dephosphorylating effect on hyperphosphorylated tau induced by Aβ42. This observation led us to assume that additional mechanisms of donepezil, including its inhibitory effect on GSK‐3β activity and/or the activation role of nicotinic acetylcholine receptors (nAChRs), might be involved. Taken together, our results suggest that the neuroprotective effects of donepezil against Aβ42‐induced neurotoxicity are mediated through activation of PP2A, but its additional mechanisms including regulation of GSK‐3β and nAChRs activity would partially contribute to its effects.

     

Protein kinase A increases the binding affinity and the Ca2+ release activity of the inositol 1,4,5-trisphosphate receptor type 3 in RINm5F cells

Background information. In endocrine cells, IP₃R (inositol 1,4,5‐trisphosphate receptor), a ligand‐gated Ca²⁺ channel, plays an important role in the control of intracellular Ca²⁺ concentration. There are three subtypes of IP₃R that are distributed differentially among cell types. RINm5F cells express almost exclusively the IP₃R‐3 subtype. The purpose of the present study was to investigate the effect of PKA (protein kinase A) on the activity of IP₃R‐3 in RINm5F cells. Results. We show that immunoprecipitated IP₃R‐3 is a good substrate for PKA. Using a back‐phosphorylation approach, we show that endogenous PKA phosphorylates IP₃R‐3 in intact RINm5F cells. [³H]IP₃ (inositol 1,4,5‐trisphosphate) binding affinity and IP₃‐induced Ca²⁺ release activity were enhanced in permeabilized cells that were pre‐treated with forskolin or PKA. The PKA‐induced enhancement of IP₃R‐3 activity was also observed in intact RINm5F cells stimulated with carbachol and epidermal growth factor, two agonists that use different receptor types to activate phospholipase C. Conclusion. The results of the present study reveal a converging step where the cAMP and the Ca²⁺ signalling systems act co‐operatively in endocrine cell responses to external stimuli.     

Luminescent properties of Ca3SiO4Cl2 co‐doped with Ce3+ and Eu2+ for near‐ultraviolet light‐emitting diodes

Ca₃SiO₄Cl₂ co‐doped with Ce³⁺,Eu²⁺ was prepared by high temperature reaction. The structure, luminescent properties and the energy transfer process of Ca₃SiO₄Cl₂: Ce³⁺,Eu²⁺ were investigated. Eu²⁺ ions can give enhanced green emission through Ce³⁺ → Eu²⁺ energy transfer in these phosphors. The green phosphor Ca_2.9775SiO₄Cl₂:0.0045Ce³⁺,0.018Eu²⁺ showed intense green emission with broader excitation in the near‐ultraviolet light range. A green light‐emitting diode (LED) based on this phosphor was made, and bright green light from this green LED could be observed by the naked eye under 20 mA current excitation. Hence it is considered to be a good candidate for the green component of a three‐band white LED. Copyright © 2015 John Wiley & Sons, Ltd.     

A Glycine soja methionine sulfoxide reductase B5a interacts with the Ca2+/CAM‐binding kinase GsCBRLK and activates ROS signaling under carbonate alkaline stress

Although research has extensively illustrated the molecular basis of plant responses to salt and high‐pH stresses, knowledge on carbonate alkaline stress is poor and the specific responsive mechanism remains elusive. We have previously characterized a Glycine soja Ca²⁺/CAM‐dependent kinase GsCBRLK that could increase salt tolerance. Here, we characterize a methionine sulfoxide reductase (MSR) B protein GsMSRB5a as a GsCBRLK interactor by using Y2H and BiFc assays. Further analyses showed that the N‐terminal variable domain of GsCBRLK contributed to the GsMSRB5a interaction. Y2H assays also revealed the interaction specificity of GsCBRLK with the wild soybean MSRB subfamily proteins, and determined that the BoxI/BoxII‐containing regions within GsMSRBs were responsible for their interaction. Furthermore, we also illustrated that the N‐terminal basic regions in GsMSRBs functioned as transit peptides, which targeted themselves into chloroplasts and thereby prevented their interaction with GsCBRLK. Nevertheless, deletion of these regions allowed them to localize on the plasma membrane (PM) and interact with GsCBRLK. In addition, we also showed that GsMSRB5a and GsCBRLK displayed overlapping tissue expression specificity and coincident expression patterns under carbonate alkaline stress. Phenotypic experiments demonstrated that GsMSRB5a and GsCBRLK overexpression in Arabidopsis enhanced carbonate alkaline stress tolerance. Further investigations elucidated that GsMSRB5a and GsCBRLK inhibited reactive oxygen species (ROS) accumulation by modifying the expression of ROS signaling, biosynthesis and scavenging genes. Summarily, our results demonstrated that GsCBRLK and GsMSRB5a interacted with each other, and activated ROS signaling under carbonate alkaline stress.