Physical and functional interaction of the active zone protein CAST/ERC2 and the β-subunit of the voltage-dependent Ca(2+) channel

In the nerve terminals, the active zone protein CAST/ERC2 forms a protein complex with the other active zone proteins ELKS, Bassoon, Piccolo, RIM1 and Munc13-1, and is thought to play an organizational and functional role in neurotransmitter release. However, it remains obscure how CAST/ERC2 regulates the Ca(2+)-dependent release of neurotransmitters. Here, we show an interaction of CAST with voltage-dependent Ca(2+) channels (VDCCs), which are essential for regulating neurotransmitter release triggered by depolarization-induced Ca(2+) influx at the active zone. Using a biochemical assay, we showed that CAST was coimmunoprecipitated with the VDCC β(4)-subunit from the mouse brain. A pull-down assay revealed that the VDCC β(4)-subunit interacted directly with at least the N- and C-terminal regions of CAST. The II-III linker of VDCC α(1)-subunit also interacted with C-terminal regions of CAST; however, the interaction was much weaker than that of β(4)-subunit. Furthermore, coexpression of CAST and VDCCs in baby hamster kidney cells caused a shift in the voltage dependence of activation towards the hyperpolarizing direction. Taken together, these results suggest that CAST forms a protein complex with VDCCs, which may regulate neurotransmitter release partly through modifying the opening of VDCCs at the presynaptic active zones.     

An investigation of the effects of Ca²+ channel inhibitors on branching and chemotropism in the oomycete Achlya bisexualis: Support for a role for Ca²+ in apical dominance

In an attempt to better understand branching and chemotropism, we describe the effects of Ca2+ channel inhibitors on these processes in Achlya bisexualis, using a branch induction technique and whole plate assays. Branching appears to be a two step process with the initial formation of a bump from which a branch emerges. Verapamil increased numbers of branches in whole plate assays and decreased the distance from the first branch to the tip. In induction assays verapamil increased the number of bumps formed, although in some hyphae it inhibited the transition from an initial bump to a branch. When a branch formed it did not affect the time taken to branch. It had no effect on chemotropism. Lanthanum (La3+) and gadolinium (Gd3+) also increased branching in whole plate assays but their effect was much less marked and they had no effect on bump/branch number in induction assays. Gd3+ decreased the time taken to branch. Both La3+ and Gd3+ increased chemotropism. These data suggest firstly that the respective inhibitors may affect different parts of the branching process and secondly that Ca2+ influx through channels may not be a requirement for branching, indeed such movements may suppress branching. This would fit with elevated Ca2+ at the tip playing a role in apical dominance.     

EAAT2 density at the astrocyte plasma membrane and Ca2 + -regulated exocytosis

We studied whether regulated exocytosis affects the glutamate transporter density in cultured astrocytes, in which the expression of a fluorescently labeled excitatory amino acid transporter 2 (EAAT2-EGFP) predominantly labeled the plasma membrane. The addition of ionomycin that elevates cytosolic Ca²⁺ strongly increased the fluorescence of FM 4-64 membrane area dye, confirming the presence of regulated exocytosis in transfected astrocytes. However, concomitant with Ca²⁺-dependent FM 4-64 fluorescence increase, ionomycin induced a significant steady-state decrease in EAAT2-EGFP fluorescence. This is likely due to a secondary inner filter effect since,(i) in the absence of FM 4-64, ionomycin stimulation was ineffective in changing the EAAT2-EGFP fluorescence, and (ii) fluorescence changes in FM 4-64 and EAAT2-EGFP were inversely correlated. To test whether subcellular EAAT2-EGFP structures are translocated from the cytoplasm to the plasma membrane during ionomycin stimulation, EAAT2-EGFP fluorescence was monitored locally at the plasma membrane and a few microns away in the adjacent cytoplasm. Measurements revealed sites with an increase in EAAT2-EGFP plasma membrane fluorescence correlated with a fluorescence decrease beneath the plasma membrane, and sites with plasma membrane fluorescence decrease correlated with fluorescence increase within the adjacent cytoplasm. The sites of rapid translocation/retrieval of EAAT2-EGFP structures to/from the plasma membrane appeared to be distributed in a punctuate pattern around the cell perimeter. The density of EAAT2-EGFP was regulated in a Ca²⁺-dependent manner, since in the absence of extracellular Ca²⁺ local translocation/retrieval events were absent, revealing rapid surface density regulation of EAAT2 in astrocytes by regulated exo/endocytosis.     

Synaptotagmin increases the dynamic range of synapses by driving Ca²+-evoked release and by clamping a near-linear remaining Ca²+ sensor

Ca2+-evoked transmitter release shows a high dynamic range over spontaneous release. We investigated the role of the Ca2+ sensor protein, Synaptotagmin2 (Syt2), in both spontaneous and Ca2+-evoked release under direct control of presynaptic [Ca2+](i), using an in vivo rescue approach at the calyx of Held. Re-expression of Syt2 rescued the highly Ca2+ cooperative release and suppressed the elevated spontaneous release seen in Syt2 KO synapses. This latter release clamping function was partially mediated by the poly-lysine motif of the C?B domain. Using an aspartate mutation in the C?B domain (D364N) in which Ca2+ triggering was abolished but release clamping remained intact, we show that Syt2 strongly suppresses the action of another, near-linear Ca2+ sensor that mediates release over a wide range of [Ca2+](i). Thus, Syt2 increases the dynamic range of synapses by driving release with a high Ca2+ cooperativity, as well as by suppressing a remaining, near-linear Ca2+ sensor.     

Enhanced store-operated Ca²+ entry and TRPC channel expression in pulmonary arteries of monocrotaline-induced pulmonary hypertensive rats

Pulmonary hypertension (PH) is associated with profound vascular remodeling and alterations in Ca(2+) homeostasis in pulmonary arterial smooth muscle cells (PASMCs). Previous studies show that canonical transient receptor potential (TRPC) genes are upregulated and store-operated Ca(2+) entry (SOCE) is augmented in PASMCs of chronic hypoxic rats and patients of pulmonary arterial hypertension (PAH). Here we further examine the involvement of TRPC and SOCE in PH with a widely used rat model of monocrotaline (MCT)-induced PAH. Rats developed severe PAH, right ventricular hypertrophy, and significant increase in store-operated TRPC1 and TRPC4 mRNA and protein in endothelium-denuded pulmonary arteries (PAs) 3 wk after MCT injection. Contraction of PA and Ca(2+) influx in PASMC evoked by store depletion using cyclopiazonic acid (CPA) were enhanced dramatically, consistent with augmented SOCE in the MCT-treated group. The time course of increase in CPA-induced contraction corresponded to that of TRPC1 expression. Endothelin-1 (ET-1)-induced vasoconstriction was also potentiated in PAs of MCT-treated rats. The response was partially inhibited by SOCE blockers, including Gd(3+), La(3+), and SKF-96365, as well as the general TRPC inhibitor BTP-2, suggesting that TRPC-dependent SOCE was involved. Moreover, the ET-1-induced contraction and Ca(2+) response in the MCT group were more susceptible to the inhibition caused by the various SOCE blockers. Hence, our study shows that MCT-induced PAH is associated with increased TRPC expression and SOCE, which are involved in the enhanced vascular reactivity to ET-1, and support the hypothesis that TRPC-dependent SOCE is an important pathway for the development of PH.     

Liprin-α/SYD-2 determines the size of dense projections in presynaptic active zones in C. elegans

Synaptic vesicle (SV) release is spatially and temporally regulated by a network of proteins that form the presynaptic active zone (AZ). The hallmark of most AZs is an electron-dense projection (DP) surrounded by SVs. Despite their importance for our understanding of triggered SV release, high-resolution analyses of DP structures are limited. Using electron microscopy, we show that DPs at Caenorhabditis elegans neuromuscular junctions (NMJs) were highly structured, composed of building units forming bays in which SVs are docked to the AZ membrane. Furthermore, larger ribbonlike DPs that were multimers of the NMJ building unit are found at synapses between inter- and motoneurons. We also demonstrate that DP size is determined by the activity of the AZ protein SYD-2/Liprin-α. Whereas loss of syd-2 function led to smaller DPs, syd-2 gain-of-function mutants displayed larger ribbonlike DPs through increased recruitment of ELKS-1/ELKS. Therefore, our data suggest that a main role of SYD-2/Liprin-α in synaptogenesis is to regulate the polymerization of DPs.     

Bassoon and the synaptic ribbon organize Ca²+ channels and vesicles to add release sites and promote refilling

At the presynaptic active zone, Ca2+ influx triggers fusion of synaptic vesicles. It is not well understood how Ca2+ channel clustering and synaptic vesicle docking are organized. Here, we studied structure and function of hair cell ribbon synapses following genetic disruption of the presynaptic scaffold protein Bassoon. Mutant synapses--mostly lacking the ribbon--showed a reduction in membrane-proximal vesicles, with ribbonless synapses affected more than ribbon-occupied synapses. Ca2+ channels were also fewer at mutant synapses and appeared in abnormally shaped clusters. Ribbon absence reduced Ca2+ channel numbers at mutant and wild-type synapses. Fast and sustained exocytosis was reduced, notwithstanding normal coupling of the remaining Ca2+ channels to exocytosis. In vitro recordings revealed a slight impairment of vesicle replenishment. Mechanistic modeling of the in vivo data independently supported morphological and functional in vitro findings. We conclude that Bassoon and the ribbon (1) create a large number of release sites by organizing Ca2+ channels and vesicles, and (2) promote vesicle replenishment.     

Liprin-α2 promotes the presynaptic recruitment and turnover of RIM1/CASK to facilitate synaptic transmission

The presynaptic active zone mediates synaptic vesicle exocytosis, and modulation of its molecular composition is important for many types of synaptic plasticity. Here, we identify synaptic scaffold protein liprin-α2 as a key organizer in this process. We show that liprin-α2 levels were regulated by synaptic activity and the ubiquitin–proteasome system. Furthermore, liprin-α2 organized presynaptic ultrastructure and controlled synaptic output by regulating synaptic vesicle pool size. The presence of liprin-α2 at presynaptic sites did not depend on other active zone scaffolding proteins but was critical for recruitment of several components of the release machinery, including RIM1 and CASK. Fluorescence recovery after photobleaching showed that depletion of liprin-α2 resulted in reduced turnover of RIM1 and CASK at presynaptic terminals, suggesting that liprin-α2 promotes dynamic scaffolding for molecular complexes that facilitate synaptic vesicle release. Therefore, liprin-α2 plays an important role in maintaining active zone dynamics to modulate synaptic efficacy in response to changes in network activity.     

Overall charge and local charge density of pectin determines the enthalpic and entropic contributions to complexation with β-lactoglobulin

The complex formation between β-lactoglobulin and pectins of varying overall charge and local charge density were investigated. Isothermal titration calorimetry experiments were carried out to determine the enthalpic contribution to the complex formation at pH 4.25 and various ionic strengths. Complex formation was found to be an exothermic process for all conditions. Combination with previously published binding constants by Sperber et al. (Sperber, B. L. H. M.; Cohen Stuart, M. A.; Schols, H. A.; Voragen, A. G. J.; Norde, W. Biomacromolecules 2009, 10, 3246-3252) allows for the determination of the changes in the Gibbs energy and the change in entropy of the system upon complex formation between β-lactoglobulin and pectin. The local charge density of pectin is found to determine the balance between enthalpic and entropic contributions. For a high local charge density pectin, the main contribution to the Gibbs energy is of an enthalpic nature, supported by a favorable entropy effect due to the release of small counterions. A pectin with a low local charge density has a more even distribution of the enthalpic and entropic part to the change of the Gibbs energy. The enthalpic part is reduced due to the lower charge density, while the relative increase of the entropic contribution is thought to be caused by a change in the location of the binding place for pectin on the β-lactoglobulin molecule. The association of the hydrophobic methyl esters on pectin with an exposed hydrophobic region on β-lg results in the release of water molecules from the hydrophobic region and surrounding the methyl esters of the pectin molecule. An increase in the ionic strength decreases the enthalpic contribution due to the shielding of electrostatic attraction in favor of the entropic contribution, supporting the idea that the release of water molecules from hydrophobic areas plays a part in the complex formation.     

Elasticity–density and viscoelasticity–density relationships at the tibia mid-diaphysis assessed from resonant ultrasound spectroscopy measurements

Cortical bone tissue is an anisotropic material characterized by typically five independent elastic coefficients (for transverse isotropy) governing shear and longitudinal deformations in the different anatomical directions. It is well established that the Young’s modulus in the direction of the bone axis of long bones has a strong relationship with mass density. It is not clear, however, whether relationships of similar strength exist for the other elastic coefficients, for they have seldom been investigated, and the results available in the literature are contradictory. The objectives of the present work were to document the anisotropic elastic properties of cortical bone at the tibia mid-diaphysis and to elucidate their relationships with mass density. Resonant ultrasound spectroscopy (RUS) was used to measure the transverse isotropic stiffness tensor of 55 specimens from 19 donors. Except for Poisson’s ratios and the non-diagonal stiffness coefficient, strong linear correlations between the different elastic coefficients \((0.7 < {r^{2}} < 0.99)\) and between these coefficients and density \((0.79 < {r^{2}} < 0.89)\) were found. Comparison with previously published data from femur specimens suggested that the strong correlations evidenced in this study may not only be valid for the mid-tibia. RUS also measures the viscous part of the stiffness tensor. An anisotropy ratio close to two was found for damping coefficients. Damping increased as the mass density decreased. The data suggest that a relatively accurate estimation of all the mid-tibia elastic coefficients can be derived from mass density. This is of particular interest (1) to design organ-scale bone models in which elastic coefficients are mapped according to Hounsfield values from computed tomography scans as a surrogate for mass density and (2) to model ultrasound propagation at the mid-tibia, which is an important site for the in vivo assessment of bone status with axial transmission techniques.     

Co- and post-translational translocation through the protein-conducting channel: analogous mechanisms at work?

Many proteins are translocated across, or integrated into, membranes. Both functions are fulfilled by the 'translocon/translocase', which contains a membrane-embedded protein-conducting channel (PCC) and associated soluble factors that drive translocation and insertion reactions using nucleotide triphosphates as fuel. This perspective focuses on reinterpreting existing experimental data in light of a recently proposed PCC model comprising a front-to-front dimer of SecY or Sec61 heterotrimeric complexes. In this new framework, we propose (i) a revised model for SRP-SR-mediated docking of the ribosome-nascent polypeptide to the PCC; (ii) that the dynamic interplay between protein substrate, soluble factors and PCC controls the opening and closing of a transmembrane channel across, and/or a lateral gate into, the membrane; and (iii) that co- and post-translational translocation, involving the ribosome and SecA, respectively, not only converge at the PCC but also use analogous mechanisms for coordinating protein translocation.     

Glu106 targeted inhibitors of ORAI1 as potential Ca2+ release-activated Ca2+ (CRAC) channel blockers – molecular modeling and docking studies

Calcium release-activated calcium modulator 1(ORAI1) is an integral component of the calcium release-activated calcium channel (CRAC) channel complex and plays a central role in regulating Ca^2?+?concentrations in T-lymphocytes. It is critical for many physiological processes, including cell-proliferation, cytokine production and activation of the immune system. Loss of ORAI1 function is linked with rheumatoid arthritis (RA) and hence pharmacological blockers of ORAI1 could be potential therapeutic agents for the treatment of RA. In this study, we have used a high-throughput screening approach to inhibit the binding of Ca^2+?toward ORAI1 and the interactions are verified through induced fit docking. The results hint that these compounds act by possibly binding with, and thereby blocking Ca²⁺-binding with ORAI1 (E106). The molecular dynamics (MD) simulations shows strong support toward the hit compounds by showing the ligand potency throughout the simulation timescale of 30?ns. We have thus identified a novel class of highly stable, potential lead compounds that directly bind with the selectivity filter region E106 and block Ca²⁺ binding on ORAI1. This resulting alteration in the pore geometry of ORAI1 due to the strong blocking mechanism of lead compounds will greatly diminish its function and the downstream activities that result from the same including decreased production of cytokines in autoimmune disorders. This study may lay the foundation for finding novel lead compounds for clinical trials that could positively modulate the course of autoimmune disorders with ORAI1 as its specific target.     

3D-QSAR and docking studies of pentacycloundecylamines at the sigma-1 (σ1) receptor

Pentacycloundecylamine (PCU) derived compounds have been shown to be promising lead structures for the development of novel drug candidates aimed at a variety of neurodegenerative and psychiatric diseases. Here we show for the first time a 3D quantitative structure–activity relationship (3D-QSAR) for a series of aza-PCU-derived compounds with activity at the sigma-1 (σ₁) receptor. A comparative molecular field analysis (CoMFA) model was developed with a partial least squares cross validated (q²) regression value of 0.6, and a non-cross validated r² of 0.9. The CoMFA model was effective at predicting the sigma-1 activities of a test set with an r² >0.7. We also describe here the docking of the PCU-derived compounds into a homology model of the sigma-1 (σ₁) receptor, which was developed to gain insight into binding of these cage compounds to the receptor. Based on docking studies we evaluated in a [³H]pentazocine binding assay an oxa-PCU, NGP1-01 (IC₅₀ = 1.78 μM) and its phenethyl derivative (IC₅₀ = 1.54 μM). Results from these studies can be used to develop new compounds with specific affinity for the sigma-1(σ₁) receptor.     

Arabidopsis annexin1 mediates the radical-activated plasma membrane Ca²+- and K+-permeable conductance in root cells

Plant cell growth and stress signaling require Ca²⁺ influx through plasma membrane transport proteins that are regulated by reactive oxygen species. In root cell growth, adaptation to salinity stress, and stomatal closure, such proteins operate downstream of the plasma membrane NADPH oxidases that produce extracellular superoxide anion, a reactive oxygen species that is readily converted to extracellular hydrogen peroxide and hydroxyl radicals, OH^•. In root cells, extracellular OH^• activates a plasma membrane Ca²⁺-permeable conductance that permits Ca²⁺ influx. In Arabidopsis thaliana, distribution of this conductance resembles that of annexin1 (ANN1). Annexins are membrane binding proteins that can form Ca²⁺-permeable conductances in vitro. Here, the Arabidopsis loss-of-function mutant for annexin1 (Atann1) was found to lack the root hair and epidermal OH^•-activated Ca²⁺- and K⁺-permeable conductance. This manifests in both impaired root cell growth and ability to elevate root cell cytosolic free Ca²⁺ in response to OH^•. An OH^•-activated Ca²⁺ conductance is reconstituted by recombinant ANN1 in planar lipid bilayers. ANN1 therefore presents as a novel Ca²⁺-permeable transporter providing a molecular link between reactive oxygen species and cytosolic Ca²⁺ in plants.     

MD simulation and experimental evidence for Mg²+ binding at the B site in human AP endonuclease 1

Apurinic/apyrimidinic endonuclease 1 (APE1), a central enzyme in the base excision repair pathway, cleaves damaged DNA in Mg(2+) dependent reaction. Despite characterization of nine X-ray crystallographic structures of human APE1, in some cases, bound to various metal ions and substrate/product, the position of the metal ion and its stoichiometry for the cleavage reaction are still being debated. While a mutation of the active site E96Q was proposed to eliminate Mg(2+) binding at the "A" site, we show experimentally that this mutant still requires Mg(2+) at concentration similar to that for the wild type enzyme to cleave the AP site in DNA. Molecular dynamics simulations of the wild type APE1, E96Q and a double missense mutant E96Q + D210N indicate that Mg(2+) placed at the A-site destabilizes the bound AP site-containing DNA. In these simulations, the H-bond chain D238-H309-AP site oxygen is broken and the substrate DNA is shifted away from its crystal structure position (1DE9). In contrast, simulations with the Mg(2+) at site B or A+B sites leave the substrate DNA at the position shown in the crystal structure (1DE9). Taken together our MD simulations and biochemical analysis suggests that Mg(2+) binding at the B site is involved in the reaction mechanism associated with endonuclease function of APE1.     

Signal integration at the level of ion channel and exocytotic function in pancreatic β-cells

Whole body energy balance is ensured by the exquisite control of insulin secretion, the dysregulation of which has serious consequences. Although a great deal has been learned about the control of insulin secretion from pancreatic β-cells in the past 30 years, there remains much to be understood about the molecular mechanisms and interactions that underlie the precise control of this process. Numerous molecular interactions at the plasma membrane mediate the excitatory and amplifying events involved in insulin secretion; this includes interactions between ion channels, signal transduction machinery, and exocytotic proteins. The present Perspectives article considers evidence that key membrane and membrane-associated proteins essential to insulin secretion are regulated in concert as a functional unit, ensuring an integrated excitatory and exocytotic response to the signals that control insulin secretion.     

Mechanism of the Ca²+-dependent interaction between S100A4 and tail fragments of nonmuscle myosin heavy chain IIA

The interaction between the calcium-binding protein S100A4 and the C-terminal fragments of nonmuscle myosin heavy chain IIA has been studied by equilibrium and kinetic methods. Using site-directed mutants, we conclude that Ca(2+) binds to the EF2 domain of S100A4 with micromolar affinity and that the K(d) value for Ca(2+) is reduced by several orders of magnitude in the presence of myosin target fragments. The reduction in K(d) results from a reduced dissociation rate constant (from 16?s(-1) to 0.3?s(-1) in the presence of coiled-coil fragments) and an increased association rate constant. Using peptide competition assays and NMR spectroscopy, we conclude that the minimal binding site on myosin heavy chain IIA corresponds to A1907-G1938; therefore, the site extends beyond the end of the coiled-coil region of myosin. Electron microscopy and turbidity assays were used to assess myosin fragment filament disassembly by S100A4. The latter assay demonstrated that S100A4 binds to the filaments and actively promotes disassembly rather than just binding to the myosin monomer and displacing the equilibrium. Quantitative modelling of these in vitro data suggests that S100A4 concentrations in the micromolar region could disassemble myosin filaments even at resting levels of cytoplasmic [Ca(2+)]. However, for Ca(2+) transients to be effective in further promoting dissociation, the elevated Ca(2+) signal must persist for tens of seconds. Fluorescence recovery after photobleaching of A431/SIP1 cells expressing green fluorescent protein-myosin IIA, immobilised on fibronectin micropatterns to control stress fibre location, yielded a recovery time constant of around 20?s, consistent with in vitro data.     

Oxalate production at different initial Pb²+ concentrations and the influence of oxalate during solid-state fermentation of straw with Phanerochaete chrysosporium

The production of oxalate at different initial Pb²⁺ concentrations during solid-state fermentation of straw with Phanerochaete chrysosporium was investigated. It was found that the maximal peak value of oxalate concentration (22.84 mM) was detected at the initial Pb²⁺ concentration of 200 mg kg⁻¹ dry straw, while the minimum (15.89 mM) at the concentration of 600 mg Pb²⁺ kg⁻¹ dry straw, and at moderate concentration of Pb²⁺ the capability of oxalic acid secretion was enhanced. In addition, it was also found that more oxalic acid accumulation went together with better Pb²⁺ passivation effect and higher manganese peroxidase (MnP) activity. The present findings will improve the understandings of the interactions of heavy metals with white-rot fungi and the role of oxalate in lignin degradation system, which could provide useful references for more efficient treatment of Pb-contaminated lignocellulosic waste.