Internal Ca2 Research Articles

Molecular Interplay Between the Sigma-1 Receptor, Steroids, and Ion Channels

Cell excitability is tightly regulated by the activity of ion channels that allow for the passage of ions across cell membranes. Ion channel activity is controlled by different mechanisms that change their gating properties, expression or abundance in the cell membrane. The latter can be achieved by forming complexes with a diversity of proteins like chaperones such as the Sigma-1 receptor (Sig-1R), which is one with unique features and exhibits a role as a ligand-operated chaperone. This molecule also displays high intracellular mobility according to its activation level since, depletion of internal Ca+2 stores or the presence of specific ligands, produce Sig-1R’s mobilization from the endoplasmic reticulum toward the plasma membrane or nuclear envelope. The function of the Sig-1R as a chaperone is regulated by synthetic and endogenous ligands, with some of these compounds being a steroids and acting as key endogenous modifiers of the actions of the Sig-1R. There are cases in the literature that exemplify the close relationship between the actions of steroids on the Sig-1R and the resulting negative or positive effects on ion channel function/abundance. Such interactions have been shown to importantly influence the physiology of mammalian cells leading to changes in their excitability. The present review focuses on describing how the Sig-1R regulates the functional properties and the expression of some sodium, calcium, potassium, and TRP ion channels in the presence of steroids and the physiological consequences of these interplays at the cellular level are also discussed.

The Plasma Membrane Channel TRPV4 Evokes Endothelium‐Dependent Relaxation via a Novel Ca2+‐Induced Ca2+ Release Acting, at the Endothelial IP3 Receptor

The vascular endothelium lines the the entire vascular network and is a critical regulator of vascular tone given its ability to sense the complex composition of vasoactive substances in the circulating blood, as well as hemodynamic stresses and mechanical forces acting on the blood vessel wall. TRPV4 is a Ca2+ permeable cation channel which is expressed in the membrane of the endothelium and is activated by osmotic, mechanical and chemical stimuli, including the pharmacological agent GSK1016790A (GSK101). TRPV4 is an important contributor to endothelial regulation of vascular tone. Ca2+ influx from outside cells is a key messenger in the signalling cascades which transduce activating stimuli sensed by the endothelium into a functional output in the underlying smooth muscle. Previous studies suggested that the TRPV4 activation evoked endothelium‐dependent relaxation solely as a result of Ca2+ influx via TRPV4. However, recently, we found the endothelium‐dependent vasorelaxation evoked by TRPV4 activation was attenuated when Ca2+ release from the internal store was prevented. Here, we investigated the link between TRPV4 activation and Ca2+ release from the internal store. In mesenteric arteries from 8–12 week old Sprague Dawley rats, we visualised Ca2+ signalling (Cal‐520/AM) in response to GSK101(20 nM) and used pharmacological agents to characterise the signalling pathway leading to vasorelaxation. In conjunction, we also measured the contractile response of the arteries in response to the vasoconstrictor phenylephrine (PE), in the absence and presence of GSK101, the endothelium and after Ca2+ store depletion. All vessels were perfused with MOPS buffer in which all drugs were diluted. Images were captured using a high‐sensitivity CCD camera and analysed using custom analysis packages written in Python.The TRPV4 activator GSK101 (20 nM) stimulated a ‘slow’ continuous global rise in intracellular Ca2+ on which ‘fast’ sporadic intracellular waves which propagated both within and between cells occurred. All Ca2+ signals (slow and fast) were inhibited by the removal of extracellular Ca2+ or by prior treatment with the TRPV inhibitors, Ruthenium Red and HC067047. On the other hand, prior depletion of the intracellular store with cyclopiazonic acid (CPA) or inhibition of phospholipase C (PLC) using U73122, or block of the IP3‐receptor with 2‐APB inhibited only the widespread propagating waves. These data suggest that TRPV4 activation caused Ca2+ influx that induced IP3‐evoked Ca2+ release and propagating Ca2+ waves.In PE pre‐constricted vessels, GSK101 (20 nM) induced a dilation that was entirely dependent upon an intact endothelium. Prior depletion of the intracellular store using CPA prevented the inhibitory effect of GSK on PE‐induced contractions indicating that vasodilation induced by the TRPV4 activator GSK101 is dependent upon IP3‐stimulated Ca2+ release from the endothelium's internal Ca2+ store. These results suggest that Ca2+ influx via the plasma membrane channel TPRV4, evokes a novel Ca2+‐induced Ca2+ release like mechanism acting at the IP3 receptor in the intact endothelium to evoke vasodilation.Support or Funding InformationBHFThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Blockade of nifedipine‐sensitive Ca2+ channels decreases manganese accumulation on the dopamine neurons

Manganese (Mn) is an essential trace element involved in many physiological processes. Mn is transported into the cells through a number of transporters. Overexposure to Mn leads to its accumulation in the basal ganglia where it induces circuit dysregulation and a Parkinson-like syndrome referred to as manganism. The mechanisms by which Mn regulates the intrinsic firing behaviors of dopamine neurons and how Mn overexposure dysregulates this activity are not well understood. In this study, we investigated Mn's modulation of the excitability, intrinsic firing activity, Ca2+ homeostasis, and voltage-gated Ca2 channel expression in mouse dopamine neurons. We found that Mn increases the spontaneous firing activity of dopamine neuron via a concentration-dependent mechanism (10–100mM). Analysis of action potential morphology revealed that Mn truncated the amplitude and narrowed the width of action potentials and reduced coefficients of variation of the interspike interval. Unexpectedly, we found neither removal of external Ca2+ (Ca2+-free), chelation of intracellular Ca2+, nor combined chelation of internal and external Ca2+ altered Mn-regulation of action potential in dopamine neurons. Therefore, we asked whether Mn alters Ca2+ currents via the voltage-gated Ca2+ channels in the dopamine neurons. We found that Mn suppressed the peak amplitude of Ca2+ channel-mediated currents, and blockade of Ca2+ channels prevented Mn-stimulation of firing rate, reduction of action potential amplitude in dopamine neurons, and reduced 54Mn uptake in the CaV1-2, CaV1-3 expressing cells (p< 0.05, n=8–10). Finally, in vivo magnetic resonance imaging (MEMRI) revealed nifidepine-blockade of Ca2+ channels decreased Mn accumulation in the midbrain region of C57BL mice (p<0.05, n=6–8). Our data suggest a potential mechanism by which Mn can induce defects in basal ganglia function, namely a marked increase the excitability of dopaminergic neurons. Mn increases the spontaneous firing activity of dopamine neurons. Blockade of L-type Ca2+ channels inhibited Mn-stimulation of spontaneous firing activity of dopamine neurons. Manganese enhanced magnetic resonance imaging (MEMRI) provides evidence of an in vivo calcium-dependent mechanism involved in midbrain Mn accumulation. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Rikkunshito Depolarizes Pacemaker Potentials of Cultured Interstitial Cells of Cajal through Ghrelin Receptors in Murine Small Intestine

Background: Rikkunshito has been used to treat gastrointestinal (GI) disorders. The purpose of this study was to investigate the effects of Rikkunshito, a traditional Japanese herbal medicine, on the pacemaker potentials of interstitial cells of Cajal (ICCs) from the small intestines of mice. Methods: We isolated ICCs from the small intestines of mice, and the whole-cell patch-clamp configuration was used to record the pacemaker potentials in cultured ICCs and membrane currents. Results: Rikkunshito depolarized ICC pacemaker potentials in a dose-dependent manner. Pretreatment with GSK1614343 or (D-Lys3)-growth hormone-releasing peptide-6 inhibited Rikkunshito-induced depolarization of pacemaker potentials. Intracellular GDP-β-S inhibited Rikkunshito-induced effects. In Ca²⁺-free solution or in the presence of thapsigargin, Rikkunshito did not depolarize pacemaker potentials. Moreover, in the presence of U-73122 or xestospongin C, Rikkunshito-induced effects were inhibited. However, in the presence of staurosporine, Go6976 or Rottlerin, Rikkunshito depolarized pacemaker potentials. Furthermore, Rikkunshito inhibited both transient receptor potentials melastatin 7 (TRPM7) and Ca²⁺-activated Cl^– channels (ANO1) currents. Conclusion: Rikkunshito depolarized pacemaker potentials of ICCs via ghrelin receptor and G protein through internal or external Ca²⁺-, phospholipase C-, and inositol triphosphate-dependent and protein kinase C-, TRPM7-, and ANO1-independent pathways. The study shows that Rikkunshito may alleviate GI motility disorders through its depolarizing effects on ICCs.

Physiological differences in photosynthetic inorganic carbon utilization between gametophytes and sporophytes of the economically important red algae Pyropia haitanensis

Despite the lifecycle of the red seaweed Pyropia/Porphyra being extensively studied, photosynthesis and the CO2 concentrating mechanisms (CCMs) have comparatively not been well studied between the gametophyte phase (leafy thallus) and the sporophyte phase (filamentous conchocelis). This study found that the net photosynthetic rates of the thalli were about twice that of the conchocelis of Py. haitanensis, and the maximal quantum yield of photosystem II (Fv/Fm) and thallus respiration were significantly higher and lower than the conchocelis, respectively. Additionally, two phases did not show significant differences in phycocyanin (PC) content, but significantly higher contents of phycoerythrin (PE), allophycocyanin (APC), and chlorophyll α (Chl α) were observed in the thalli compared with the conchocelis. The external carbonic anhydrase (eCA) activity of the thalli was also higher than that of the conchocelis, but no differences in internal CA (iCA) activity were detected between the two phases. As for carbon acquisition for photosynthesis, the major carbon source of the thalli was seawater HCO3−, which was absorbed via eCA-catalyzed conversion to CO2. Conversely, the conchocelis used HCO3− transporters to directly absorb seawater HCO3−, which contributed equally to photosynthesis compared with the eCA- or iCA-mediated pathway. The discovery of different CCMs between the thallus and conchocelis phases could further our understanding of lifecycle regulation in Pyropia. In addition, a decrease in pH from 9 to 6 significantly increased the photosynthetic rates of both the thalli and conchocelis by about 100% and 600%, respectively. Thus, both phases have potential for carbon capture from flue gases and carbon sequestration.

Bioelectrical brain cortex activity in intellectually gifted children of primary and middle school age in solving cognitive tasks

The current review provides a historical perspective on the evolution of hypothesized mechanisms for senescent neurophysiology, focused on the CA1 region of the hippocampus, and the relationship of senescent neurophysiology to impaired hippocampal-dependent memory. Senescent neurophysiology involves processes linked to calcium (Ca2+) signaling including an increase in the Ca2+-dependent afterhyperpolarization (AHP), decreasing pyramidal cell excitability, hyporesponsiveness of N-methyl-D-aspartate (NMDA) receptor function, and a shift in Ca2+-dependent synaptic plasticity. Dysregulation of intracellular Ca2+ and downstream signaling of kinase and phosphatase activity lies at the core of senescent neurophysiology. Ca2+-dysregulation involves a decrease in Ca2+ influx through NMDA receptors and an increase release of Ca2+ from internal Ca2+ stores. Recent work has identified changes in redox signaling, arising in middle-age, as an initiating factor for senescent neurophysiology. The shift in redox state links processes of aging, oxidative stress and inflammation, with functional changes in mechanisms required for episodic memory. The link between age-related changes in Ca2+ signaling, epigenetics and gene expression is an exciting area of research. Pharmacological and behavioral intervention, initiated in middle-age, can promote memory function by initiating transcription of neuroprotective genes and rejuvenating neurophysiology. However, with more advanced age, or under conditions of neurodegenerative disease, epigenetic changes may weaken the link between environmental influences and transcription, decreasing resilience of memory function.

Light control of G protein signaling pathways by a novel photopigment.

Channelopsins and photo-regulated ion channels make it possible to use light to control electrical activity of cells. This powerful approach has lead to a veritable explosion of applications, though it is limited to changing membrane voltage of the target cells. An enormous potential could be tapped if similar opto-genetic techniques could be extended to the control of chemical signaling pathways. Photopigments from invertebrate photoreceptors are an obvious choice—as they do not bleach upon illumination -however, their functional expression has been problematic. We exploited an unusual opsin, pScop2, recently identified in ciliary photoreceptors of scallop. Phylogenetically, it is closer to vertebrate opsins, and offers the advantage of being a bi-stable photopigment. We inserted its coding sequence and a fluorescent protein reporter into plasmid vectors and demonstrated heterologous expression in various mammalian cell lines. HEK 293 cells were selected as a heterologous system for functional analysis, because wild type cells displayed the largest currents in response to the G-protein activator, GTP-γ-S. A line of HEK cells stably transfected with pScop2 was generated; after reconstitution of the photopigment with retinal, light responses were obtained in some cells, albeit of modest amplitude. In native photoreceptors pScop2 couples to Go; HEK cells express poorly this G-protein, but have a prominent Gq/PLC pathway linked to internal Ca mobilization. To enhance pScop2 competence to tap into this pathway, we swapped its third intracellular loop—important to confer specificity of interaction between 7TMDRs and G-proteins—with that of a Gq-linked opsin which we cloned from microvillar photoreceptors present in the same retina. The chimeric construct was evaluated by a Ca fluorescence assay, and was shown to mediate a robust mobilization of internal calcium in response to illumination. The results project pScop2 as a potentially powerful optogenetic tool to control signaling pathways.

From the Literature

From the Literature

The involvement of calmodulin and protein kinases in the upstream of cytosolic and nucleic calcium signaling induced by hypoosmotic shock in tobacco cells

ABSTRACTChanges in Ca2+ concentrations in cytosol ([Ca2+]C) or nucleus ([Ca2+]N) may play some vital roles in plants under hypoosmotic shock (Hypo-OS). Here, we observed that Hypo-OS induces biphasic increases in [Ca2+]C and [Ca2+]N in two tobacco cell lines (BY-2) expressing apoaequorin either in the cytosol or in the nucleus. Both [Ca2+]C and [Ca2+]N were sensitively modulated by the inhibitors of calmodulin and protein kinases, supporting the view that calmodulin suppresses the 1st peaks and and protein kinases enhance 2nd peaks in [Ca2+]C and [Ca2+]N. Data also suggested that the 1st and 2nd events depend on the internal and extracellular Ca2+ sources, respectively.

Ryanodine receptor Ca2+ release channel post-translational modification: Central player in cardiac and skeletal muscle disease.

Calcium release from internal stores is a quintessential event in excitation-contraction coupling in cardiac and skeletal muscle. The ryanodine receptor Ca2+ release channel is embedded in the internal sarcoplasmic reticulum Ca2+ store, which releases Ca2+ into the cytoplasm, enabling contraction. Ryanodine receptors form the hub of a macromolecular complex extending from the extracellular space to the sarcoplasmic reticulum lumen. Ryanodine receptor activity is influenced by the integrated effects of associated co-proteins, ions, and post-translational phosphor and redox modifications. In healthy muscle, ryanodine receptors are phosphorylated and redox modified to basal levels, to support cellular function. A pathological increase in the degree of both post-translational modifications disturbs intracellular Ca2+ signalling, and is implicated in various cardiac and skeletal disorders. This review summarises our current understanding of the mechanisms linking ryanodine receptor post-translational modification to heart failure and skeletal myopathy and highlights the challenges and controversies within the field.

The ER Ca2+ sensor STIM1 can activate osteoblast and odontoblast differentiation in mineralized tissues

ABSTRACTBone and dentin development requires temporal and spatial deposition of calcium phosphate mineral. A host of proteins works in concert to contribute to this tightly regulated process while malfunction in this scheme often leads to pathological defects. We have reported earlier that DMP1 stimulation of preosteoblasts leads to calcium release from internal Ca2+ stores and this store depletion is sensed by the ER Ca2+ sensor STIM1 (stromal interaction molecule 1). In this study, we first assessed the temporal and spatial localization of STIM1 protein during the development of bone and dentin by immunohistochemical methods. We further analyzed the function of STIM1 by establishing a stable MC3T3-E1 cell-line by overexpressing STIM1 (MC3T3-E1/STIM1 OE). Under mineralizing conditions, STIM1 overexpressing cells showed increased calcium deposits with higher expression of key osteogenic markers, such as Runx2 and type I collagen, BMP4 when compared with the control cells. Our results demonstrate that during mineralized matrix formation STIM1, the key ER sensor protein, can promote cellular differentiation in the presence of extracellular calcium.

Determination of the Stoichiometry between α- and γ1 Subunits of the BK Channel Using LRET

Two families of accessory proteins, β and γ, modulate BK channel gating and pharmacology. Notably, in the absence of internal Ca2+, the γ1 subunit promotes a large shift of the BK conductance-voltage curve to more negative potentials. However, very little is known about how α- and γ1 subunits interact. In particular, the association stoichiometry between both subunits is unknown. Here, we propose a method to answer this question using lanthanide resonance energy transfer. The method assumes that the kinetics of lanthanide resonance energy transfer-sensitized emission of the donor double-labeled α/γ1 complex is the linear combination of the kinetics of the sensitized emission in single-labeled complexes. We used a lanthanide binding tag engineered either into the α- or the γ1 subunits to bind Tb+3 as the donor. The acceptor (BODIPY) was attached to the BK pore-blocker iberiotoxin. We determined that γ1 associates with the α-subunit with a maximal 1:1 stoichiometry. This method could be applied to determine the stoichiometry of association between proteins within heteromultimeric complexes.

Modulation of Pacemaker Potentials in Murine Small Intestinal Interstitial Cells of Cajal by Gamisoyo-San, a Traditional Chinese Herbal Medicine

Background: The Gamisoyo-san (GSS) has been used for ­improving the gastrointestinal (GI) symptoms. The purpose of this study was to investigate the effects of GSS, a traditional Chinese herbal medicine, on the pacemaker potentials of mouse small intestinal interstitial cells of Cajal (ICCs). Methods: ICCs from the small intestines were dissociated and cultured. Whole-cell patch-clamp configuration was used to record pacemaker potentials and membrane currents. Results: GSS depolarized ICC pacemaker potentials in a dose-dependent manner. Pretreatment with 4-diphenylacetoxypiperidinium iodide completely inhibited GSS-induced pacemaker potential depolarizations. Intracellular GDP-β-S inhibited GSS-induced effects, and in the presence of U-73122, GSS-induced effects were inhibited. Also, GSS in the presence of a Ca²⁺-free solution or thapsigargin did not depolarize pacemaker potentials. However, in the presence of calphostin C, GSS slightly depolarized pacemaker potentials. Furthermore, GSS inhibited both transient receptor potential melastatin7 and Ca²⁺-activated Cl^– channel (anoctamin1) currents. Conclusion: GSS depolarized pacemaker potentials of ICCs via G protein and muscarinic M₃ receptor signaling pathways and through internal or external Ca²⁺-, phospholipase C-, and protein kinase C-dependent and transient receptor potential melastatin 7-, and anoctamin 1-independent pathways. The study shows that GSS may regulate GI tract motility, suggesting that GSS could be a basis for developing novel prokinetic agents for treating GI motility dysfunctions.

Orai1 downregulation impairs lymphocyte function in type 2 diabetes mellitus

Background/AimsIt has been suggested that diabetes is associated with immune dysfunction, in which Ca2+ signaling malfunction in lymphocyte may contributes most. However, the pattern of the Ca2+ signal disorder and the mechanism(s) that explains the change are unclear. Here, in this study we aimed to investigate possible changes and mechanism(s) accounting for the internal Ca2+ signals in diabetic T lymphocyte upon stimulation. Methods and resultsUsing Fura-2-AM, we found a significant decrease in Ca2+ influx induced by thapsigargin (TG) and anti-CD3 antibody (OKT3) in T lymphocytes from blood of both diabetes patients and animals. Furthermore, a downregulated Orai1 protein expression, but not mRNA, was also observed in these cells using western blot and qRT-PCR, respectively. In addition, in high-glucose and agonist treated Jurkat T cells, Ca2+ entry and the release of interleukin-2 (IL-2) were also decreased. Orai1 expression reduced, while stromal interaction molecule 1 (STIM1) and other downstream proteins remained unchanged. ConclusionThis study demonstrates that the declined Orai1 expression, at least partly, contributes to the downregulated Ca2+ entry during lymphocyte excitation, providing an important mechanism for T lymphocyte malfunction in diabetes.

A New Model for Understanding the Egg Cell Surface at Fertilization

The cell signaling pathway initiating fertilization has not been fully elucidated in any species. Several downstream components in the pathway leading to an internal Ca2+ release have been found; however, not much is known regarding the participation of proteins on the surface of the sperm and egg. The starfish Patiria miniata is an excellent model organism to study this process because they produce a significant quantity of large synchronized oocytes that are nearly transparent and rigid enough to hold their spherical shape despite the removal of extracellular layers. Recently, lipid rafts have been found on the surface of a variety of eggs, but their role at fertilization is unknown. Lipid rafts are small dynamic regions enriched with cholesterol and sphingolipids, which hold clusters of membrane bound proteins. Lipid rafts are found in a variety of cells and they are critical in certain signaling processes, such as the activation of T cells. This study develops the starfish egg as a model system for the study of lipid rafts and for the discovery of cell surface proteins necessary for fertilization. To visualize egg surface proteins in living cells, oocytes were labeled with a membrane impermeant form of biotin and imaged during fertilization by laser scanning confocal microscopy. Evidence of an increase in protein at the fertilization cone, which marks the site of sperm‐egg fusion, was seen. To further understand how these surface proteins were behaving at fertilization, samples prepared from the biotinylated eggs were subjected to gel electrophoresis followed by affinity interaction with streptavidin‐HRP to visualize the labeled proteins. This showed the presence of multiple egg surface proteins, ranging in size from 75 to 250 kDa in great quantity, as well as a few proteins ranging from 30 to 75kDa. Interestingly, biotinylated proteins were also recovered from the surrounding seawater following fertilization, suggesting the action of a protease that cleaves egg cell surface proteins. Experiments with protease inhibitors suggest that this protease activity is necessary for fertilization, leading to the idea that the protease may be involved in sperm‐egg fusion and/or signaling. To examine the idea that signaling may be initiated at the moment of sperm‐egg interaction, and to determine if lipid rafts could be involved, a detergent‐based lipid raft isolation assay was developed for the starfish egg. The eggs are separated into cytosolic, soluble membrane, and insoluble membrane (lipid rafts) fractions. The fractions were subjected to western blotting for tyrosine‐phosphorylated proteins, which were found almost exclusively in the insoluble fraction of the unfertilized egg. Therefore, the lipid rafts contain as of yet unidentified signaling molecules that could play a role in the initiation of fertilization. Understanding this mechanism at the molecular level in starfish can to eventually lead to understanding infertility issues as well as offer new possible non‐hormonal prophylactics in humans.Support or Funding InformationThis research was funded by the NSF and Florida Institute of TechnologyThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Chloroplast Ca2+ Fluxes into and across Thylakoids Revealed by Thylakoid-Targeted Aequorin Probes.

Chloroplasts require a fine-tuned control of their internal Ca2+ concentration, which is crucial for many aspects of photosynthesis and for other chloroplast-localized processes. Increasing evidence suggests that calcium regulation within chloroplasts also may influence Ca2+ signaling pathways in the cytosol. To investigate the involvement of thylakoids in Ca2+ homeostasis and in the modulation of chloroplast Ca2+ signals in vivo, we targeted the bioluminescent Ca2+ reporter aequorin as a YFP fusion to the lumen and the stromal surface of thylakoids in Arabidopsis (Arabidopsis thaliana). Thylakoid localization of aequorin-based probes in stably transformed lines was confirmed by confocal microscopy, immunogold labeling, and biochemical analyses. In resting conditions in the dark, free Ca2+ levels in the thylakoid lumen were maintained at about 0.5 μm, which was a 3- to 5-fold higher concentration than in the stroma. Monitoring of chloroplast Ca2+ dynamics in different intrachloroplast subcompartments (stroma, thylakoid membrane, and thylakoid lumen) revealed the occurrence of stimulus-specific Ca2+ signals, characterized by unique kinetic parameters. Oxidative and salt stresses initiated pronounced free Ca2+ changes in the thylakoid lumen. Localized Ca2+ increases also were observed on the thylakoid membrane surface, mirroring transient Ca2+ changes observed for the bulk stroma, but with specific Ca2+ dynamics. Moreover, evidence was obtained for dark-stimulated intrathylakoid Ca2+ changes, suggesting a new scenario for light-to-dark-induced Ca2+ fluxes inside chloroplasts. Hence, thylakoid-targeted aequorin reporters can provide new insights into chloroplast Ca2+ storage and signal transduction. These probes represent novel tools with which to investigate the role of thylakoids in Ca2+ signaling networks within chloroplasts and plant cells.

Mechanism of Regulation of Gi/o-Mediated TRPC4 Activation by Intracellular Protons

Transient Receptor Potential Canonical 4 (TRPC4) protein forms non-selective cation channels activated downstream from receptors that signal through heterotrimeric G proteins. Although the receptor-operated TRPC channel activation has mostly been attributed to Gq/11 proteins or receptor tyrosine kinases through stimulation of phospholipase C (PLC) isoforms β and γ, or internal Ca2+ store depletion, our recently work suggests that TRPC4 channels are particularly coupled to pertussis toxin-sensitive Gi/o proteins, with a codependence on PLCδ1. We showed that the Gi/o-mediated TRPC4 activation is dually dependent on and bimodally regulated by phosphatidylinositol 4, 5-bisphosphate (PIP2), the substrate hydrolyzed by PLC, and intracellular Ca2+, the level of which is increased following PLC stimulation due to inositol 1,4,5-trisphosphate induced internal Ca2+ release and Ca2+ influx through TRPC4 itself. As a byproduct of PLC-mediated PIP2 hydrolysis, protons have been shown to play an important role in the activation of Drosophila TRP channels. However, how intracellular pH affects mammalian TRPC channels was unclear. Using patch-clamp recordings of HEK293 cells heterologously coexpressing mouse TRPC4β and Gi/o-coupled µ opioid receptor, here we investigated the effect of intracellular protons on Gi/o-mediated TRPC4 activation. We found that lowering cytosolic pH greatly accelerated the rate of TRPC4 activation without altering the maximal current density and this effect was dependent on intracellular Ca2+ elevation. However, protons did not accelerate channel activation via a direct action on TRPC4, as shown by the inhibitory effect of low pH on channel activation by Englerin A, a direct TRPC4 agonist. We propose that protons exert their effect through sensitization of PLCδ1 to Ca2+, which in turn promotes TRPC4 opening and further PLCδ activities via a positive feedback mechanism.

Preoperative hip rotation moments do not predict long-term development after femoral derotation osteotomy in children with cerebral palsy

BackgroundFemoral derotation osteotomy (FDO) is the standard treatment for internal rotation gait (IRG) in children with cerebral palsy (CP) although high rates of recurrence have been reported recently. Various factors associated with recurrence could be identified, but no predictor named. Research questionsDoes FDO lead to a change of internal transversal hip moments?Are preoperative internal transversal hip moments a predictor for recurrence of IRG? Methods41 children with spastic bilateral CP and 72 limbs that received a FDO (10.4 ± 2.7 years at surgery) were included retrospectively. Kinematic data were analyzed pre- (2 ± 3 months), postoperatively (12 ± 3 months) and at long-term follow-up (at least five years postoperatively; 84 ± 13 months), internal transversal hip moments were analyzed pre- and postoperatively. ResultsThe maximum peaks of the internal hip rotation moment during loading response decreased significantly (p = 0.003). The minimum during the second half of the stance phase increased significantly (p = 0.004) and the initially internal externally rotating moment changed to an internal internally rotating moment. No correlation between changes in hip rotation from postoperatively to the long-term follow-up and the preoperative internal hip rotation moment could be identified. SignificanceFDO leads to changes in internal hip rotation moments. Preoperative internal hip rotation moments can’t be used as predicting factor for recurrence of IRG. The data suggest, that recurrence of IRG depends less on patient specific motion patterns, but more on the time point of surgery and the therapy of all concomitant deformities during SEMLS.

Optogenetics-Inspired Tunable Synaptic Functions in Memristors.

Two-terminal memristors with internal Ca2+-like dynamics can be used to faithfully emulate biological synaptic functions and have been intensively studied for neural network implementations. Inspired by the optogenetic technique that utilizes light to tune the Ca2+ dynamics and subsequently the synaptic plasticity, we develop a CH3NH3PbI3 (MAPbI3)-based memristor that exhibits light-tunable synaptic behaviors. Specifically, we show that by increasing the formation energy of iodine vacancy (VI·/VI×), light illumination can be used to control the VI·/VI× generation and annihilation dynamics, resembling light-controlled Ca2+ influx in biological synapses. We demonstrate that the memory formation and memory loss behaviors in the memristors can be modified by controlling the intensity and the wavelength of the illuminated light. Coincidence detection of electrical and light stimulations is also implemented in the memristive device with real-time (≤20 ms) response to light illumination. These results open options to modify the synaptic plasticity effects in memristor-based neuromorphic systems and can lead to the development of electronic systems that can faithfully emulate diverse biological processes.

Color Doppler imaging study in patients with primary open-angle glaucoma treated with timolol 0.5% and carteolol 2.

To evaluate with color Doppler imaging (CDI), in patients with primary open-angle glaucoma (PDAG), the possible influence on ocular hemodynamics of a beta-blocking agent with intrinsic sympathomimetic acitivity (carteolol 2%) compared to a beta-blocker agent without this activity. A study was carried out on 20 patients, with bilateral POaG, intraocular pressure (IOP) 20 mmHg, all treated twice a day with timolol maleate 0.5% ophthalmic solution. The visual field was evaluated (Octopus 2000 perimeter, G1 program) examining the mean sensitivity (MS) and the mean defect (MD). CDI was carried out to evaluate the resistance index of the internal carotid artery (ICA), the ophthalmic artery (OA), the central retinal artery (CRA), and the short posterior ciliary arteries (SPCA). After these examinations, the therapy was changed to carteolol 2% twice a day. After six months of treatment the examinations were repeated. The data were analysed statistically using Student's t test. The mean intraocular pressure during treatment with timolol 0.5% was 16.7 1.67 mmHg and 16.33 1.72 mmHg after treatment with carteolol 2%, the difference not being significant (p=0.494). After six months of treatment with carteolol 2% the MS increased significantly from 22.4 2.5 dB to 24.1 1.8 dB (p=0.018), and the mean defect (MD) fell from 5.3 0.8 dB to 4.7 0.6 dB (p=0.011). There was no significant difference in the resistance index of the CA, the OA and the CRA with the two treatments, whereas the resistance index of the SPCA dropped significantly, from 0.80 0.05 to 0.77 0.02 (p = 0.017). CDI did not show significant differences in the resistance indexes of the internal CA, the OA, and the CRA after treatment with carteolol 2% but the resistance index of the SPCA was significantly reduced. Carteolol 2% induced significant changes in the perimetric indexes examined, with an increase in ms and a decrease in md. These findings suggest that the intrinsic sympathomimetic activity of carteolol may reduce peripheral vascular resistance of the SCA, thus improving perfusion of the optic nerve head, with a protective effect on visual function. (Eur J Ophthalmol 2001; 11: 240-4).