Functional interaction between TRPC1 channel and connexin-43 protein: a novel pathway underlying S1P action on skeletal myogenesis

We recently demonstrated that skeletal muscle differentiation induced by sphingosine 1-phosphate (S1P) requires gap junctions and transient receptor potential canonical 1 (TRPC1) channels. Here, we searched for the signaling pathway linking the channel activity with Cx43 expression/function, investigating the involvement of the Ca²⁺-sensitive protease, m-calpain, and its targets in S1P-induced C2C12 myoblast differentiation. Gene silencing and pharmacological inhibition of TRPC1 significantly reduced Cx43 up-regulation and Cx43/cytoskeletal interaction elicited by S1P. TRPC1-dependent functions were also required for the transient increase of m-calpain activity/expression and the subsequent decrease of PKCα levels. Remarkably, Cx43 expression in S1P-treated myoblasts was reduced by m-calpain-siRNA and enhanced by pharmacological inhibition of classical PKCs, stressing the relevance for calpain/PKCα axis in Cx43 protein remodeling. The contribution of this pathway in myogenesis was also investigated. In conclusion, these findings provide novel mechanisms by which S1P regulates myoblast differentiation and offer interesting therapeutic options to improve skeletal muscle regeneration.     

Arachidonic acid release by ionomycin and phorbol ester is similar in C127 epithelial cells expressing wild-type or mutated (ΔF508) cystic fibrosis transmembrane conductance regulator

The Ca²⁺ ionophore ionomycin induced cytosolic [Ca²⁺]_i elevation as well as strong activation of Cl⁻ efflux in mouse mammary epithelial cell lines expressing wild-type or mutated (deletion of phenylalaline 508) cystic fibrosis transmembrane conductance regulator (CFTR) or vector. Ionomycin-induced Cl⁻ efflux was abolished by the intracellular Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid, whereas both activators and inhibitors of phospholipase A₂ had no effect, indicating the involvement of Ca²⁺-dependent Cl^- channels. Stimulation of arachidonic acid release by ionomycin and phorbol ester was not significantly different between wild-type or mutated cell lines, whereas vector-transfected cells exhibited a significant higher release, which was shown to be due to larger amount of immunoreactive cytosolic phospholipase A₂. These results indicate that phospholipase A₂ activity of C127 cells was not influenced by the presence of wild-type or mutated CFTR. Received 27 April 1999; received after revision 11 June 1999; accepted 23 July 1999     

The selective BH4-domain biology of Bcl-2-family members: IP3Rs and beyond

Anti-apoptotic Bcl-2-family members not only neutralize pro-apoptotic proteins but also directly regulate intracellular Ca²⁺ signaling from the endoplasmic reticulum (ER), critically controlling cellular health, survival, and death initiation. Furthermore, distinct Bcl-2-family members may selectively regulate inositol 1,4,5-trisphosphate receptor (IP₃R): Bcl-2 likely acts as an endogenous inhibitor of the IP₃R, preventing pro-apoptotic Ca²⁺ transients, while Bcl-X_L likely acts as an endogenous IP₃R-sensitizing protein promoting pro-survival Ca²⁺ oscillations. Furthermore, distinct functional domains in Bcl-2 and Bcl-X_L may underlie the divergence in IP₃R regulation. The Bcl-2 homology (BH) 4 domain, which targets the central modulatory domain of the IP₃R, is likely to be Bcl-2’s determining factor. In contrast, the hydrophobic cleft targets the C-terminal Ca²⁺-channel tail and might be more crucial for Bcl-X_L’s function. Furthermore, one amino acid critically different in the sequence of Bcl-2’s and Bcl-X_L’s BH4 domains underpins their selective effect on Ca²⁺ signaling and distinct biological properties of Bcl-2 versus Bcl-X_L. This difference is evolutionary conserved across five classes of vertebrates and may represent a fundamental divergence in their biological function. Moreover, these insights open novel avenues to selectively suppress malignant Bcl-2 function in cancer cells by targeting its BH4 domain, while maintaining essential Bcl-X_L functions in normal cells. Thus, IP₃R-derived molecules that mimic the BH4 domain’s binding site on the IP₃R may function synergistically with BH3-mimetic molecules selectivity suppressing Bcl-2’s proto-oncogenic activity. Finally, a more general role for the BH4 domain on IP₃Rs, rather than solely anti-apoptotic, may not be excluded as part of a complex network of molecular interactions.     

Involvement of thiols in the induction of inward current induced by silver in frog skeletal muscle membrane

Exposure of voltage-clamped frog skeletal muscle fibres to silver caused a maintained inward current which could be carried by Ca²⁺, Mg²⁺ or Na⁺. Inorganic Ca²⁺ channel blockers and dithiothreitol (SH reducing agent) diminished this current, but a Na⁺ channel blocker did not. Thus, silver activates the Ca²⁺ channel by acting on SH groups in a Ca²⁺ channel protein.     

Complex modulation of Cav3.1 T-type calcium channel by nickel

Nickel is considered to be a selective blocker of low-voltage-activated T-type calcium channel. Recently, the Ni²⁺-binding site with critical histidine-191 (H191) within the extracellular IS3–IS4 domain of the most Ni²⁺-sensitive Ca_v3.2 T-channel isoform has been identified. All calcium channels are postulated to also have intrapore-binding site limiting maximal current carried by permeating divalent cations (PDC) and determining the blockade by non-permeating ones. However, the contribution of the two sites to the overall Ni²⁺ effect and its dependence on PDC remain uncertain. Here we compared Ni²⁺ action on the wild-type “Ni²⁺-insensitive” Ca_v3.1_w/t channel and Ca_v3.1_Q172H mutant having glutamine (Q) equivalent to H191 of Ca_v3.2 replaced by histidine. Each channel was expressed in Xenopus oocytes, and Ni²⁺ blockade of Ca²⁺, Sr²⁺, or Ba²⁺ currents was assessed by electrophysiology. Inhibition of Ca_v3.1_w/t by Ni²⁺ conformed to two sites binding. Ni²⁺ binding with high-affinity site (IC₅₀ = 0.03–3 μM depending on PDC) produced maximal inhibition of 20–30 % and was voltage-dependent, consistent with its location within the channel’s pore. Most of the inhibition (70–80 %) was produced by Ni²⁺ binding with low-affinity site (IC₅₀ = 240–700 μM). Q172H-mutation mainly affected low-affinity binding (IC₅₀ = 120–160 μM). The IC₅₀ of Ni²⁺ binding with both sites in the Ca_v3.1_w/t and Ca_v3.1_Q172H was differentially modulated by PDC, suggesting a varying degree of competition of Ca²⁺, Sr²⁺, or Ba²⁺ with Ni²⁺. We conclude that differential Ni²⁺-sensitivity of T-channel subtypes is determined only by H-containing external binding sites, which, in the absence of Ni²⁺, may be occupied by PDC, influencing in turn the channel’s permeation.     

Ca<Superscript>2+</Superscript> signals,cell membrane disintegration,and activation of TMEM16F during necroptosis

Activated receptor-interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain like (MLKL) are essential components of the necroptotic pathway. Phosphorylated MLKL (pMLKL) is thought to induce membrane leakage, leading to cell swelling and disintegration of the cell membrane. However, the molecular identity of the necroptotic membrane pore remains unclear, and the role of pMLKL for membrane permeabilization is currently disputed. We observed earlier that the phospholipid scramblase and ion channel TMEM16F/anoctamin 6 cause large membrane currents, cell swelling, and cell death when activated by a strong increase in intracellular Ca²⁺. We, therefore, asked whether TMEM16F is also central to necroptotic cell death and other cellular events during necroptosis. Necroptosis was induced by TNFα, smac mimetic, and Z-VAD (TSZ) in NIH3T3 fibroblasts and the four additional cell lines HT₂₉, 16HBE, H441, and L929. Time-dependent changes in intracellular Ca²⁺, cell morphology, and membrane currents were recorded. TSZ induced a small and only transient oscillatory rise in intracellular Ca²⁺, which was paralleled by the activation of outwardly rectifying Cl^? currents, which were typical for TMEM16F/ANO6. Ca²⁺ oscillations were due to Ca²⁺ release from endoplasmic reticulum, and were independent of extracellular Ca²⁺. The initial TSZ-induced cell swelling was followed by cell shrinkage. Using typical channel blockers and siRNA-knockdown, the Cl^? currents were shown to be due to the activation of ANO6. However, the knockdown of ANO6 or inhibitors of ANO6 did not inhibit necroptotic cell death. The present data demonstrate the activation of ANO6 during necroptosis, which, however, is not essential for cell death.     

Mechanism of inhibition of IgE-dependent histamine release from rat mast cells by xestobergsterol A from the Okinawan marine spongeXestospongia bergquistia

Histamine release from rat peritoneal mast cells induced by anti-IgE was essentially complete within 4–5 min. Xestobergsterol A and B, which are constituents of the Okinawan marine spongeXestospongia bergquistia Fromont, dose-dependently inhibited anti-IgE-induced histamine release from rat mast cells. The IC₅₀ values of xestobergsterol A and B for histamine release in mast cells activated by anti-IgE were 0.07 and 0.11 M, respectively. Anti-IgE stimulated PI-PLC activity in a mast cell membrane preparation. Xestobergsterol A dose-dependently inhibited the generation of IP3 and membrane-bound PI-PLC activity. Moreover, xestobergsterol A inhibited Ca²⁺-mobilization from intracellular Ca²⁺-stores as well as histamine release in mast cells activated by anti-IgE. On the other hand, xestobergsterol B did not inhibit the membrane-bound and cytosolic PI-PLC activity, IP3 generation or the initial rise in [Ca²⁺]_i in mast cells activated by anti-IgE. These results suggest that the mechanism of inhibition by xestobergsterol A of the initial rise in [Ca²⁺]_i, of the generation of IP3, and of histamine release induced by anti-IgE, was through the inhibition of PI-PLC activity.     

Mechanical and biochemical characterization of the contraction elicited by a calcium-independent myosin light chain kinase in chemically skinned smooth muscle

Summary The contraction induced by a Ca²⁺-independent myosin light chain kinase (MLCK-) was characterized in terms of isometric force (F_o), immediate elastic recoil (SE), unloaded shortening velocity (V_us), shortening under a constant load and ATPase activity of chemically skinned smooth muscle preparations. These parameters were compared to those measured in a Ca²⁺-induced contraction to assess the nature of cross bridge interaction in the MLCK-induced contraction. F_o developed in chicken gizzard fibers as well as SE were similar in contractions elicited by either agent. V_us in the contraction induced by MLCK-(0.36 mg/ml) was similar though averaged 39.3±8.9% less than V_us induced by Ca²⁺ (1.6x10^–6M) in the control fibers. Addition of Ca²⁺ (1.6x10^–6M) to a contraction induced by MLCK-resulted in small increases in both F_o and V_us. Shortening under a constant load was similar for both types of contractions. The contraction induced by MLCK-was accompanied by an increased rate of ATP hydrolysis. The MLCK-induced contraction is thus kinetically similar though not identical to a contraction induced by Ca²⁺. We conclude that with respect to actin-myosin interaction, MLCK- and Ca²⁺-induced contractions are similar.     

The continuing disappearance of “pure” Ca2+ buffers

Advances in the understanding of a class of Ca²⁺-binding proteins usually referred to as “Ca²⁺ buffers” are reported. Proteins historically embraced within this group include parvalbumins (α and β), calbindin-D9k, calbindin-D28k and calretinin. Within the last few years a wealth of data has accumulated that allow a better understanding of the functions of particular family members of the >240 identified EF-hand Ca²⁺-binding proteins encoded by the human genome. Studies often involving transgenic animal models have revealed that they exert their specific functions within an intricate network consisting of many proteins and cellular mechanisms involved in Ca²⁺ signaling and Ca²⁺ homeostasis, and are thus an essential part of the Ca²⁺ homeostasome. Recent results indicate that calbindin-D28k, possibly also calretinin and oncomodulin, the mammalian β parvalbumin, might have additional Ca²⁺ sensor functions, leaving parvalbumin and calbindin-D9k as the only “pure” Ca²⁺ buffers. Received 10 September 2008; received after revision 15 October 2008; accepted 4 November 2008     

Structure and function of a calmodulin-dependent smooth muscle myosin light chain kinase

Summary In smooth muscle the M_r 20,000 light chain of myosin is phosphorylated by a calmodulin-dependent protein kinase. It consists of 2 subunits: calmodulin, an acidic protein of M_r 17,000 that binds 4 moles of Ca²⁺; and a larger protein of M_r circa 130,000. Activation of the kinase is dependent upon their association in the presence of Ca²⁺. Cyclic AMP-dependent protein kinase phosphorylation of the myosin light chain kinase occurs at 2 sites. It decreases the affinity of the kinase for calmodulin and a reduction in the rate of light chain phosphorylation occurs. The kinase has an overall asymmetric shape composed of a globular head and tail region for the skeletal muscle enzyme. Trypsin digestion of this kinase releases a fragment of M_r 36,000 from the globular region that contains the catalytic and calmodulin binding sites. Chymotrypsin digestion of the kinase from smooth muscle generates a fragment of M_r 80,000 that does not contain the calmodulin binding or cyclic AMP-dependent protein kinase phosphorylation sites. It is a Ca²⁺-independent form of the kinase that phosphorylates the light chain of myosin. These structural features indicate a regulatory role for the kinase in smooth muscle phosphorylation and contraction.     

The role of calcium in aggregation and development ofDictyostelium

Changes in cytosolic Ca²⁺ play an important role in a wide array of cell types and the control of its concentration depends upon the interplay of many cellular constituents. Resting cells maintain cytosolic calcium ([Ca²⁺]_i) at a low level in the face of steep gradients of extracellular and sequestered Ca²⁺. Many different signals can provoke the opening of calcium channels in the plasma membrane or in intracellular compartments and cause rapid influx of Ca²⁺ into the cytosol and elevation of [Ca²⁺]_i. After such stimulation Ca²⁺ ATPases located in the plasma membrane and in the membranes of intracellular stores rapidly return [Ca²⁺]_i to its basal level. Such responses to elevation of [Ca²⁺]_i are a part of an important signal transduction mechanism that uses calcium (often via the binding protein calmodulin) to mediate a variety of cellular actions responsive to outside influences.     

Cytosolic free Ca2+ in insulin secreting cells and its regulation by isolated organelles

Summary The role of Ca²⁺ in secretagogue-induced insulin release is documented not only by the measurements of⁴⁵Ca fluxes in pancreatic islets, but also, by direct monitoring of cytosolic free Ca²⁺, [Ca²⁺]_i. As demonstrated, using the fluorescent indicator quin 2, glyceraldehyde, carbamylcholine and alanine raise [Ca²⁺]_i in the insulin secreting cell line RINm5F, whereas glucose has a similar effect in pancreatic islet cells. The regulation of cellular Ca²⁺ homeostasis by organelles from a rat insulinoma, was investigated with a Ca²⁺ selective electrode. The results suggest that both the endoplasmic reticulum and the mitochondria participate in this regulation, albeit at different Ca²⁺ concentrations. By contrast, the secretory granules do not appear to be involved in the short-term regulation of [Ca²⁺]_i. Evidence is presented that inositol 1,4,5-trisphosphate, which is shown to mobilize Ca²⁺ from the endoplasmic reticulum, is acting as an intracellular mediator in the stimulation of insulin release.     

Cytosolic calcium in platelet activation

Experiments with permeabilised platelets, and with intact platelets loaded with fluorescent Ca²⁺-indicators, over the past several years have greatly extended our knowledge and understanding of cytosolic Ca²⁺ as a platelet activator and its interactions with other cytosolic regulators. This article outlines insights, gained from the use of the fluorescent dyes, into maintenance and restoration of basal [Ca²⁺]_i, mechanisms of receptor-mediated Ca²⁺-mobilisation and quantitation of [Ca²⁺]_i/response relations in intact human platelets.     

Pyk2 cytonuclear localization: mechanisms and regulation by serine dephosphorylation

Cytonuclear signaling is essential for long-term alterations of cellular properties. Several pathways involving regulated nuclear accumulation of Ser/Thr kinases have been described but little is known about cytonuclear trafficking of tyrosine kinases. Proline-rich tyrosine kinase 2 (Pyk2) is a cytoplasmic non-receptor tyrosine kinase enriched in neurons and involved in functions ranging from synaptic plasticity to bone resorption, as well as in cancer. We previously showed the Ca²⁺-induced, calcineurin-dependent, nuclear localization of Pyk2. Here, we characterize the molecular mechanisms of Pyk2 cytonuclear localization in transfected PC12 cells. The 700–841 linker region of Pyk2 recapitulates its depolarization-induced nuclear accumulation. This region includes a nuclear export motif regulated by phosphorylation at residue S778, a substrate of cAMP-dependent protein kinase and calcineurin. Nuclear import is controlled by a previously identified sequence in the N-terminal domain and by a novel nuclear targeting signal in the linker region. Regulation of cytonuclear trafficking is independent of Pyk2 activity. The region regulating nuclear localization is absent from the non-neuronal shorter splice isoform of Pyk2. Our results elucidate the mechanisms of Ca²⁺-induced nuclear accumulation of Pyk2. They also suggest that Pyk2 nuclear accumulation is a novel type of signaling response that may contribute to specific long-term adaptations in neurons.     

Proinsulin C-peptide and its analogues induce intracellular Ca2+ increases in human renal tubular cells

Based on the findings that proinsulin C-peptide binds specifically to cell membranes, we investigated the effects of C-peptide and related molecules on the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in human renal tubular cells using the indicator fura-2/AM. The results show that human C-peptide and its C-terminal pentapeptide (positions 27–31, EGSLQ), but not the des (27–31) C-peptide or randomly scrambled C-peptide, elicit a transient increase in [Ca²⁺]_i. Rat C-peptide and rat C-terminal pentapeptide also induce a [Ca²⁺]_i response in human tubular cells, while a human pentapeptide analogue with Ala at position 1 gives no [Ca²⁺]_i response, and those with Ala at positions 2–5 induce responses with different amplitudes. These results define a species cross-reactivity for C-peptide and demonstrate the importance of Glu at position 1 of the pentapeptide. Preincubation of cells with pertussis toxin abolishes the effect on [Ca²⁺]_i by both C-peptide and the pentapeptide. These results are compatible with previous data on C-peptide binding to cells and activation of Na⁺,K⁺ATPase. Combined, all data show that C-peptide is a bioactive peptide and suggest that it elicits changes in [Ca²⁺]_i via G-protein-coupled pathways, giving downstream enzyme effects. Received 13 May 2002; accepted 16 May 2002     

Inhibition ofE. coli ATPase activity by a troponin component,TN-I,and by mitochondrial ATPase inhibitor

Summary The enzymic activity of Mg²⁺-or Ca²⁺-stimulated ATPase fromEscherichia coli was inhibited by one of the troponin components, TN-I, and by mitochondrial ATPase inhibitor (F₁-inhibitor). The inhibitory ability of component TN-I against Mg²⁺-stimulated ATPase activity was lost after digestion of component TN-I with trypsin. The Mg²⁺-stimulated ATPase activity inhibited by component TN-I was completely restored by the addition of another troponin component, TN-C.     

CD23 (the low-affinity IgE receptor) as a C-type lectin: a multidomain and multifunctional molecule

This review, regards the low-affinity receptor CD23 as a C-type lectin and compares it with other C-type lectins and C-type lectin-like receptors. C-type lectins such as the asialoglycoprotein receptor, as well as the dendritic cell immunoreceptor and the dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin on dendritic cell lectin, possess amino acid sequences which interact with Ca⁺⁺ and sugar, and many of them possess an endocytosis signal sequence that includes tyrosine or serine in the cytoplasmic region. In contrast, natural killer receptors lack the Ca⁺⁺ and sugar-binding amino acids but conserve homologous cysteines in the form of C-type lectin, and possess an immunoreceptor tyrosine-based inhibitory motif in the cytoplasmic region which inhibits killer activity when they recognize the self major histocompatibility (MHC) class I molecule. Since human CD23a form has a similar amino acid sequence, the possibility that this sequence is an endocytosis signal or an ITIM is discussed. The function of the reverse RGD and RGD-binding inhibitory peptide in human CD23 from the point of view of the relation between a C-type lectin and MHC class II molecules is also considered. Received 21 May 2001; received after revision 28 November 2001; accepted 29 November 2001     

Neuronal calcium signaling: function and dysfunction

Calcium (Ca²⁺) is an universal second messenger that regulates the most important activities of all eukaryotic cells. It is of critical importance to neurons as it participates in the transmission of the depolarizing signal and contributes to synaptic activity. Neurons have thus developed extensive and intricate Ca²⁺ signaling pathways to couple the Ca²⁺ signal to their biochemical machinery. Ca²⁺ influx into neurons occurs through plasma membrane receptors and voltage-dependent ion channels. The release of Ca²⁺ from the intracellular stores, such as the endoplasmic reticulum, by intracellular channels also contributes to the elevation of cytosolic Ca²⁺. Inside the cell, Ca²⁺ is controlled by the buffering action of cytosolic Ca²⁺-binding proteins and by its uptake and release by mitochondria. The uptake of Ca²⁺ in the mitochondrial matrix stimulates the citric acid cycle, thus enhancing ATP production and the removal of Ca²⁺ from the cytosol by the ATP-driven pumps in the endoplasmic reticulum and the plasma membrane. A Na⁺/Ca²⁺ exchanger in the plasma membrane also participates in the control of neuronal Ca²⁺. The impaired ability of neurons to maintain an adequate energy level may impact Ca²⁺ signaling: this occurs during aging and in neurodegenerative disease processes. The focus of this review is on neuronal Ca²⁺ signaling and its involvement in synaptic signaling processes, neuronal energy metabolism, and neurotransmission. The contribution of altered Ca²⁺ signaling in the most important neurological disorders will then be considered.