TRPM7 is regulated by halides through its kinase domain

Transient receptor potential melastatin 7 (TRPM7) is a divalent-selective cation channel fused to an atypical α-kinase. TRPM7 is a key regulator of cell growth and proliferation, processes accompanied by mandatory cell volume changes. Osmolarity-induced cell volume alterations regulate TRPM7 through molecular crowding of solutes that affect channel activity, including magnesium (Mg²⁺), Mg-nucleotides and a further unidentified factor. Here, we assess whether chloride and related halides can act as negative feedback regulators of TRPM7. We find that chloride and bromide inhibit heterologously expressed TRPM7 in synergy with intracellular Mg²⁺ ([Mg²⁺]_i) and this is facilitated through the ATP-binding site of the channel’s kinase domain. The synergistic block of TRPM7 by chloride and Mg²⁺ is not reversed during divalent-free or acidic conditions, indicating a change in protein conformation that leads to channel inactivation. Iodide has the strongest inhibitory effect on TRPM7 at physiological [Mg²⁺]_i. Iodide also inhibits endogenous TRPM7-like currents as assessed in MCF-7 breast cancer cells, where upregulation of SLC5A5 sodium-iodide symporter enhances iodide uptake and inhibits cell proliferation. These results indicate that chloride could be an important factor in modulating TRPM7 during osmotic stress and implicate TRPM7 as a possible molecular mechanism contributing to the anti-proliferative characteristics of intracellular iodide accumulation in cancer cells.     

TRPM6 kinase activity regulates TRPM7 trafficking and inhibits cellular growth under hypomagnesic conditions

The channel kinases TRPM6 and TRPM7 are both members of the melastatin-related transient receptor potential (TRPM) subfamily of ion channels and the only known fusions of an ion channel pore with a kinase domain. TRPM6 and TRPM7 form functional, tetrameric channel complexes at the plasma membrane by heteromerization. TRPM6 was previously shown to cross-phosphorylate TRPM7 on threonine residues, but not vice versa. Genetic studies demonstrated that TRPM6 and TRPM7 fulfill non-redundant functions and that each channel contributes uniquely to the regulation of Mg²⁺ homeostasis. Although there are indications that TRPM6 and TRPM7 can influence each other’s cellular distribution and activity, little is known about the functional relationship between these two channel-kinases. In the present study, we examined how TRPM6 kinase activity influences TRPM7 serine phosphorylation, intracellular trafficking, and cell surface expression of TRPM7, as well as Mg²⁺-dependent cellular growth. We found TRPM7 serine phosphorylation via the TRPM6 kinase, but no TRPM6 serine phosphorylation via the TRPM7 kinase. Intracellular trafficking of TRPM7 was altered in HEK-293 epithelial kidney cells and DT40 B cells in the presence of TRPM6 with intact kinase activity, independently of the availability of extracellular Mg²⁺, but TRPM6/7 surface labeling experiments indicate comparable levels of the TRPM6/7 channels at the plasma membrane. Furthermore, using a complementation approach in TRPM7-deficient DT40 B-cells, we demonstrated that wild-type TRPM6 inhibited cell growth under hypomagnesic cell culture conditions in cells co-expressing TRPM6 and TRPM7; however, co-expression of a TRPM6 kinase dead mutant had no effect—a similar phenotype was also observed in TRPM6/7 co-expressing HEK-293 cells. Our results provide first clues about how heteromer formation between TRPM6 and TRPM7 influences the biological activity of these ion channels. We show that TRPM6 regulates TRPM7 intracellular trafficking and TRPM7-dependent cell growth. All these effects are dependent upon the presence of an active TRPM6 kinase domain. Dysregulated Mg²⁺-homeostasis causes or exacerbates many pathologies. As TRPM6 and TRPM7 are expressed simultaneously in numerous cell types, understanding how their relationship impacts regulation of Mg²⁺-uptake is thus important knowledge.     

Modulation of signal transduction through the cellular prion protein is linked to its incorporation in lipid rafts

Because expressed at a significant level at the membrane of human T cells, we made the hypothesis that the cellular prion protein (PrP^c) could behave as a receptor, and be responsible for signal transduction. PrP^c engagement by specific antibodies was observed to induce an increase in cytosolic calcium concentration and led to enhanced activity of Src protein tyrosine kinases. Antibodies to CD4 and CD59 did not influence calcium fluxes or signaling. The effect was maximal after the formation of a network involving avidin and biotinylated antibody to PrP^c and was inhibited after raft disruption. PrP^c localization was not restricted to rafts in resting cells but engagement was a prerequisite for signaling induction, with concomitant PrP^c recruitment into rafts. These results suggest a role for PrP^c in signaling pathways, and show that lateral redistribution of the protein into rafts is important for subsequent signal transduction.Received 22 July 2004; received after revision 10 September 2004; accepted 7 October 2004     

Glycoprotein misfolding in the endoplasmic reticulum: identification of released oligosaccharides reveals a second ER-associated degradation pathway for Golgi-retrieved proteins

Endoplasmic reticulum-associated degradation (ERAD) is a key cellular process whereby misfolded proteins are removed from the endoplasmic reticulum (ER) for subsequent degradation by the ubiquitin/proteasome system. In the present work, analysis of the released, free oligosaccharides (FOS) derived from all glycoproteins undergoing ERAD, has allowed a global estimation of the mechanisms of this pathway rather than following model proteins through degradative routes. Examining the FOS produced in endomannosidase-compromised cells following α-glucosidase inhibition has revealed a mechanism for clearing Golgi-retrieved glycoproteins that have failed to enter the ER quality control cycle. The Glc₃Man₇GlcNAc₂ FOS species has been shown to be produced in the ER lumen by a mechanism involving a peptide: N-glycanase-like activity, and its production was sensitive to disruption of Golgi-ER trafficking. The detection of this oligosaccharide was unaffected by the overexpression of EDEM1 or cytosolic mannosidase, both of which increased the production of previously characterised cytosolically localised FOS. The lumenal FOS identified are therefore distinct in their production and regulation compared to FOS produced by the conventional route of misfolded glycoproteins directly removed from the ER. The production of such lumenal FOS is indicative of a novel degradative route for cellular glycoproteins that may exist under certain conditions.     

Poly(ADP-ribose)glycohydrolase is an upstream regulator of Ca<Superscript>2+</Superscript> fluxes in oxidative cell death

Oxidative DNA damage to cells activates poly(ADP-ribose)polymerase-1 (PARP-1) and the poly(ADP-ribose) formed is rapidly degraded to ADP-ribose by poly(ADP-ribose)glycohydrolase (PARG). Here we show that PARP-1 and PARG control extracellular Ca²⁺ fluxes through melastatin-like transient receptor potential 2 channels (TRPM2) in a cell death signaling pathway. TRPM2 activation accounts for essentially the entire Ca²⁺ influx into the cytosol, activating caspases and causing the translocation of apoptosis inducing factor (AIF) from the inner mitochondrial membrane to the nucleus followed by cell death. Abrogation of PARP-1 or PARG function disrupts these signals and reduces cell death. ADP-ribose-loading of cells induces Ca²⁺ fluxes in the absence of oxidative damage, suggesting that ADP-ribose is the key metabolite of the PARP-1/PARG system regulating TRPM2. We conclude that PARP-1/PARG control a cell death signal pathway that operates between five different cell compartments and communicates via three types of chemical messengers: a nucleotide, a cation, and proteins.     

The alpha-kinase family: an exceptional branch on the protein kinase tree

The alpha-kinase family represents a class of atypical protein kinases that display little sequence similarity to conventional protein kinases. Early studies on myosin heavy chain kinases in Dictyostelium discoideum revealed their unusual propensity to phosphorylate serine and threonine residues in the context of an alpha-helix. Although recent studies show that some members of this family can also phosphorylate residues in non-helical regions, the name alpha-kinase has remained. During evolution, the alpha-kinase domains combined with many different functional subdomains such as von Willebrand factor-like motifs (vWKa) and even cation channels (TRPM6 and TRPM7). As a result, these kinases are implicated in a large variety of cellular processes such as protein translation, Mg²⁺ homeostasis, intracellular transport, cell migration, adhesion, and proliferation. Here, we review the current state of knowledge on different members of this kinase family and discuss the potential use of alpha-kinases as drug targets in diseases such as cancer.     

The in vitro characterization of the inhibition of mouse brain protein kinase-C by retinoids and their receptors

Summary The mechanism of the in vitro inhibition of Ca²⁺-, phosphatidylserine-dependent protein kinase C (PK-C)² by the purifiedholo (ligand-saturated) forms of cellular retinol-binding protein (cRBP) and cellular retinoic acid-binding protein (cRABP) was studied. We report here that i) the PK-C-inhibitory action ofholo-cRBP andholo-cRABP is due to their respective ligands, all-trans-retinol and all-trans-retinoic acid; ii) the reduced phosphorylation of theholo-retinoid-binding proteins and brain cytosolic proteins is not the result of a retinoid-induced soluble phosphatase or protease activity; iii) retinoids reduce PK-C affinity for calcium and phosphatidylserine in vitro; and iv) the structure-function activity of the retinoids and the specific interaction of these effect of retinoids on plasma membrane-associated PK-C activity pays a significant role in defining the early epigenetic aspects of PK-C-dependent tumor promotion and may be a physiological mechanism by which retinoids induce terminal differentiation in cell types that do not express soluble retinoid-binding proteins.We would like to thank Dr L.M. De Luca (NIH, USA) for his contribution of retinylphosphate, Dr H.N. Bhagavan (Hoffmann-La Roche) for his contribution of the arotinoids, and Merrill-Dow Corp. for their contribution of difluoromethylornithine. This work was supported by NIH Grants CA-34968, CA-07175, CA-22484, and CA-09020.     

Insulin-producing cells in the brain of adult Drosophila are regulated by the serotonin 5-HT1A receptor

Insulin signaling regulates lifespan, reproduction, metabolic homeostasis, and resistance to stress in the adult organism. In Drosophila, there are seven insulin-like peptides (DILP1–7). Three of these (DILP2, 3 and 5) are produced in median neurosecretory cells of the brain, designated IPCs. Previous work has suggested that production or release of DILPs in IPCs can be regulated by a factor secreted from the fat body as well as by neuronal GABA or short neuropeptide F. There is also evidence that serotonergic neurons may regulate IPCs. Here, we investigated mechanisms by which serotonin may regulate the IPCs. We show that the IPCs in adult flies express the 5-HT_1A, but not the 5-HT_1B or 5-HT₇ receptors, and that processes of serotonergic neurons impinge on the IPC branches. Knockdown of 5-HT_1A in IPCs by targeted RNA interference (RNAi) leads to increased sensitivity to heat, prolonged recovery after cold knockdown and decreased resistance to starvation. Lipid metabolism is also affected, but no effect on growth was seen. Furthermore, we show that DILP2-immunolevels in IPCs increase after 5-HT_1A knockdown; this is accentuated by starvation. Heterozygous 5-HT_1A mutant flies display the same phenotype in all assays, as seen after targeted 5-HT_1A RNAi, and flies fed the 5-HT_1A antagonist WAY100635 display reduced lifespan at starvation. Our findings suggest that serotonin acts on brain IPCs via the 5-HT_1A receptor, thereby affecting their activity and probably insulin signaling. Thus, we have identified a second inhibitory pathway regulating IPC activity in the Drosophila brain.     

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.     

Der Einfluss von Acetylcholin auf den Calciumumsatz ruhender und kontrahierender Vorhofmuskulaturin vitro

Summary Acetylcholine 5 × 10^–8 g/ml reduces the Ca⁴⁵ uptake of the beating left atria of guinea-pig; the tissue calcium is not altered. In resting atria, acetylcholine 5 × 10^–7 g/ml has no influence upon the calcium content and Ca⁴⁵ uptake. It is concluded that acetylcholine acts by shortening the action potential duration and thereby reduces the release of cellular calcium per excitation.

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Mit grosszügiger Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Breathing at high altitude

Acclimatization to long-term hypoxia takes place at high altitude and allows gradual improvement of the ability to tolerate the hypoxic environment. An important component of this process is the hypoxic ventilatory acclimatization (HVA) that develops over several days. HVA reveals profound cellular and neurochemical re-organization occurring both in the peripheral chemoreceptors and in the central nervous system (in brainstem respiratory groups). These changes lead to an enhanced activity of peripheral chemoreceptor and re-inforce the central translation of peripheral inputs to efficient respiratory motor activity under the steady low O₂ pressure. We will review the cellular processes underlying these changes with a particular emphasis on changes of neurotransmitter function and ion channel properties in peripheral chemoreceptors, and present evidence that low O₂ level acts directly on brainstem nuclei to induce cellular changes contributing to maintain a high tonic respiratory drive under chronic hypoxia. (This study is part of a multi-author review.)     

The in vitro characterization of the inhibition of mouse brain protein kinase-C by retinoids and their receptors

The mechanism of the in vitro inhibition of Ca2+-, phosphatidylserine-dependent protein kinase C (PK-C)2 by the purified holo (ligand-saturated) forms of cellular retinol-binding protein (cRBP) and cellular retinoic acid-binding protein (cRABP) was studied. We report here that the PK-C-inhibitory action of holo-cRBP and holo-cRABP is due to their respective ligands, all-trans-retinol and all-trans-retinoic acid; the reduced phosphorylation of the holo-retinoid-binding proteins and brain cytosolic proteins is not the result of a retinoid-induced soluble phosphatase or protease activity; retinoids reduce PK-C affinity for calcium and phosphatidylserine in vitro; and the structure-function activity of the retinoids and the specific interaction of these compounds with their binding proteins are important in blocking the activity of PK-C. These observations suggest that the inhibitory effect of retinoids on plasma membrane-associated PK-C activity pays a significant role in defining the early epigenetic aspects of PK-C-dependent tumor promotion and may be a physiological mechanism by which retinoids induce terminal differentiation in cell types that do not express soluble retinoid-binding proteins.     

Oxidative stress triggers neuronal caspase-independent death: Endonuclease G involvement in programmed cell death-type III

To characterize neuronal death, primary cortical neurons (C57/Black 6 J mice) were exposed to hydrogen peroxide (H₂O₂) and staurosporine. Both caused cell shrinkage, nuclear condensation, DNA fragmentation and loss of plasma membrane integrity. Neither treatment induced caspase-7 activity, but caspase-3 was activated by staurosporine but not H₂O₂. Each treatment caused redistribution from mitochondria of both endonuclease G (Endo G) and cytochrome c. Neurons knocked down for Endo G expression using siRNA showed reduction in both nuclear condensation and DNA fragmentation after treatment with H₂O₂, but not staurosporine. Endo G suppression protected cells against H₂O₂-induced cell death, while staurosporine-induced death was merely delayed. We conclude that staurosporine induces apoptosis in these neurons, but severe oxidative stress leads to Endo G-dependent death, in the absence of caspase activation (programmed cell death-type III). Therefore, oxidative stress triggers in neurons a form of necrosis that is a systematic cellular response subject to molecular regulation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

New antimicrobial activity for the catecholamine release-inhibitory peptide from chromogranin A

Catestatin (bCGA_344–364), an endogenous peptide of bovine chromogranin A, was initially characterized for its effect on the inhibition of catecholamine release from chromaffin cells. Catestatin and its active domain (bCGA_344–358) were identified in chromaffin cells and in secretion medium. The present study identified a potent antimicrobial activity of bCGA_344–358 in the lowmicromolar range against bacteria, fungi and yeasts, without showing any haemolytic activity. Confocal laser microscopy demonstrated penetration of the rhodaminated peptide into the cell membranes of fungi and yeasts and its intracellular accumulation. Time-lapse videomicroscopy showed arrest of fungal growth upon penetration of the labelled peptide into a fungal filament. We identified several catestatin-containing fragments in the stimulated secretion medium of human polymorphonuclear neutrophils, suggesting the N-terminal sequence of catestatin (bCGA_344–358) (named cateslytin) as a novel component of innate immunity.Received 21 October 2004; received after revision 6 December 2004; accepted 7 December 2004     

Der Einfluss von Calcium auf die Brenzcatechinaminfreisetzung

Summary (1) Experiments on isolated perfused suprarenals of cattle have shown that the acetylcholine-induced release of catechol amines, but not that of phenylethylamine, is dependent on calcium, since a perfusion with calcium-free Tyrode's solution abolishes the action of acetylcholine and not that of phenylethylamine. (2) Inincubation experiments with isolated chromaffin granules, calcium produces a dose-dependent, significant release of catechol amines even in physiological concentrations (2.5 mM). (3) Acetylcholine does not release catechol amines from isolated granules, either in the presence or in the absence of calcium.

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Forschungs-Stipendiat der A. von Humboldt-Stiftung.

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Ausgeführt mit Unterstützung der Deutschen Forschungsgemeinschaft.     

Triadin: a multi-protein family for which purpose?

Triadin is a protein first identified as a member of the muscle calcium release complex, involved in calcium release for muscle contraction. However, its precise function in this complex is still undefined. Recently, triadin has been shown to be a multi-protein family, with different distribution of the various splice variants within the sarcoplasmic reticulum, raising the possibility of multiple functions for this family of polypeptides. Such functions may include involvement in excitation-contraction coupling, in triad targeting, in structural function or in muscle differentiation. The putative role(s) of triadin(s) will be discussed here.Received 5 May 2004; received after revision 4 June 2004; accepted 7 June 2004     

Cadmium-induced autophagy and apoptosis are mediated by a calcium signaling pathway

The cytotoxicity of cadmium (Cd) induced autophagy and apoptosis in MES-13 cells was determined by flow cytometry. Autophagy was also assessed by formation of autophagosomes and processing of LC3. Pharmacological inhibition of autophagy resulted in increased of cell viability, suggesting autophagy plays a role in cell death in Cd-treated mesangial cells. Cd also induced a rapid elevation in cytosolic calcium ([Ca²⁺]_i ), and modulation of [Ca²⁺]_i via treatment with IP ₃R inhibitor or knockdown of calcineurin resulted in a change in the proportion of cell death, suggesting that the release of calcium from the ER plays a crucial role in Cd-induced cell death. Inhibition of Cd-induced ERK activation by PD 98059 suppressed Cd-induced autophagy, and BAPTA-AM eliminated activation of ERK. BAPTA-AM also inhibited Cd-induced mitochondrial depolarization and activation of caspases. These findings demonstrated that Cd induces both autophagy and apoptosis through elevation of [Ca²⁺]_i, followed by Ca²⁺-ERK and Ca²⁺-mitochondria-caspase signaling pathways. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Received 05 July 2008; received after revision 25 August 2008; accepted 17 September 2008     

A llama-derived gelsolin single-domain antibody blocks gelsolin–G-actin interaction

RNA interference has tremendously advanced our understanding of gene function but recent reports have exposed undesirable side-effects. Recombinant Camelid single-domain antibodies (VHHs) provide an attractive means for studying protein function without affecting gene expression. We raised VHHs against gelsolin (GsnVHHs), a multifunctional actin-binding protein that controls cellular actin organization and migration. GsnVHH-induced delocalization of gelsolin to mitochondria or the nucleus in mammalian cells reveals distinct subpopulations including free gelsolin and actin-bound gelsolin complexes. GsnVHH 13 specifically recognizes Ca²⁺-activated gelsolin (K _d ~10 nM) while GsnVHH 11 binds gelsolin irrespective of Ca²⁺ (K _d ~5 nM) but completely blocks its interaction with G-actin. Both GsnVHHs trace gelsolin in membrane ruffles of EGF-stimulated MCF-7 cells and delay cell migration without affecting F-actin severing/capping or actin nucleation activities by gelsolin. We conclude that VHHs represent a potent way of blocking structural proteins and that actin nucleation by gelsolin is more complex than previously anticipated.     

Neurosteroid enhances glutamate release in rat prelimbic cortex via activation of α1-adrenergic and σ1 receptors

The present paper studied the effect and mechanism of neurosteroid pregnenolone sulfate (PREGS) on spontaneous glutamate release using electrophysiological and biochemical methods combined with a pharmacological approach. The results suggested that PREGS had a selective enhancing effect on spontaneous glutamate release in the prelimbic cortex and the hippocampus but not in the striatum. The effect of PREGS in the prelimbic cortex appeared to be via modulation of ₁-adrenergic and ₁ receptors, but in the hippocampus it might be dependent on ₁ receptors only. The activation of ₁-adrenergic receptors synergized ₁ receptor activation in the prelimbic cortex. Intracellular calcium released from the endoplasmic reticulum, protein kinase C, adenylyl cyclase and protein kinase A played a key role in the effect of PREGS. Intracellular calcium, protein kinase C and adenylyl cyclase might be upstream events in the activation of protein kinase A after PREGS.Received 7 January 2005; received after revision 19 February 2005; accepted 22 February 2005 Available online 29 March 2005     

RTA2 is involved in calcineurin-mediated azole resistance and sphingoid long-chain base release in Candida albicans

The calcineurin pathway has been reported to be essential for the development of azole resistance in Candida albicans. The depletion or ectopic over-expression of RTA2 increased or decreased susceptibility of C. albicans to azoles, respectively. CaCl₂- induced activation of the calcineurin pathway in wildtype C. albicans promoted resistance to azoles, while the Ca ²⁺ chelator (EGTA), calcineurin inhibitors (FK506 and cyclosporin A) and the deletion of RTA2 blocked the resistance-promoting effects of CaCl₂. Furthermore, we found that RTA2 was up-regulated in a calcineurin-dependent manner. The depletion of RTA2 also made the cell membrane of C. albicans liable to be destroyed by azoles and RTA2 over-expression attenuated the destroying effects. Finally, the disruption of RTA2 caused an increased accumulation of dihydrosphingosine (DHS), one of the two sphingolipid long-chain bases, by decreasing release of DHS. In conclusion, our findings suggest that RTA2 is involved in calcineurin-mediated azole resistance and sphingoid long-chain base release in C. albicans. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Received 14 July 2008; received after revision 29 August 2008; accepted 16 September 2008