TRPM7 residue S1269 mediates cAMP dependence of Ca2+ influx.

The nonspecific divalent cation channel TRPM7 (transient receptor potential-melastatin-like 7) is involved in many Ca2+ and Mg2+-dependent cellular processes, including survival, proliferation and migration. TRPM7 expression predicts metastasis and recurrence in breast cancer and several other cancers. In cultured cells, it can induce an invasive phenotype by promoting Ca2+-mediated epithelial-mesenchymal transition. We previously showed that in neuroblastoma cells that overexpress TRPM7 moderately, stimulation with Ca2+-mobilizing agonists leads to a characteristic sustained influx of Ca2+. Here we report that sustained influx through TRPM7 is abruptly abrogated by elevating intracellular levels of cyclic adenosine monophosphate (cAMP). Using pharmacological inhibitors and overexpression studies we show that this blockage is mediated by the cAMP effector Protein Kinase A (PKA). Mutational analysis demonstrates that the Serine residue S1269, which is present proximal to the coiled-coil domain within the protein c-terminus, is responsible for sensitivity to cAMP.

Impaired mechanical, heat, and cold nociception in a murine model of genetic TACE/ADAM17 knockdown.

TNF-α-converting enzyme, a member of the ADAM (A disintegrin and metalloproteinase) protease family and also known as ADAM17, regulates inflammation and regeneration in health and disease. ADAM17 targets are involved in pain development and hypersensitivity in animal models of inflammatory and neuropathic pain. However, the role of ADAM17 in the pain pathway is largely unknown. Therefore, we used the hypomorphic ADAM17 (ADAM17ex/ex) mouse model to investigate the importance of ADAM17 in nociceptive behavior, morphology, and function of primary afferent nociceptors. ADAM17ex/ex mice were hyposensitive to noxious stimulation, showing elevated mechanical thresholds as well as impaired heat and cold sensitivity. Despite these differences, skin thickness and innervation were comparable to controls. Although dorsal root ganglia of ADAM17ex/ex mice exhibited normal morphology of peptidergic and nonpeptidergic neurons, a small but significant reduction in the number of isolectin ß-4-positive neurons was observed. Functional electrical properties of unmyelinated nociceptors showed differences in resting membrane potential, afterhyperpolarization, and firing patterns in specific subpopulations of sensory neurons in ADAM17ex/ex mice. However, spinal cord morphology and microglia activity in ADAM17ex/ex mice were not altered. Our data suggest that ADAM17 contributes to the processing of painful stimuli, with a complex mode of action orchestrating the function of neurons along the pain pathway.-Quarta, S., Mitric, M., Kalpachidou, T., Mair, N., Schiefermeier-Mach, N., Andratsch, M., Qi, Y., Langeslag, M., Malsch, P., Rose-John, S., Kress, M. Impaired mechanical, heat, and cold nociception in a murine model of genetic TACE/ADAM17 knockdown.

Identification of Chloride Channels CLCN3 and CLCN5 Mediating the Excitatory Cl- Currents Activated by Sphingosine-1-Phosphate in Sensory Neurons.

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid involved in numerous physiological and pathophysiological processes. We have previously reported a S1P-induced nocifensive response in mice by excitation of sensory neurons via activation of an excitatory chloride current. The underlying molecular mechanism for the S1P-induced chloride conductance remains elusive. In the present study, we identified two CLCN voltage-gated chloride channels, CLCN3 and CLCN5, which mediated a S1P-induced excitatory Cl- current in sensory neurons by combining RNA-seq, adenovirus-based gene silencing and whole-cell electrophysiological voltage-clamp recordings. Downregulation of CLCN3 and CLCN5 channels by adenovirus-mediated delivery of shRNA dramatically reduced S1P-induced Cl- current and membrane depolarization in sensory neurons. The mechanism of S1P-induced activation of the chloride current involved Rho GTPase but not Rho-associated protein kinase. Although S1P-induced potentiation of TRPV1-mediated ionic currents also involved Rho-dependent process, the lack of correlation of the S1P-activated Cl- current and the potentiation of TRPV1 by S1P suggests that CLCN3 and CLCN5 are necessary components for S1P-induced excitatory Cl- currents but not for the amplification of TRPV1-mediated currents in sensory neurons. This study provides a novel mechanistic insight into the importance of bioactive sphingolipids in nociception.

Reduced excitability of gp130-deficient nociceptors is associated with increased voltage-gated potassium currents and Kcna4 channel upregulation.

Neuropathic pain and pain arising from local inflammation are characterized by increased release of inflammatory mediators like interleukin-6 (IL-6) by immune cells. The levels of IL-6 is increased in various painfull conditions and correlates with the severity of thermal and mechanical hypersensitivity. Deletion of the IL-6 signal transducer glycoprotein 130 (gp130) reduces inflammation associated with hypersensitivity to thermal and mechanical stimuli. In this study, we show that nociceptor-specific deletion of gp130 alters excitability parameters that are linked to changes in the potassium conductance. In SNS-gp130(-/-) sensory neurons, the resting membrane potential was reduced. Moreover the repolarization speed of the action potential and afterhypolarization was augmented, however, voltage-gated Na(+) and Ca(2+) current were not obviously altered. The main difference between gp130-deficient and control neurons was a significant increase in the conductance of both delayed rectifier as well as A-type potassium currents. Taqman RT-PCR analysis revealed significantly higher levels of Kcna4 mRNA, encoding A-type Kv1.4 potassium channel, in neuron cultures from SNS-gp130(-/-) versus control mice, which may account for the electrophysiological data. No difference in other voltage-gated ion channel mRNAs was observed. The present data show for the first time increased A-type K(+) currents and expression of voltage-gated potassium channel Kcna4 (Kv1.4) in SNS-gp130(-/-) nociceptors. This suggests that gp130 acts as a break for the expression of potassium channels and important regulator hub for nociceptor excitability.

TRPM7 triggers Ca2+ sparks and invadosome formation in neuroblastoma cells.

Cell migration depends on the dynamic formation and turnover of cell adhesions and is tightly controlled by actomyosin contractility and local Ca2+ signals. The divalent cation channel TRPM7 (Transient Receptor Potential cation channel, subfamily Melastatin, member 7) has recently received much attention as a regulator of cell adhesion, migration and (localized) Ca2+ signaling. Overexpression and knockdown of TRPM7 affects actomyosin contractility and the formation of cell adhesions such as invadosomes and focal adhesions, but the role of TRPM7-mediated Ca2+ signals herein is currently not understood. Using Total Internal Reflection Fluorescence (TIRF) Ca2+ fluorometry and a novel automated analysis routine we have addressed the role of Ca2+ in the control of invadosome dynamics in N1E-115 mouse neuroblastoma cells. We find that TRPM7 promotes the formation of highly repetitive and localized Ca2+ microdomains or "Ca2+ sparking hotspots" at the ventral plasma membrane. Ca2+ sparking appears strictly dependent on extracellular Ca2+ and is abolished by TRPM7 channel inhibitors such as waixenicin-A. TRPM7 inhibition also induces invadosome dissolution. However, invadosome formation is (functionally and spatially) dissociated from TRPM7-mediated Ca2+ sparks. Rather, our data indicate that TRPM7 affects actomyosin contractility and invadosome formation independent of Ca2+ influx.

Identification of voltage-gated K(+) channel beta 2 (Kvß2) subunit as a novel interaction partner of the pain transducer Transient Receptor Potential Vanilloid 1 channel (TRPV1).

The Transient Receptor Potential Vanilloid 1 (TRPV1, vanilloid receptor 1) ion channel plays a key role in the perception of thermal and inflammatory pain, however, its molecular environment in dorsal root ganglia (DRG) is largely unexplored. Utilizing a panel of sequence-directed antibodies against TRPV1 protein and mouse DRG membranes, the channel complex from mouse DRG was detergent-solubilized, isolated by immunoprecipitation and subsequently analyzed by mass spectrometry. A number of potential TRPV1 interaction partners were identified, among them cytoskeletal proteins, signal transduction molecules, and established ion channel subunits. Based on stringent specificity criteria, the voltage-gated K(+) channel beta 2 subunit (Kvß2), an accessory subunit of voltage-gated K(+) channels, was identified of being associated with native TRPV1 channels. Reverse co-immunoprecipitation and antibody co-staining experiments confirmed TRPV1/Kvß2 association. Biotinylation assays in the presence of Kvß2 demonstrated increased cell surface expression levels of TRPV1, while patch-clamp experiments resulted in a significant increase of TRPV1 sensitivity to capsaicin. Our work shows, for the first time, the association of a Kvß subunit with TRPV1 channels, and suggests that such interaction may play a role in TRPV1 channel trafficking to the plasma membrane.

Spatiotemporal regulation of chloride intracellular channel protein CLIC4 by RhoA.

Chloride intracellular channel (CLIC) 4 is a soluble protein structurally related to omega-type glutathione-S-transferases (GSTs) and implicated in various biological processes, ranging from chloride channel formation to vascular tubulogenesis. However, its function(s) and regulation remain unclear. Here, we show that cytosolic CLIC4 undergoes rapid but transient translocation to discrete domains at the plasma membrane upon stimulation of G(13)-coupled, RhoA-activating receptors, such as those for lysophosphatidic acid, thrombin, and sphingosine-1-phosphate. CLIC4 recruitment is strictly dependent on Galpha(13)-mediated RhoA activation and F-actin integrity, but not on Rho kinase activity; it is constitutively induced upon enforced RhoA-GTP accumulation. Membrane-targeted CLIC4 does not seem to enter the plasma membrane or modulate transmembrane chloride currents. Mutational analysis reveals that CLIC4 translocation depends on at least six conserved residues, including reactive Cys35, whose equivalents are critical for the enzymatic function of GSTs. We conclude that CLIC4 is regulated by RhoA to be targeted to the plasma membrane, where it may function not as an inducible chloride channel but rather by displaying Cys-dependent transferase activity toward a yet unknown substrate.

Parathyroid hormone activates TRPV5 via PKA-dependent phosphorylation.

Low extracellular calcium (Ca(2+)) promotes release of parathyroid hormone (PTH), which acts on multiple organs to maintain overall Ca(2+) balance. In the distal part of the nephron, PTH stimulates active Ca(2+) reabsorption via the adenylyl cyclase-cAMP-protein kinase A (PKA) pathway, but the molecular target of this pathway is unknown. The transient receptor potential vanilloid 5 (TRPV5) channel constitutes the luminal gate for Ca(2+) entry in the distal convoluted tubule and has several putative PKA phosphorylation sites. Here, we investigated the effect of PTH-induced cAMP signaling on TRPV5 activity. Using fluorescence resonance energy transfer, we studied cAMP and Ca(2+) dynamics during PTH stimulation of HEK293 cells that coexpressed the PTH receptor and TRPV5. PTH increased cAMP levels, followed by a rise in TRPV5-mediated Ca(2+) influx. PTH (1 to 31) and forskolin, which activate the cAMP pathway, mimicked the stimulation of TRPV5 activity. Remarkably, TRPV5 activation was limited to conditions of strong intracellular Ca(2+) buffering. Cell surface biotinylation studies demonstrated that forskolin did not affect TRPV5 expression on the cell surface, suggesting that it alters the single-channel activity of a fixed number of TRPV5 channels. Application of the PKA catalytic subunit, which phosphorylated TRPV5, directly increased TRPV5 channel open probability. Alanine substitution of threonine-709 abolished both in vitro phosphorylation and PTH-mediated stimulation of TRPV5. In summary, PTH activates the cAMP-PKA signaling cascade, which rapidly phosphorylates threonine-709 of TRPV5, increasing the channel's open probability and promoting Ca(2+) reabsorption in the distal nephron.

Calcium channel kinetics of melanotrope cells in Xenopus laevis depend on environmental stimulation.

We have tested the hypothesis that the type and kinetics of voltage-activated Ca(2+) channels in a neuroendocrine cell depend on the cell's long-term external input. For this purpose, the presence and kinetics of both low (LVA) and high-voltage-activated (HVA) L-type Ca(2+) channels have been assessed in melanotrope pituitary cells of the amphibian Xenopus laevis. The secretory activity of this cell type can readily be manipulated in vivo by changing the animal's environmental light condition, from a black to a white background. We here show that, compared to white background-adapted Xenopus, melanotropes from black background-adapted frogs have (1) a much larger size, as revealed by their 2.5 times larger membrane capacitance (P<0.001), (2) a 2 times higher HVA current density (P<0.05), (3) a clearly smaller Ca(2+)-dependent inactivation (10%; P<0.05), (4) L-type channels with 5 times slower activation and inactivation kinetics (P<0.05), and (5) slower kinetics of L-type channels that become faster and more similar to those in white-background adapted cells when the intracellular Ca(2+)-buffering capacity is reduced. Furthermore, white-adapted melanotropes possess LVA-type Ca(2+) channels, which are lacking from cells from black-adapted animals. The melanotrope calmodulin mRNA level does not differ between the two adaptation states. These results indicate that HVA L-type channel kinetics differ in relation to environmentally induced changes in cellular secretory state, probably mediated via intracellular Ca(2+)-buffering, whereas the occurrence of LVA Ca(2+) channels may depend on environmentally controlled channel gene expression.

Myosin II and mechanotransduction: a balancing act.

Adherent cells respond to mechanical properties of the surrounding extracellular matrix. Mechanical forces, sensed at specialized cell-matrix adhesion sites, promote actomyosin-based contraction within the cell. By manipulating matrix rigidity and adhesion strength, new roles for actomyosin contractility in the regulation of basic cellular functions, including cell proliferation, migration and stem cell differentiation, have recently been discovered. These investigations demonstrate that a balance of forces between cell adhesion on the outside and myosin II-based contractility on the inside of the cell controls many aspects of cell behavior. Disturbing this balance contributes to the pathogenesis of various human diseases. Therefore, elaborate signaling networks have evolved that modulate myosin II activity to maintain tensional homeostasis. These include signaling pathways that regulate myosin light chain phosphorylation as well as myosin II heavy chain interactions.

Activation of TRPM7 channels by phospholipase C-coupled receptor agonists.

TRPM7 is a ubiquitously expressed nonspecific cation channel that has been implicated in cellular Mg(2+) homeostasis. We have recently shown that moderate overexpression of TRPM7 in neuroblastoma N1E-115 cells elevates cytosolic Ca(2+) levels and enhances cell-matrix adhesion. Furthermore, activation of TRPM7 by phospholipase C (PLC)-coupled receptor agonists caused a further increase in intracellular Ca(2+) levels and augmented cell adhesion and spreading in a Ca(2+)-dependent manner (1). Regulation of the TRPM7 channel is not well understood, although it has been reported that PIP(2) hydrolysis closes the channel. Here we have examined the regulation of TRPM7 by PLC-coupled receptor agonists such as bradykinin, lysophosphatidic acid, and thrombin. Using FRET assays for second messengers, we have shown that the TRPM7-dependent Ca(2+) increase closely correlates with activation of PLC. Under non-invasive "perforated patch clamp" conditions, we have found similar activation of TRPM7 by PLC-coupled receptor agonists. Although we could confirm that, under whole-cell conditions, the TRPM7 currents were significantly inhibited following PLC activation, this PLC-dependent inhibition was only observed when [Mg(2+)](i) was reduced below physiological levels. Thus, under physiological ionic conditions, TRPM7 currents were activated rather than inhibited by PLC-activating receptor agonists.