scholarly journals The role of calmodulin-binding sites in the regulation of the Drosophila TRPL cation channel expressed in Xenopus laevis oocytes by Ca2+, inositol 1,4,5-trisphosphate and GTP-binding proteins

1998 ◽  
Vol 330 (3) ◽  
pp. 1149-1158 ◽  
Author(s):  
Ling LAN ◽  
Helen BRERETON ◽  
J. Greg BARRITT
Keyword(s):  
Binding Sites ◽  
Transient Receptor Potential ◽  
Receptor Potential ◽  
Protein S ◽  
Tryptophan Residue ◽  
Xenopus Laevis Oocytes ◽  
Calmodulin Binding ◽  
Gtp Binding ◽  
Inositol 1,4,5 Trisphosphate ◽  
Stimulation Of

The roles of calmodulin-binding sites in the regulation by Ca2+, inositol 1,4,5-trisphosphate (InsP3) and GTP-binding regulatory proteins (G-proteins) of the Drosophila melanogaster TRPL (transient-receptor-potential-like) non-specific Ca2+ channel were investigated. Wild-type TRPL protein and two mutant forms, TRPL (W713G) and TRPL (W814G), in which a key tryptophan residue in each of the two putative calmodulin-binding sites (Sites 1 and 2, respectively) was replaced by glycine, were expressed heterologously in Xenopuslaevis oocytes. Immunofluorescence studies indicated that the expressed TRPL, TRPL (W713G) and TRPL (W814G) proteins are located at the plasma membrane. TRPL oocytes (oocytes injected with trpl cRNA) and TRPL (W814G) oocytes [oocytes injected with trpl (W814G) cRNA] exhibited substantially greater rates of basal (constitutive) Ca2+ inflow (measured using fluo-3 and the Ca2+ add-back protocol) than mock-injected oocytes (mock oocytes). In TRPL (W713G) oocytes, this difference was abolished. In TRPL and TRPL (W814G) [oocytes injected with trpl (W713G) cRNA], but not in TRPL (W713G) oocytes, basal Ca2+ inflow was inhibited by W13, an inhibitor of calmodulin action. Calmodulin (3 μM intracellular) inhibited basal Ca2+ inflow in TRPL but not in TRPL (W713G) or TRPL (W814G) oocytes. Staurosporin, an inhibitor of protein kinase C (PKC), inhibited, while PMA (an activator of PKC) stimulated, basal Ca2+ inflow in TRPL oocytes. In oocytes incubated in the presence of PMA (to suppress Ca2+ inflow through endogenous receptor-activated Ca2+ channels), the InsP3-induced stimulation of Ca2+ inflow through TRPL channels was more clearly evident than in oocytes incubated in the absence of PMA. InsP3 caused a significant stimulation of Mn2+ inflow in TRPL but not in mock oocytes. Rates of InsP3-stimulated Ca2+ inflow through the TRPL, TRPL (W713G) and TRPL (W814G) channels were similar. The ability of GTPγS to stimulate Ca2+ inflow through TRPL channels was inhibited by 50% in TRPL (W713G) oocytes but was unaffected in TRPL (W814G) oocytes. It is concluded that, in the environment of the Xenopus oocyte, the Drosophila TRPL channel is activated by (a) interaction with Ca2+/calmodulin at calmodulin-binding Site 1; (b) PKC; (c) InsP3 in a process that does not involve Ca2+ and calmodulin; and (d) a trimeric G-protein(s) through both a Ca2+/calmodulin-dependent and a Ca2+/calmodulin-independent mechanism.

Inhibition of TASK-1 potassium channel by phospholipase C

2001 ◽  
Vol 281 (2) ◽  
pp. C700-C708 ◽  
Author(s):  
Gábor Czirják ◽  
Gábor L. Petheő ◽  
András Spät ◽  
Péter Enyedi
Keyword(s):  
Phospholipase C ◽  
Lysophosphatidic Acid ◽  
Protein S ◽  
Receptor Activation ◽  
Xenopus Laevis Oocytes ◽  
Ang Ii ◽  
Inositol 1,4,5 Trisphosphate ◽  
Glomerulosa Cells ◽  
Pore Domain ◽  
Stimulation Of

The two-pore-domain K+ channel, TASK-1, was recently shown to be a target of receptor-mediated regulation in neurons and in adrenal glomerulosa cells. Here, we demonstrate that TASK-1 expressed in Xenopus laevis oocytes is inhibited by different Ca2+-mobilizing agonists. Lysophosphatidic acid, via its endogenous receptor, and ANG II and carbachol, via their heterologously expressed ANG II type 1a and M1 muscarinic receptors, respectively, inhibit TASK-1. This effect can be mimicked by guanosine 5′- O-(3-thiotriphosphate), indicating the involvement of GTP-binding protein(s). The phospholipase C inhibitor U-73122 reduced the receptor-mediated inhibition of TASK-1. Downstream signals of phospholipase C action (inositol 1,4,5-trisphosphate, cytoplasmic Ca2+ concentration, and diacylglycerol) do not mediate the inhibition. Unlike the Gq-coupled receptors, stimulation of the Gi-activating M2 muscarinic receptor coexpressed with TASK-1 results in an only minimal decrease of the TASK-1 current. However, additional coexpression of phospholipase C-β2 (which is responsive also to Giβγ-subunits) renders M2 receptor activation effective. This indicates the significance of phospholipase C activity in the receptor-mediated inhibition of TASK-1.

scholarly journals Identification and characterization of two distinct calmodulin-binding sites in the Trpl ion-channel protein of Drosophila melanogaster

1996 ◽  
Vol 314 (2) ◽  
pp. 497-503 ◽  
Author(s):  
Coral G. WARR ◽  
Leonard E. KELLY
Keyword(s):  
Protein Kinase ◽  
Ion Channel ◽  
Binding Sites ◽  
Transient Receptor Potential ◽  
Receptor Potential ◽  
Free Form ◽  
Calmodulin Binding ◽  
Dependent Protein ◽  
Transient Receptor

Two putative light-sensitive ion channels have been isolated from Drosophila, encoded by the transient-receptor-potential (trp) and transient-receptor-potential-like (trpl) genes. The cDNA encoding the Trpl protein was initially isolated on the basis that the expressed protein binds calmodulin. Using both fusion proteins and a synthetic peptide, we now show that two calmodulin-binding sites are present in the C-terminal domain of the Trpl protein, CBS-1 and CBS-2. CBS-1 binds calmodulin in a Ca2+-dependent fashion, requiring Ca2+ concentrations above 0.3–0.5 μM for calmodulin binding. In contrast, CBS-2 binds the Ca2+-free form of calmodulin, with dissociation occurring at Ca2+ concentrations between 5 and 25 μM. Phosphorylation of a serine residue within a peptide encompassing CBS-1 by cyclic AMP-dependent protein kinase (PKA) abolishes calmodulin binding, and phosphorylation of the adjacent serine by protein kinase C appears to modulate this phosphorylation by PKA. Interpretation of these findings provides a novel model for ion-channel gating and modulation in response to changing levels of intracellular Ca2+.

scholarly journals The transient receptor potential, TRP4, cation channel is a novel member of the family of calmodulin binding proteins

2001 ◽  
Vol 355 (3) ◽  
pp. 663-670 ◽  
Author(s):  
Claudia TROST ◽  
Christiane BERGS ◽  
Nina HIMMERKUS ◽  
Veit FLOCKERZI
Keyword(s):  
Amino Acid ◽  
Ion Channels ◽  
Transient Receptor Potential ◽  
Direct Interaction ◽  
Receptor Potential ◽  
Amino Acid Residues ◽  
Glutathione S Transferase ◽  
Calmodulin Binding ◽  
Transient Receptor

The mammalian gene products, transient receptor potential (trp)1 to trp7, are related to the Drosophila TRP and TRP-like ion channels, and are candidate proteins underlying agonist-activated Ca2+-permeable ion channels. Recently, the TRP4 protein has been shown to be part of native store-operated Ca2+-permeable channels. These channels, most likely, are composed of other proteins in addition to TRP4. In the present paper we report the direct interaction of TRP4 and calmodulin (CaM) by: (1) retention of in vitro translated TRP4 and of TRP4 protein solubilized from bovine adrenal cortex by CaM–Sepharose in the presence of Ca2+, and (2) TRP4–glutathione S-transferase pull-down experiments. Two domains of TRP4, amino acid residues 688–759 and 786–848, were identified as being able to interact with CaM. The binding of CaM to both domains occurred only in the presence of Ca2+ concentrations above 10µM, with half maximal binding occurring at 16.6µM (domain 1) and 27.9µM Ca2+ (domain 2). Synthetic peptides, encompassing the two putative CaM binding sites within these domains and covering amino acid residues 694–728 and 829–853, interacted directly with dansyl–CaM with apparent Kd values of 94–189nM. These results indicate that TRP4/Ca2+-CaM are parts of a signalling complex involved in agonist-induced Ca2+ entry.

scholarly journals Efficient and Flexible Synthesis of New Photoactivatable Propofol Analogs

2020 ◽  
Author(s):  
Kenneth Skinner ◽  
Joseph Wzorek ◽  
Daniel Kahne ◽  
Rachelle Gaudet
Keyword(s):  
Binding Sites ◽  
Transient Receptor Potential ◽  
Receptor Potential ◽  
General Anesthetic ◽  
Bioorthogonal Chemistry ◽  
Functional Assays ◽  
Pain Pathway ◽  
Synthetic Routes ◽  
Transient Receptor ◽  
Proof Of Principle

Propofol is a widely used general anesthetic, which acts by binding to and modulating several neuronal ion channels. We describe the synthesis of photoactivatable propofol analogs functionalized with an alkyne handle for bioorthogonal chemistry. Such tools are useful for detecting and isolating photolabeled proteins. We designed expedient and flexible synthetic routes to three new diazirine-based crosslinkable propofol derivatives, two of which have alkyne handles. As a proof of principle, we show that these compounds activate heterologously expressed Transient Receptor Potential Ankyrin 1 (TRPA1), a key ion channel of the pain pathway, with a similar potency as propofol in fluorescence-based functional assays. This work demonstrates that installation of the crosslinkable and clickable group on a short nonpolar spacer at the para position of propofol does not affect TRPA1 activation, supporting the utility of these chemical tools in identifying and characterizing potentially druggable binding sites in propofolinteracting proteins.

scholarly journals Polyamines regulate intestinal epithelial restitution through TRPC1-mediated Ca2+ signaling by differentially modulating STIM1 and STIM2

2012 ◽  
Vol 303 (3) ◽  
pp. C308-C317 ◽  
Author(s):  
Jaladanki N. Rao ◽  
Navneeta Rathor ◽  
Ran Zhuang ◽  
Tongtong Zou ◽  
Lan Liu ◽  
...  
Keyword(s):  
Cell Migration ◽  
Intestinal Epithelial Cell ◽  
Transient Receptor Potential ◽  
Receptor Potential ◽  
Intestinal Epithelial ◽  
Canonical Transient Receptor Potential ◽  
Pathological Conditions ◽  
Epithelial Restitution ◽  
Transient Receptor ◽  
Stimulation Of

Early epithelial restitution occurs as a consequence of intestinal epithelial cell (IEC) migration after wounding, and its defective regulation is implicated in various critical pathological conditions. Polyamines stimulate intestinal epithelial restitution, but their exact mechanism remains unclear. Canonical transient receptor potential-1 (TRPC1)-mediated Ca2+ signaling is crucial for stimulation of IEC migration after wounding, and induced translocation of stromal interaction molecule 1 (STIM1) to the plasma membrane activates TRPC1-mediated Ca2+ influx and thus enhanced restitution. Here, we show that polyamines regulate intestinal epithelial restitution through TRPC1-mediated Ca2+ signaling by altering the ratio of STIM1 to STIM2. Increasing cellular polyamines by ectopic overexpression of the ornithine decarboxylase (ODC) gene stimulated STIM1 but inhibited STIM2 expression, whereas depletion of cellular polyamines by inhibiting ODC activity decreased STIM1 but increased STIM2 levels. Induced STIM1/TRPC1 association by increasing polyamines enhanced Ca2+ influx and stimulated epithelial restitution, while decreased formation of the STIM1/TRPC1 complex by polyamine depletion decreased Ca2+ influx and repressed cell migration. Induced STIM1/STIM2 heteromers by polyamine depletion or STIM2 overexpression suppressed STIM1 membrane translocation and inhibited Ca2+ influx and epithelial restitution. These results indicate that polyamines differentially modulate cellular STIM1 and STIM2 levels in IECs, in turn controlling TRPC1-mediated Ca2+ signaling and influencing cell migration after wounding.

scholarly journals Activation of TRPV1 channels leads to stimulation of NKCC1 cotransport in the lens

2018 ◽  
Vol 315 (6) ◽  
pp. C793-C802 ◽  
Author(s):  
Mohammad Shahidullah ◽  
Amritlal Mandal ◽  
Nicholas A. Delamere
Keyword(s):  
Ion Transport ◽  
Transient Receptor Potential ◽  
Ion Homeostasis ◽  
Receptor Potential ◽  
Transient Receptor Potential Vanilloid ◽  
Signaling Mechanism ◽  
Trpv1 Channels ◽  
Trpv1 Antagonist ◽  
Transient Receptor ◽  
Stimulation Of

Lens ion homeostasis is crucial in maintaining water content and, in turn, refractive index and transparency of the multicellular syncytium-like structure. New information is emerging on the regulation of ion transport in the lens by mechanisms that rely on transient receptor potential vanilloid (TRPV) ion channels. We found recently that TRPV1 activation leads to Ca2+/PKC-dependent ERK1/2 signaling. Here, we show that the TRPV1 agonist capsaicin (100 nM) and hyperosmotic solution (350 vs. 300 mosM) each caused an increase of bumetanide-inhibitable Rb uptake by intact porcine lenses and Na-K-2Cl cotransporter 1 (NKCC1) phosphorylation in the lens epithelium. The TRPV1 antagonist A889425 (1 µM) abolished the increases of Rb uptake and NKCC1 phosphorylation in response to hyperosmotic solution. Exposing lenses to hyperosmotic solution in the presence of MEK/ERK inhibitor U0126 (10 µM) or the with-no-lysine kinase (WNK) inhibitor WNK463 (1 µM) also prevented NKCC1 phosphorylation and the Rb uptake responses to hyperosmotic solution. WNK463 did not prevent the increase in ERK1/2 phosphorylation that occurs in response to capsaicin or hyperosmotic solution, suggesting that ERK1/2 activation occurs before WNK activation in the sequence of signaling events. Taken together, the evidence indicates that activation of TRPV1 is a critical early step in a signaling mechanism that responds to a hyperosmotic stimulus, possibly lens shrinkage. By activating ERK1/2 and WNK, TRPV1 activation leads to NKCC1 phosphorylation and stimulation of NKCC1-mediated ion transport.

Importance of melastatin-like transient receptor potential 7 and magnesium in the stimulation of osteoblast proliferation and migration by platelet-derived growth factor

2009 ◽  
Vol 297 (2) ◽  
pp. C360-C368 ◽  
Author(s):  
Elie Abed ◽  
Robert Moreau
Keyword(s):  
Growth Factor ◽  
Transient Receptor Potential ◽  
Receptor Potential ◽  
Platelet Derived Growth Factor ◽  
Human Osteoblast ◽  
Osteoblast Proliferation ◽  
Transient Receptor ◽  
And Migration ◽  
Stimulation Of ◽  
Proliferation And Migration

Bone is a dynamic tissue that is continuously being remodeled throughout life. Specialized cells called osteoclasts transiently break down old bone (resorption process) at multiple sites as other cells known as osteoblasts are replacing it with new tissue (bone formation). Usually, both resorption and formation processes are in balance and thereby maintain skeletal strength and integrity. This equilibrium is assured by the coordination of proliferation, migration, differentiation, and secretory functions of the osteoblasts, which are essential for adequate formation and resorption processes. Disturbances of this equilibrium may lead to decreased bone mass (osteoporosis), increased bone fragility, and susceptibility to fractures. Epidemiological studies have linked insufficient dietary magnesium (Mg2+) intake in humans with low bone mass and osteoporosis. Here, we investigated the roles of Mg2+ and melastatin-like transient receptor potential 7 (TRPM7), known as Mg2+ channels, in human osteoblast cell proliferation and migration induced by platelet-derived growth factor (PDGF), which has been involved in the bone remodeling process. PDGF promoted an influx of Mg2+, enhanced cell migration, and stimulated the gene expression of TRPM7 channels in human osteoblast MG-63 cells. The stimulation of osteoblast proliferation and migration by PDGF was significantly reduced under culture conditions of low extracellular Mg2+ concentrations. Silencing TRPM7 expression in osteoblasts by specific small interfering RNA prevented the induction by PDGF of Mg2+ influx, proliferation, and migration. Our results indicate that extracellular Mg2+ and TRPM7 are important for PDGF-induced proliferation and migration of human osteoblasts. Thus Mg2+ deficiency, a common condition among the general population, may be associated with altered osteoblast functions leading to inadequate bone formation and the development of osteoporosis.

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