scholarly journals Ca2+-calmodulin inhibits Ca2+ release mediated by type-1, -2 and -3 inositol trisphosphate receptors

2000 ◽  
Vol 345 (2) ◽  
pp. 357-363 ◽  
Author(s):  
Charles E. ADKINS ◽  
Stephen A. MORRIS ◽  
Humbert DE SMEDT ◽  
Ilse SIENAERT ◽  
Katalin TÖRÖK ◽  
...  
Keyword(s):  
Calcineurin Inhibitors ◽  
Cell Types ◽  
Receptor Subtypes ◽  
Glutathione S Transferase ◽  
Calmodulin Binding ◽  
Insp3 Receptors ◽  
Adenophostin A ◽  
A Site ◽  
Insp3 Receptor

InsP3 binding to type-1, but not type-3, InsP3 receptors is inhibited by calmodulin in a Ca2+-independent fashion [Cardy and Taylor (1998) Biochem. J. 334, 447-455], and Ca2+ mobilization by type-1 InsP3 receptors of cerebellum is inhibited by calmodulin [Patel, Morris, Adkins, O'Beirne and Taylor (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 11627-11632]. Using cell types expressing predominantly type-1, -2 or -3 InsP3 receptors, we show that InsP3-evoked Ca2+ mobilization from each is similarly inhibited by calmodulin. In SH-SY5Y cells, which express largely type-1 receptors, calmodulin (IC50 ≈ 15 μM) inhibited InsP3-evoked Ca2+ release only in the presence of Ca2+. The inhibition was unaffected by calcineurin inhibitors. The effect of calmodulin did not result from enhanced metabolism of InsP3 because calmodulin also decreased the sensitivity of the Ca2+ stores to adenophostin A, a non-metabolizable InsP3-receptor agonist. Protein kinase A-catalysed phosphorylation of type-1 InsP3 receptors was unaffected by Ca2+-calmodulin. Using a scintillation proximity assay to measure 125I-calmodulin binding to glutathione S-transferase-fusion proteins, we identified two regions of the type-1 InsP3 receptor (cyt1, residues -6 to 159; and cyt11, residues 1499-1649) that bound 125I-calmodulin. The higher-affinity site (cyt11) was also photoaffinity labelled with N-hydroxysuccinimidyl-4-azidobenzoate (HSAB)-calmodulin. We speculate that Ca2+-independent binding of calmodulin to a site within the first 159 residues of the type-1 InsP3 receptor inhibits InsP3 binding and may thereby regulate the kinetics of Ca2+ release. Ca2+-dependent inhibition of Ca2+ release by calmodulin is mediated by a different site: it may reside on an accessory protein that associates with all three receptor subtypes, or Ca2+-calmodulin binding to a site lying between residues 1499 and 1649 of the type-1 receptor may inhibit Ca2+ release from any tetrameric receptor that includes a type-1 subunit.

scholarly journals Type 3 inositol trisphosphate receptors in RINm5F cells are biphasically regulated by cytosolic Ca2+ and mediate quantal Ca2+ mobilization

1999 ◽  
Vol 344 (1) ◽  
pp. 55-60 ◽  
Author(s):  
Jane E. SWATTON ◽  
Stephen A. MORRIS ◽  
Thomas J. A. CARDY ◽  
Colin W. TAYLOR
Keyword(s):  
Single Type ◽  
Receptor Subtype ◽  
Receptor Subtypes ◽  
Rinm5f Cells ◽  
Intact Cells ◽  
Single Receptor ◽  
Insp3 Receptors ◽  
Type 3 ◽  
Insp3 Receptor

There are three subtypes of mammalian Ins(1,4,5)P3 (InsP3) receptor, each of which forms an intracellular Ca2+ channel. Biphasic regulation of InsP3 receptors by cytosolic Ca2+ is well documented in cells expressing predominantly type 1 or type 2 InsP3 receptors and might contribute to the regenerative recruitment of Ca2+ release events and to limiting their duration in intact cells. The properties of type 3 receptors are less clear. Bilayer recording from InsP3 receptors of RIN-5F cells, cells in which the InsP3 receptors are likely to be largely type 3, recently suggested that the receptors are not inhibited by Ca2+ [Hagar, Burgstahler, Nathanson and Ehrlich (1998) Nature (London) 296, 81-84]. By using antipeptide antisera that either selectively recognized each InsP3 receptor subtype or interacted equally well with all subtypes, together with membranes from Spodoptera frugiperda (Sf9) cells expressing only single receptor subtypes to calibrate the immunoblotting, we quantified the relative levels of expression of type 1 (17%) and type 3 (77%) InsP3 receptors in RINm5F cells. In unidirectional 45Ca2+ efflux experiments from permeabilized RINm5F cells, submaximal concentrations of InsP3 released only a fraction of the InsP3-sensitive Ca2+ stores, indicating that responses to InsP3 are quantal. Increasing the cytosolic free [Ca2+] ([Ca2+]i) from approx. 4 to 186 nM increased the sensitivity of the Ca2+ stores to InsP3: the EC50 decreased from 281±15 to 82±2 nM. Further increases in [Ca2+]i massively decreased the sensitivity of the stores to InsP3, by almost 10-fold when [Ca2+]i was 2.4 μM, and by more than 3000-fold when it was 100 μM. The inhibition caused by 100 μM Ca2+ was fully reversed within 60 s of the restoration of [Ca2+]i to 186 nM. The effect of submaximal InsP3 concentrations on Ca2+ mobilization from permeabilized RINm5F cells is therefore biphasically regulated by cytosolic Ca2+. We conclude that type 3 InsP3 receptors of RINm5F cells mediate quantal Ca2+ release and they are biphasically regulated by cytosolic Ca2+, either because a single type 1 subunit within the tetrameric receptor confers the Ca2+ inhibition or because the type 3 subtype is itself directly inhibited by Ca2+.

scholarly journals Isolation and characterization of vascular smooth muscle inositol 1,4,5-trisphosphate receptor

1996 ◽  
Vol 316 (1) ◽  
pp. 295-302 ◽  
Author(s):  
Md. Omedul ISLAM ◽  
Yutaka YOSHIDA ◽  
Takaki KOGA ◽  
Masayasu KOJIMA ◽  
Kenji KANGAWA ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Biochemical Properties ◽  
Pcr Analysis ◽  
Binding Property ◽  
Dependent Manner ◽  
Calmodulin Binding ◽  
Isolation And Characterization ◽  
Inositol 1,4,5 Trisphosphate ◽  
Insp3 Receptor

myo-Inositol 1,4,5-trisphosphate (InsP3) receptor of porcine aorta was purified to near homogeneity and its biochemical properties were compared with those of cerebellar InsP3 receptor of the same animal species. The aortic InsP3 receptor consisted of equal amounts of two polypeptides with slightly differing molecular masses of around 240 kDa and was found to possess a single population of InsP3-binding site (Kd of 1.2 nM). The InsP3 receptor purified from porcine cerebellum was also comprised of two polypeptides. However, the molecular mass was slightly but definitely larger, being 250 kDa, and the amounts of the two polypeptides were not equal. The aortic InsP3 receptor cross-reacted with polyclonal antibody specific to type 1 InsP3 receptor as did the cerebellar InsP3 receptor. The aortic InsP3 receptor bound to calmodulin–Sepharose in a Ca2+-dependent manner, while the cerebellar InsP3 receptor did not. Reverse transcriptase-PCR analysis revealed two splicing variants of the type 1 InsP3 receptor in porcine aortic smooth muscle distinct from those of the type 1 InsP3 receptor of porcine cerebellum. The possible relevance of this difference to difference in calmodulin-binding property was discussed.

scholarly journals Purification and characterization of the human type 1 Ins(1,4,5)P3 receptor from platelets and comparison with receptor subtypes in other normal and transformed blood cells

1995 ◽  
Vol 312 (2) ◽  
pp. 499-503 ◽  
Author(s):  
F O'Rourke ◽  
E Matthews ◽  
M B Feinstein
Keyword(s):  
T Cell ◽  
Cell Lines ◽  
Blood Cells ◽  
Gel Filtration ◽  
Receptor Protein ◽  
Receptor Subtypes ◽  
Human Form ◽  
Single Class ◽  
Insp3 Receptor

We report the first purification of a native human form of the Ins(1,4,5)P3 (InsP3) receptor. This receptor, isolated from platelets, has an apparent molecular mass on SDS/PAGE of 252 kDa and is chromatographed by gel filtration as an oligomer of about 1 x 10(6) kDa. [3H]InsP3 bound to a single class of sites on the purified receptor protein with a Kd of 27 nM and a Bmax. of 2.2 nmol/mg of protein. The platelet InsP3 receptor, like the rodent cerebellar receptors, was identified immunochemically as a type 1 receptor, but unlike its brain counterparts bound poorly to concanavalin A and other lectins and was not significantly phosphorylated by protein kinase A. All cultured megakaryocytic leukaemia cell lines (e.g. Dami, CHRF-288 and Meg-01) and HEL cells were also immunopositive for type 1 receptor, which was substantially increased in some cases by DMSO or phorbol 12-myristate 13-acetate (PMA) which induce further megakaryocytic differentiation. Normal mixed lymphocyte and granulocyte fractions and an enriched T-cell fraction from human blood had measurable InsP3-binding activity, but no detectable type 1 protein. In contrast, Jurkat E6-1 (T-cell lymphoma) cells and the transformed B-cell line RPMI 8392 were immunopositive for type 1 receptor. HL-60 (human promyelocytic leukaemia) cells had no detectable type 1 receptor unless they were stimulated to differentiate along monocyte/macrophage lines by PMA. We conclude that: (1) of the major normal blood cells only platelets contain type 1 InsP3 receptors; (2) some neoplastic transformed blood cell lines also express type 1 receptors, in contrast to their normal counterparts; and (3) increased levels of type 1 InsP3 receptor are induced in some transformed cells under conditions that favour their further terminal differentiation.

scholarly journals Determinants of adenophostin A binding to inositol trisphosphate receptors

2002 ◽  
Vol 367 (1) ◽  
pp. 113-120 ◽  
Author(s):  
Stephen A. MORRIS ◽  
Edmund P. NEROU ◽  
Andrew M. RILEY ◽  
Barry V.L. POTTER ◽  
Colin W. TAYLOR
Keyword(s):  
Binding Domain ◽  
Sf9 Cells ◽  
Receptor Subtypes ◽  
Ip3 Receptors ◽  
Ip3 Receptor ◽  
High Affinity ◽  
Recombinant Type ◽  
Adenophostin A ◽  
Binding Core

Inositol 1,4,5-trisphosphate (IP3) receptors from cerebellum and recombinant type 1 IP3 receptors expressed in Sf9 cells had indistinguishable affinities for IP3 (Kd = 6.40±0.48nM) and adenophostin A (Kd = 0.89±0.05nM). In cytosol-like medium, each of the three mammalian IP3 receptor subtypes when expressed in Sf9 cells bound adenophostin A with greater affinity than IP3. It has been suggested that adenophostin A binds with high affinity only in the presence of ATP, but we found that adenophostin A similarly displaced [3H]IP3 from type 1 IP3 receptors whatever the ATP concentration. N-terminal fragments of the type 1 receptor were expressed with and without the S1 splice site; its removal had no effect on [3H]IP3 binding to the 1—604 protein, but abolished binding to the 224—604 protein. The 1—604 fragment and full-length receptor bound adenophostin A with the same affinity, but the fragment had 3-fold greater affinity for IP3, suggesting that C-terminal residues selectively inhibit IP3 binding. The 224—604S1+ fragment bound IP3 and adenophostin A with increased affinity, but as with the 1—604 fragment it bound adenophostin A with only 2-fold greater affinity than IP3. High-affinity binding of adenophostin A may be partially determined by its 2′-phosphate interacting more effectively than the 1-phosphate of IP3 with residues within the IP3-binding core. This may account for the 2-fold greater affinity of adenophostin A relative to IP3 for the minimal IP3-binding domain. In addition we suggest that C-terminal residues, which impede access of IP3, may selectively interact with adenophostin A to allow it unhindered access to the IP3-binding domain.

scholarly journals Characterization of Escherichia coliType 1 Pilus Mutants with Altered Binding Specificities

2001 ◽  
Vol 183 (13) ◽  
pp. 4099-4102 ◽  
Author(s):  
Sandra L. Harris ◽  
Patricia A. Spears ◽  
Edward A. Havell ◽  
Terri S. Hamrick ◽  
John R. Horton ◽  
...  
Keyword(s):  
Amino Acid ◽  
Cell Types ◽  
Binding Pocket ◽  
Temperature Dependent ◽  
Type 1 Pili ◽  
Adhesin Protein ◽  
A Site ◽  
Single Lesion

ABSTRACT PCR mutagenesis and a unique enrichment scheme were used to obtain two mutants, each with a single lesion in fimH, the chromosomal gene that encodes the adhesin protein (FimH) ofEscherichia coli type 1 pili. These mutants were noteworthy in part because both were altered in the normal range of cell types bound by FimH. One mutation altered an amino acid at a site previously shown to be involved in temperature-dependent binding, and the other altered an amino acid lining the predicted FimH binding pocket.

CELL AND TISSUE THERAPY OF HEART

2019 ◽  
pp. 77-84
Keyword(s):  
Extracellular Matrix ◽  
Cell Types ◽  
Heart Tissue ◽  
Decellularized Extracellular Matrix ◽  
Tissue Therapy ◽  
Essential Components ◽  
Long Time ◽  
Different Populations ◽  
A Site ◽  
Recipient Tissue

The strategy of heart tissue engineering is simple enough: first remove all the cells from a organ then take the protein scaffold left behind and repopulate it with stem cells immunologically matched to the patient in need. While various suc- cessful methods for decellularization have been developed, and the feasibility of using decellularized whole hearts and extracellular matrix to support cells has been demonstrated, the reality of creating whole hearts for transplantation and of clinical application of decellularized extracellular matrix-based scaffolds will require much more research. For example, further investigations into how lineage-restricted progenitors repopulate the decellularized heart and differentiate in a site-specific manner into different populations of the native heart would be essential. The scaffold heart does not have to be human. Pig hearts carries all the essential components of the extracellular matrix. Through trial and error, scaling up the concentration, timing and pressure of the detergents, researchers have refined the decellularization process on hundreds of hearts and other organs, but this is only the first step. Further, the framework must be populated with human cells. Most researchers in the field use a mixture of two or more cell types, such as endothelial precursor cells to line blood vessels and muscle progenitors to seed the walls of the chambers. The final challenge is one of the hardest: vasculariza- tion, placing a engineered heart into a living animal, integration with the recipient tissue, and keeping it beating for a long time. Much remains to be done before a bioartificial heart is available for transplantation in humans.

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 The distribution, induction and isoenzyme profile of glutathione S-transferase and glutathione peroxidase in isolated rat liver parenchymal, Kupffer and endothelial cells

1989 ◽  
Vol 264 (3) ◽  
pp. 737-744 ◽  
Author(s):  
P Steinberg ◽  
H Schramm ◽  
L Schladt ◽  
L W Robertson ◽  
H Thomas ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Glutathione Peroxidase ◽  
Rat Liver ◽  
Peroxidase Activity ◽  
Cell Types ◽  
Transferase Activity ◽  
Glutathione Peroxidase Activity ◽  
Glutathione S Transferase ◽  
Liver Parenchymal ◽  
Parenchymal Cells

The distribution and inducibility of cytosolic glutathione S-transferase (EC 2.5.1.18) and glutathione peroxidase (EC 1.11.1.19) activities in rat liver parenchymal, Kupffer and endothelial cells were studied. In untreated rats glutathione S-transferase activity with 1-chloro-2,4-dinitrobenzene and 4-hydroxynon-2-trans-enal as substrates was 1.7-2.2-fold higher in parenchymal cells than in Kupffer and endothelial cells, whereas total, selenium-dependent and non-selenium-dependent glutathione peroxidase activities were similar in all three cell types. Glutathione S-transferase isoenzymes in parenchymal and non-parenchymal cells isolated from untreated rats were separated by chromatofocusing in an f.p.l.c. system: all glutathione S-transferase isoenzymes observed in the sinusoidal lining cells were also detected in the parenchymal cells, whereas Kupffer and endothelial cells lacked several glutathione S-transferase isoenzymes present in parenchymal cells. At 5 days after administration of Arocolor 1254 glutathione S-transferase activity was only enhanced in parenchymal cells; furthermore, selenium-dependent glutathione peroxidase activity decreased in parenchymal and non-parenchymal cells. At 13 days after a single injection of Aroclor 1254 a strong induction of glutathione S-transferase had taken place in all three cell types, whereas selenium-dependent glutathione peroxidase activity remained unchanged (endothelial cells) or was depressed (parenchymal and Kupffer cells). Hence these results clearly establish that glutathione S-transferase and glutathione peroxidase are differentially regulated in rat liver parenchymal as well as non-parenchymal cells. The presence of glutathione peroxidase and several glutathione S-transferase isoenzymes capable of detoxifying a variety of compounds in Kupffer and endothelial cells might be crucial to protect the liver from damage by potentially hepatotoxic substances.

scholarly journals Human Immunodeficiency Virus Type 1 Replication in the Absence of Integrase-Mediated DNA Recombination: Definition of Permissive and Nonpermissive T-Cell Lines

2001 ◽  
Vol 75 (17) ◽  
pp. 7944-7955 ◽  
Author(s):  
Noriko Nakajima ◽  
Richard Lu ◽  
Alan Engelman
Keyword(s):  
Human Immunodeficiency Virus ◽  
Cell Lines ◽  
Dna Recombination ◽  
Cell Types ◽  
Class Ii ◽  
Class I ◽  
Immunodeficiency Virus ◽  
T Cell Lines ◽  
Hiv 1

ABSTRACT Functional retroviral integrase protein is thought to be essential for productive viral replication. Yet, previous studies differed on the extent to which integrase mutant viruses expressed human immunodeficiency virus type 1 (HIV-1) genes from unintegrated DNA. Although one reason for this difference was that class II integrase mutations pleiotropically affected the viral life cycle, another reason apparently depended on the identity of the infected cell. Here, we analyzed integrase mutant viral infectivities in a variety of cell types. Single-round infectivity of class I integration-specific mutant HIV-1 ranged from <0.03 to 0.3% of that of the wild type (WT) across four different T-cell lines. Based on this approximately 10-fold influence of cell type on mutant gene expression, we examined class I and class II mutant replication kinetics in seven different cell lines and two primary cell types. Unexpectedly, some cell lines supported productive class I mutant viral replication under conditions that restricted class II mutant growth. Cells were defined as permissive, semipermissive, or nonpermissive based on their ability to support the continual passage of class I integration-defective HIV-1. Mutant infectivity in semipermissive and permissive cells as quantified by 50% tissue culture infectious doses, however, was only 0.0006 to 0.005% of that of WT. Since the frequencies of mutant DNA recombination in these lines ranged from 0.023 to <0.093% of the WT, we conclude that productive replication in the absence of integrase function most likely required the illegitimate integration of HIV-1 into host chromosomes by cellular DNA recombination enzymes.

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