Masahiro Enomoto

Pulmonary hypertension in adult Alk1 heterozygous mice due to oxidative stress

AIMSnMutations in the ALK1 gene, coding for an endothelial-specific receptor of the transforming growth factor-β superfamily, are the underlying cause of hereditary haemorrhagic telangiectasia type 2, but are also associated with familial pulmonary hypertension (PH). We assessed the lung vasculature of mice with a heterozygous deletion of Alk1 (Alk1(+/-)) for disease manifestations and levels of reactive O(2) species (ROS) implicated in both disorders.nnnMETHODS AND RESULTSnSeveral signs of PH, including elevated right ventricular (RV) systolic pressure leading to RV hypertrophy, reduced vascular density, and increased thickness and outward remodelling of pulmonary arterioles, were observed in 8- to 18-week-old Alk1(+/-) mice relative to wild-type littermate controls. Higher ROS lung levels were also documented. At 3 weeks, Alk1(+/-) mice were indistinguishable from controls and were prevented from subsequently developing PH when treated with the anti-oxidant Tempol for 6 weeks, confirming a role for ROS in pathogenesis. Levels of NADPH oxidases and superoxide dismutases were higher in adults than newborns, but unchanged in Alk1(+/-) mice vs. controls. Prostaglandin metabolites were also normal in adult Alk1(+/-) lungs. In contrast, NO production was reduced, while endothelial NO synthase (eNOS)-dependent ROS production was increased in adult Alk1(+/-) mice. Pulmonary near resistance arteries from adult Alk1(+/-) mice showed less agonist-induced force and greater acetylcholine-induced relaxation; the later was normalized by catalase or Tempol treatment.nnnCONCLUSIONnThe increased pulmonary vascular remodelling in Alk1(+/-) mice leads to signs of PH and is associated with eNOS-dependent ROS production, which is preventable by anti-oxidant treatment.

Ryanodine receptor calcium release channels: lessons from structure-function studies.

Ryanodine receptors (RyRs) are the largest known ion channels. They are Ca2+ release channels found primarily on the sarcoplasmic reticulum of myocytes. Several hundred mutations in RyRs are associated with skeletal or cardiomyocyte disease in humans. Many of these mutations can now be mapped onto the high resolution structures of individual RyR domains and on full‐length tetrameric cryo‐electron microscopy structures. A closely related Ca2+ release channel, the inositol 1,4,5‐trisphospate receptor (IP3R), shows a conserved structural architecture at the N‐terminus, suggesting that both channels evolved from an ancestral unicellular RyR/IP3R. The functional insights provided by recent structural studies for both channels will aid in the development of rationale treatments for a myriad of Ca2+‐signaled malignancies.

Inositol 1,4,5‐trisphosphate receptor regulates replication, differentiation, infectivity and virulence of the parasitic protist Trypanosoma cruzi

In animals, inositol 1,4,5‐trisphosphate receptors (IP3Rs) are ion channels that play a pivotal role in many biological processes by mediating Ca2+ release from the endoplasmic reticulum. Here, we report the identification and characterization of a novel IP3R in the parasitic protist, Trypanosoma cruzi, the pathogen responsible for Chagas disease. DT40 cells lacking endogenous IP3R genes expressing T.u2009cruzi IP3R (TcIP3R) exhibited IP3‐mediated Ca2+ release from the ER, and demonstrated receptor binding to IP3. TcIP3R was expressed throughout the parasite life cycle but the expression level was much lower in bloodstream trypomastigotes than in intracellular amastigotes or epimastigotes. Disruption of two of the three TcIP3R gene loci led to the death of the parasite, suggesting that IP3R is essential for T.u2009cruzi. Parasites expressing reduced or increased levels of TcIP3R displayed defects in growth, transformation and infectivity, indicating that TcIP3R is an important regulator of the parasites life cycle. Furthermore, mice infected with T.u2009cruzi expressing reduced levels of TcIP3R exhibited a reduction of disease symptoms, indicating that TcIP3R is an important virulence factor. Combined with the fact that the primary structure of TcIP3R has low similarity to that of mammalian IP3Rs, TcIP3R is a promising drug target for Chagas disease.

Structural insights into endoplasmic reticulum stored calcium regulation by inositol 1,4,5-trisphosphate and ryanodine receptors

The two major calcium (Ca²⁺) release channels on the sarco/endoplasmic reticulum (SR/ER) are inositol 1,4,5-trisphosphate and ryanodine receptors (IP3Rs and RyRs). They play versatile roles in essential cell signaling processes, and abnormalities of these channels are associated with a variety of diseases. Structural information on IP3Rs and RyRs determined using multiple techniques including X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy and cryo-electron microscopy (EM), has significantly advanced our understanding of the mechanisms by which these Ca²⁺ release channels function under normal and pathophysiological circumstances. In this review, structural advances on the understanding of the mechanisms of IP3R and RyR function and dysfunction are summarized. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.

Intracellular calcium channels: inositol-1,4,5-trisphosphate receptors.

The inositol-1,4,5-trisphosphate receptors (InsP3Rs) are the major intracellular Ca(2+)-release channels in cells. Activity of InsP3Rs is essential for elementary and global Ca(2+) events in the cell. There are three InsP3Rs isoforms that are present in mammalian cells. In this review we will focus primarily on InsP3R type 1. The InsP3R1 is a predominant isoform in neurons and it is the most extensively studied isoform. Combination of biophysical and structural methods revealed key mechanisms of InsP3R function and modulation. Cell biological and biochemical studies lead to identification of a large number of InsP3R-binding proteins. InsP3Rs are involved in the regulation of numerous physiological processes, including learning and memory, proliferation, differentiation, development and cell death. Malfunction of InsP3R1 play a role in a number of neurodegenerative disorders and other disease states. InsP3Rs represent a potentially valuable drug target for treatment of these disorders and for modulating activity of neurons and other cells. Future studies will provide better understanding of physiological functions of InsP3Rs in health and disease.

CaBP1, a neuronal Ca2+ sensor protein, inhibits inositol trisphosphate receptors by clamping intersubunit interactions

Calcium-binding protein 1 (CaBP1) is a neuron-specific member of the calmodulin superfamily that regulates several Ca2+ channels, including inositol 1,4,5-trisphosphate receptors (InsP3Rs). CaBP1 alone does not affect InsP3R activity, but it inhibits InsP3-evoked Ca2+ release by slowing the rate of InsP3R opening. The inhibition is enhanced by Ca2+ binding to both the InsP3R and CaBP1. CaBP1 binds via its C lobe to the cytosolic N-terminal region (NT; residues 1–604) of InsP3R1. NMR paramagnetic relaxation enhancement analysis demonstrates that a cluster of hydrophobic residues (V101, L104, and V162) within the C lobe of CaBP1 that are exposed after Ca2+ binding interact with a complementary cluster of hydrophobic residues (L302, I364, and L393) in the β-domain of the InsP3-binding core. These residues are essential for CaBP1 binding to the NT and for inhibition of InsP3R activity by CaBP1. Docking analyses and paramagnetic relaxation enhancement structural restraints suggest that CaBP1 forms an extended tetrameric turret attached by the tetrameric NT to the cytosolic vestibule of the InsP3R pore. InsP3 activates InsP3Rs by initiating conformational changes that lead to disruption of an intersubunit interaction between a “hot-spot” loop in the suppressor domain (residues 1–223) and the InsP3-binding core β-domain. Targeted cross-linking of residues that contribute to this interface show that InsP3 attenuates cross-linking, whereas CaBP1 promotes it. We conclude that CaBP1 inhibits InsP3R activity by restricting the intersubunit movements that initiate gating.

L-ornithine derived polyamines in cystic fibrosis airways.

Increased arginase activity contributes to airway nitric oxide (NO) deficiency in cystic fibrosis (CF). Whether down-stream products of arginase activity contribute to CF lung disease is currently unknown. The objective of this study was to test whether L-ornithine derived polyamines are present in CF airways and contribute to airway pathophysiology. Polyamine concentrations were measured in sputum of patients with CF and in healthy controls, using liquid chromatography-tandem mass spectrometry. The effect of spermine on airway smooth muscle mechanical properties was assessed in bronchial segments of murine airways, using a wire myograph. Sputum polyamine concentrations in stable CF patients were similar to healthy controls for putrescine and spermidine but significantly higher for spermine. Pulmonary exacerbations were associated with an increase in sputum and spermine levels. Treatment for pulmonary exacerbations resulted in decreases in arginase activity, L-ornithine and spermine concentrations in sputum. The changes in sputum spermine with treatment correlated significantly with changes in L-ornithine but not with sputum inflammatory markers. Incubation of mouse bronchi with spermine resulted in an increase in acetylcholine-induced force and significantly reduced nitric oxide-induced bronchial relaxation. The polyamine spermine is increased in CF airways. Spermine contributes to airways obstruction by reducing the NO-mediated smooth muscle relaxation.

Themes and Variations in ER/SR Calcium Release Channels: Structure and Function

Calcium (Ca(2+)) release from reticular stores is a vital regulatory signal in eukaryotes. Recent structural data on large NH(2)-terminal regions of IP(3)Rs and RyRs and their tetrameric arrangement in the full-length context reveal striking mechanistic similarities in Ca(2+) release channel function. A common ancestor found in unicellular genomes underscores the fundamentality of these elements to Ca(2+) release channels.

Blockage of Spontaneous Ca2+ Oscillation Causes Cell Death in Intraerythrocitic Plasmodium falciparum

Malaria remains one of the world’s most important infectious diseases and is responsible for enormous mortality and morbidity. Resistance to antimalarial drugs is a challenging problem in malaria control. Clinical malaria is associated with the proliferation and development of Plasmodium parasites in human erythrocytes. Especially, the development into the mature forms (trophozoite and schizont) of Plasmodium falciparum (P. falciparum) causes severe malaria symptoms due to a distinctive property, sequestration which is not shared by any other human malaria. Ca2+ is well known to be a highly versatile intracellular messenger that regulates many different cellular processes. Cytosolic Ca2+ increases evoked by extracellular stimuli are often observed in the form of oscillating Ca2+ spikes (Ca2+ oscillation) in eukaryotic cells. However, in lower eukaryotic and plant cells the physiological roles and the molecular mechanisms of Ca2+ oscillation are poorly understood. Here, we showed the observation of the inositol 1,4,5-trisphospate (IP3)-dependent spontaneous Ca2+ oscillation in P. falciparum without any exogenous extracellular stimulation by using live cell fluorescence Ca2+ imaging. Intraerythrocytic P. falciparum exhibited stage-specific Ca2+ oscillations in ring form and trophozoite stages which were blocked by IP3 receptor inhibitor, 2-aminoethyl diphenylborinate (2-APB). Analyses of parasitaemia and parasite size and electron micrograph of 2-APB-treated P. falciparum revealed that 2-APB severely obstructed the intraerythrocytic maturation, resulting in cell death of the parasites. Furthermore, we confirmed the similar lethal effect of 2-APB on the chloroquine-resistant strain of P. falciparum. To our best knowledge, we for the first time showed the existence of the spontaneous Ca2+ oscillation in Plasmodium species and clearly demonstrated that IP3-dependent spontaneous Ca2+ oscillation in P. falciparum is critical for the development of the blood stage of the parasites. Our results provide a novel concept that IP3/Ca2+ signaling pathway in the intraerythrocytic malaria parasites is a promising target for antimalarial drug development.

Mechanistic basis of bell-shaped dependence of inositol 1,4,5-trisphosphate receptor gating on cytosolic calcium

The inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) is an intracellular Ca2+ release channel, and its opening is controlled by IP3 and Ca2+. A single IP3 binding site and multiple Ca2+ binding sites exist on single subunits, but the precise nature of the interplay between these two ligands in regulating biphasic dependence of channel activity on cytosolic Ca2+ is unknown. In this study, we visualized conformational changes in IP3R evoked by various concentrations of ligands by using the FRET between two fluorescent proteins fused to the N terminus of individual subunits. IP3 and Ca2+ have opposite effects on the FRET signal change, but the combined effect of these ligands is not a simple summative response. The bell-shaped Ca2+ dependence of FRET efficiency was observed after the subtraction of the component corresponding to the FRET change evoked by Ca2+ alone from the FRET changes evoked by both ligands together. A mutant IP3R containing a single amino acid substitution at K508, which is critical for IP3 binding, did not exhibit this bell-shaped Ca2+ dependence of the subtracted FRET efficiency. Mutation at E2100, which is known as a Ca2+ sensor, resulted in ∼10-fold reduction in the Ca2+ dependence of the subtracted signal. These results suggest that the subtracted FRET signal reflects IP3R activity. We propose a five-state model, which implements a dual-ligand competition response without complex allosteric regulation of Ca2+ binding affinity, as the mechanism underlying the IP3-dependent regulation of the bell-shaped relationship between the IP3R activity and cytosolic Ca2+.