IP3R Type Research Articles

A structure-based computational model of IP3R1 incorporating Ca and IP3 regulation

The inositol 1,4,5-triphosphate receptor (IP3R) mediates Ca release in many cell types and is pivotal to a wide range of cellular processes. High-resolution cryoelectron microscopy studies have provided new structural details of IP3R type 1 (IP3R1), showing that channel function is determined by the movement of various domains within and between each of its four subunits. Channel properties are regulated by ligands, such as Ca and IP3, which bind at specific sites and control the interactions between these domains. However, it is not known how the various ligand-binding sites on IP3R1 interact to control the opening of the channel. In this study, we present a coarse-grained model of IP3R1 that accounts for the channel architecture and the location of specific Ca- and IP3-binding sites. This computational model accounts for the domain-domain interactions within and between the four subunits that form IP3R1, and it also describes how ligand binding regulates these interactions. Using a kinetic model, we explore how two Ca-binding sites on the cytosolic side of the channel interact with the IP3-binding site to regulate the channel open probability. Our primary finding is that the bell-shaped open probability of IP3R1 provides constraints on the relative strength of these regulatory binding sites. In particular, we argue that a specific Ca-binding site, whose function has not yet been established, is very likely a channel antagonist. Additionally, we apply our model to show that domain-domain interactions between neighboring subunits exert control over channel cooperativity and dictate the nonlinear response of the channel to Ca concentration. This suggests that specific domain-domain interactions play a pivotal role in maintaining the channel’s stability, and a disruption of these interactions may underlie disease states associated with Ca dysregulation.

Evaluation of IP3R3 Gene Silencing Effect on Pyruvate Dehydrogenase (PDH) Enzyme Activity in Breast Cancer Cells with and Without Estrogen Receptor.

Inositol 1,4,5-trisphosphate receptor (IP3R), a critical calcium ion (Ca2+) regulator, plays a vital role in breast cancer (BC) metabolism. Dysregulated IP3R in BC cells can drive abnormal growth or cell death. Estradiol increases IP3R type 3 (IP3R3) levels in BC, promoting cell proliferation and metabolic changes, including enhanced pyruvate dehydrogenase (PDH) activity, which, when reduced, leads to cell apoptosis. The study silenced IP3R3 to assess its impact on PDH. The study used IP3R3 small interfering RNA (siRNA) to target Michigan Cancer Foundation-7 (MCF-7) and MDA-MB-231 cell lines. Transfection success was confirmed by flow cytometry. Cell viability and gene silencing were evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and real-time quantitative polymerase chain reaction (PCR) assays. Protein expression and cellular activity were analyzed through western blotting and PDH activity measurement. Transfecting MCF-7 and MDA-MB-231 cells with IP3R3 siRNA achieved a 65% transfection rate without significant toxicity. IP3R3 gene silencing effectively reduced IP3R3 messenger RNA (mRNA) and protein levels in both cell lines, leading to decreased PDH enzyme activity, especially in MDA-MB-231 cells. The study highlights a link between high IP3R3 gene silencing and reduced PDH activity, with higher IP3R3 expression in estrogen-independent (MDA-MB-231) compared to estrogen-dependent (MCF-7) cell lines. This suggests a potential impact on BC metabolism and tumor growth via regulation of PDH activity.

Ligand sensitivity of type-1 inositol 1,4,5-trisphosphate receptor is enhanced by the D2594K mutation

Inositol 1,4,5-trisphosphate receptor (IP3R) and ryanodine receptor (RyR) are homologous cation channels that mediate release of Ca2+ from the endoplasmic/sarcoplasmic reticulum (ER/SR) and thereby are involved in many physiological processes. In previous studies, we determined that when the D2594 residue, located at or near the gate of the IP3R type 1, was replaced by lysine (D2594K), a gain of function was obtained. This mutant phenotype was characterized by increased IP3 sensitivity. We hypothesized the IP3R1-D2594 determines the ligand sensitivity of the channel by electrostatically affecting the stability of the closed and open states. To test this possibility, the relationship between the D2594 site and IP3R1 regulation by IP3, cytosolic, and luminal Ca2+ was determined at the cellular, subcellular, and single-channel levels using fluorescence Ca2+ imaging and single-channel reconstitution. We found that in cells, D2594K mutation enhances the IP3 ligand sensitivity. Single-channel IP3R1 studies revealed that the conductance of IP3R1-WT and -D2594K channels is similar. However, IP3R1-D2594K channels exhibit higher IP3 sensitivity, with substantially greater efficacy. In addition, like its wild type (WT) counterpart, IP3R1-D2594K showed a bell-shape cytosolic Ca2+-dependency, but D2594K had greater activity at each tested cytosolic free Ca2+ concentration. The IP3R1-D2594K also had altered luminal Ca2+ sensitivity. Unlike IP3R1-WT, D2594K channel activity did not decrease at low luminal Ca2+ levels. Taken together, our functional studies indicate that the substitution of a negatively charged residue by a positive one at the channels’ pore cytosolic exit affects the channel’s gating behavior thereby explaining the enhanced ligand-channel’s sensitivity.

IP3R-driven increases in mitochondrial Ca2+ promote neuronal death in NPC disease

Ca2+ is the most ubiquitous second messenger in neurons whose spatial and temporal elevations are tightly controlled to initiate and orchestrate diverse intracellular signaling cascades. Numerous neuropathologies result from mutations or alterations in Ca2+ handling proteins; thus, elucidating molecular pathways that shape Ca2+ signaling is imperative. Here, we report that loss-of-function, knockout, or neurodegenerative disease-causing mutations in the lysosomal cholesterol transporter, Niemann-Pick Type C1 (NPC1), initiate a damaging signaling cascade that alters the expression and nanoscale distribution of IP3R type 1 (IP3R1) in endoplasmic reticulum membranes. These alterations detrimentally increase Gq-protein coupled receptor-stimulated Ca2+ release and spontaneous IP3R1 Ca2+ activity, leading to mitochondrial Ca2+ cytotoxicity. Mechanistically, we find that SREBP-dependent increases in Presenilin 1 (PS1) underlie functional and expressional changes in IP3R1. Accordingly, expression of PS1 mutants recapitulate, while PS1 knockout abrogates Ca2+ phenotypes. These data present a signaling axis that links the NPC1 lysosomal cholesterol transporter to the damaging redistribution and activity of IP3R1 that precipitates cell death in NPC1 disease and suggests that NPC1 is a nanostructural disease.

Inositol Trisphosphate Receptors and Nuclear Calcium in Atrial Fibrillation.

The mechanisms underlying atrial fibrillation (AF), the most common clinical arrhythmia, are poorly understood. Nucleoplasmic Ca2+ regulates gene expression, but the nature and significance of nuclear Ca2+-changes in AF are largely unknown. To elucidate mechanisms by which AF alters atrial-cardiomyocyte nuclear Ca2+ ([Ca2+]Nuc) and CaMKII (Ca2+/calmodulin-dependent protein kinase-II)-related signaling. Atrial cardiomyocytes were isolated from control and AF dogs (kept in AF by atrial tachypacing [600 bpm × 1 week]). [Ca2+]Nuc and cytosolic [Ca2+] ([Ca2+]Cyto) were recorded via confocal microscopy. Diastolic [Ca2+]Nuc was greater than [Ca2+]Cyto under control conditions, while resting [Ca2+]Nuc was similar to [Ca2+]Cyto; both diastolic and resting [Ca2+]Nuc increased with AF. IP3R (Inositol-trisphosphate receptor) stimulation produced larger [Ca2+]Nuc increases in AF versus control cardiomyocytes, and IP3R-blockade suppressed the AF-related [Ca2+]Nuc differences. AF upregulated nuclear protein expression of IP3R1 (IP3R-type 1) and of phosphorylated CaMKII (immunohistochemistry and immunoblot) while decreasing the nuclear/cytosolic expression ratio for HDAC4 (histone deacetylase type-4). Isolated atrial cardiomyocytes tachypaced at 3 Hz for 24 hours mimicked AF-type [Ca2+]Nuc changes and L-type calcium current decreases versus 1-Hz-paced cardiomyocytes; these changes were prevented by IP3R knockdown with short-interfering RNA directed against IP3R1. Nuclear/cytosolic HDAC4 expression ratio was decreased by 3-Hz pacing, while nuclear CaMKII phosphorylation was increased. Either CaMKII-inhibition (by autocamtide-2-related peptide) or IP3R-knockdown prevented the CaMKII-hyperphosphorylation and nuclear-to-cytosolic HDAC4 shift caused by 3-Hz pacing. In human atrial cardiomyocytes from AF patients, nuclear IP3R1-expression was significantly increased, with decreased nuclear/nonnuclear HDAC4 ratio. MicroRNA-26a was predicted to target ITPR1 (confirmed by luciferase assay) and was downregulated in AF atrial cardiomyocytes; microRNA-26a silencing reproduced AF-induced IP3R1 upregulation and nuclear diastolic Ca2+-loading. AF increases atrial-cardiomyocyte nucleoplasmic [Ca2+] by IP3R1-upregulation involving miR-26a, leading to enhanced IP3R1-CaMKII-HDAC4 signaling and L-type calcium current downregulation. Graphic Abstract: A graphic abstract is available for this article.

Luminal Calcium Control of Type-1 Inositol 1,4,5-Trisphosphate Receptor

Inositol 1,4,5-trisphosphate receptor (IP3R) and ryanodine receptor (RyR) are homologous endo/sarcoplasmic reticulum Ca2+ release channels. IP3R and RyR are tightly controlled by both cytosolic and luminal Ca2+. The negatively charged residue E4872 in RyR2 (cardiac isoform) electrostatically interacts with luminal Ca2+, explaining RyR2's luminal Ca2+ dependency (Chen et al., 2014). The corresponding residue in IP3R type 1 is D2594, but its role (if any) in IP3R function is unknown. We hypothesized the IP3R1-D2594 also electrostatically interacts with luminal Ca2+ to determine IP3R1 luminal Ca2+ sensitivity. To test this possibility, we created a mouse knock-in model harboring the D2594K mutation in which the negatively charged IP3R1-D2594 residue was replaced by lysine, a positively charged residue. IP3R1-WT and -D2594K cerebella microsomes were isolated and reconstituted into planar lipid bilayers. Single channel IP3R1 studies showed the conductance properties of IP3R1-WT and -D2594K channels are similar. The IP3R1-D2594K channels exhibited WT-like IP3 affinity, but had substantially greater IP3 efficacy. The IP3R1-D2594K also had altered luminal Ca2+ sensitivity. Unlike IP3R1-WT, D2594K channel activity did not decrease at low luminal Ca2+ levels. In IP3R1-WT channels, the decrease in activity at these low luminal Ca2+ levels (i.e. IP3R1 luminal Ca2+ sensitivity) depends on the membrane potential. Taken together, these results indicate the IP3R1-D2594 residue, as it does in RyR2, electrostatically interacts with luminal Ca2+, explaining at least in part, the IP3R's sensitivity to luminal Ca2+. This newly identified mechanism together with luminal Ca2+ feed-through may explain the luminal control of IP3R1.

Haloperidol Affects Plasticity of Differentiated NG-108 Cells Through \u03c31R/IP3R1 Complex

Haloperidol is an antipsychotic agent that primarily acts as an antagonist of D2 dopamine receptors. Besides other receptor systems, it targets sigma 1 receptors (σ1Rs) and inositol 1,4,5-trisphosphate receptors (IP3Rs). Aim of this work was to investigate possible changes in IP3Rs and σ1Rs resulting from haloperidol treatment and to propose physiological consequences in differentiated NG-108 cells, i.e., effect on cellular plasticity. Haloperidol treatment resulted in up-regulation of both type 1 IP3Rs (IP3R1s) and σ1Rs at mRNA and protein levels. Haloperidol treatment did not alter expression of other types of IP3Rs. Calcium release from endoplasmic reticulum (ER) mediated by increased amount of IP3R1s elevated cytosolic calcium and generated ER stress. IP3R1s were bound to σ1Rs, and translocation of this complex from ER to nucleus occurred in the group of cells treated with haloperidol, which was followed by increased nuclear calcium levels. Haloperidol-induced changes in cytosolic, reticular, and nuclear calcium levels were similar when specific σ1 blocker -BD 1047- was used. Changes in calcium levels in nucleus, ER, and cytoplasm might be responsible for alterations in cellular plasticity, because length of neurites increased and number of neurites decreased in haloperidol-treated differentiated NG-108 cells.

IP3 receptor mutations and brain diseases in human and rodents

The inositol 1,4,5-trisphosphate receptor (IP3 R) is a huge Ca2+ channel that is localized at the endoplasmic reticulum. The IP3 R releases Ca2+ from the endoplasmic reticulum upon binding to IP3 , which is produced by various extracellular stimuli through phospholipase C activation. All vertebrate organisms have three subtypes of IP3 R genes, which have distinct properties of IP3 -binding and Ca2+ sensitivity, and are differently regulated by phosphorylation and by their associated proteins. Each cell type expresses the three subtypes of IP3 R in a distinct proportion, which is important for creating and maintaining spatially and temporally appropriate intracellular Ca2+ level patterns for the regulation of specific physiological phenomena. Of the three types of IP3 Rs, the type 1 receptor (IP3 R1) is dominantly expressed in the brain and is important for brain function. Recent emerging evidence suggests that abnormal Ca2+ signals from the IP3 R1 are closely associated with human brain pathology. In this review, we focus on the recent advances in our knowledge of the regulation of IP3 R1 and its functional implication in human brain diseases, as revealed by IP3 R mutation studies and analysis of human disease-associated genes. This article is part of the mini review series "60th Anniversary of the Japanese Society for Neurochemistry".

1H NMR Metabolomics Reveals Association of High Expression of Inositol 1, 4, 5 Trisphosphate Receptor and Metabolites in Breast Cancer Patients

1H NMR is used to detect alterations in metabolites and their linkage to metabolic processes in a number of pathological conditions including breast cancer. Inositol 1, 4, 5 trisphosphate (IP3R) receptor is an intracellular calcium channel known to regulate metabolism and cellular bioenergetics. Its expression is up regulated in a number of cancers. However, its linkage to metabolism in disease conditions has not been evaluated. This study was designed to determine the association if any, of these metabolites with altered expression of IP3R in breast cancer. We used 1H NMR to identify metabolites in the serum of breast cancer patients (n = 27) and performed Real-time Polymerase Chain Reaction analysis for quantifying the expression of IP3R type 3 and type 2 in tissues from breast cancer patients (n = 40). Principal Component Analysis (PCA) and Partial Least Square-Discriminant Analysis (PLS-DA) clearly distinguished patients with high/low IP3R expression from healthy subjects. The present study revealed high expression of IP3R type 2 and type 3 in human breast tumor tissue compared to adjacent non-tumorous tissue. Moreover, patients with ≥ 2-fold increase in IP3R (high IP3R group) had significantly higher concentration of metabolic intermediates compared to those with < 2-fold increase in IP3R (low IP3R group). We observed an increase in lipoprotein content and the levels of metabolites like lactate, lysine and alanine and a decrease in the levels of pyruvate and glucose in serum of high IP3R group patients when compared to those in healthy subjects. Receiver operating characteristic (ROC) curve analysis was performed to show the clinical utility of metabolites. In addition to the human studies, functional relevance of IP3Rs in causing metabolic disruption was observed in MCF-7 and MDA MB-231 cells. Results from our studies bring forth the importance of metabolic (or metabolomics) profiling of serum by 1H NMR in conjunction with tissue expression studies for characterizing breast cancer patients. The results from this study provide new insights into relationship of breast cancer metabolites with IP3R.

E platinum, a newly synthesized platinum compound, induces apoptosis through ROS-triggered ER stress in gastric carcinoma cells.

Gastric cancer (GC) is still one of the leading causes of death in cancer-related diseases. In this study, we aimed to investigate the antitumor effect of E Platinum, a newly platinum-based chemotherapeutic agent bearing the basic structure of Oxaliplatin, in a variety of gastric carcinoma cells and the underlying mechanisms. We demonstrated that E Platinum significantly induced apoptosis in gastric cancer cells via mitochondrial apoptotic pathway as a result of increased reactive oxygen species (ROS). We also found that E Platinum enhanced Ca2+ flux out from the endoplasmic reticulum by increasing the protein expression of IP3R type 1 (IP3R1) and decreasing the expression of ERp44. Dysfunction of Ca2+ homeostasis in endoplasmic reticulum (ER) leads to accumulation of unfolded proteins and ER stress. Mechanically, E Platinum increased ER stress associated protein expression such as GRP78, p-PERK, p-eIF2α, ATF4, and CHOP. However, knocking down CHOP reversed E Platinum-induced apoptosis by blocking mitochondrial apoptotic pathway. Furthermore, 10 mg/kg of E Platinum significantly suppressed BGC-823 tumor growth in vivo without toxicity, which correlated with induction of apoptosis and expression of ER stress related proteins in tumor tissues. Taken together, E Platinum inhibited tumor growth and induced apoptosis by ROS-mediated ER stress activation both in vitro and in vivo. Our study indicated that E Platinum may be a potential and effective treatment for gastric cancer in clinical. © 2016 Wiley Periodicals, Inc.

IP3R1 deficiency in the cerebellum/brainstem causes basal ganglia-independent dystonia by triggering tonic Purkinje cell firings in mice

The type 1 inositol 1,4,5- trisphosphate receptor (IP3R1) is a Ca2+ channel on the endoplasmic reticulum and is a predominant isoform in the brain among the three types of IP3Rs. Mice lacking IP3R1 show seizure-like behavior; however the cellular and neural circuit mechanism by which IP3R1 deletion causes the abnormal movements is unknown. Here, we found that the conditional knockout mice lacking IP3R1 specifically in the cerebellum and brainstem experience dystonia and show that cerebellar Purkinje cell (PC) firing patterns were coupled to specific dystonic movements. Recordings in freely behaving mice revealed epochs of low and high frequency PC complex spikes linked to body extension and rigidity, respectively. Remarkably, dystonic symptoms were independent of the basal ganglia, and could be rescued by inactivation of the cerebellum, inferior olive or in the absence of PCs. These findings implicate IP3R1-dependent PC firing patterns in cerebellum in motor coordination and the expression of dystonia through the olivo-cerebellar pathway.

FKBP52 is involved in the regulation of SOCE channels in the human platelets and MEG 01 cells

Immunophilins are FK506-binding proteins that have been involved in the regulation of calcium homeostasis, either by modulating Ca2+ channels located in the plasma membrane or in the rough endoplasmic reticulum (RE). We have investigated whether immunophilins would participate in the regulation of stored-operated Ca2+ entry (SOCE) in human platelets and MEG 01. Both cell types were loaded with fura-2 for determining cytosolic calcium concentration changes ([Ca2+]c), or stimulated and fixed to evaluate the protein interaction profile by performing immunoprecipitation and western blotting. We have found that incubation of platelets with FK506 increases Ca2+ mobilization. Thapsigargin (TG)-evoked, Thr-evoked SOCE and TG-evoked Mn2+ entry resulted in significant reduction by treatment of platelets with immunophilin antagonists. We confirmed by immunoprecipitation that immunophilins interact with transient receptor potential channel 1 (TRPC1) and Orai1 in human platelets. FK506 and rapamycin reduced the association between TRPC1 and Orai1 with FK506 binding protein (52) (FKBP52) in human platelets, and between TRPC1 and the type II IP3R, which association is known to be crucial for the maintenance of SOCE in human platelets. FKBP52 role in SOCE activation was confirmed by silencing FKBP52 using SiRNA FKBP52 in MEG 01 as demonstrated by single cell configuration imaging technique. TRPC1 silencing and depletion of cell of TRPC1 and FKBP52 simultaneously, impair activation of SOCE evoked by TG in MEG 01. Finally, in MEG 01 incubated with FK506 we observed a reduction in TRPC1/FKBP52 coupling, and similarly, FKBP52 silencing reduced the association between IP3R type II and TRPC1 during SOCE. All together, these results demonstrate that immunophilins participate in the regulation of SOCE in human platelets.

IP3 Receptor Type 2 Deficiency Is Associated with a Secretory Defect in the Pancreatic Acinar Cell and an Accumulation of Zymogen Granules

Acute pancreatitis is a painful, life-threatening disorder of the pancreas whose etiology is often multi-factorial. It is of great importance to understand the interplay between factors that predispose patients to develop the disease. One such factor is an excessive elevation in pancreatic acinar cell Ca2+. These aberrant Ca2+ elevations are triggered by release of Ca2+ from apical Ca2+ pools that are gated by the inositol 1,4,5-trisphosphate receptor (IP3R) types 2 and 3. In this study, we examined the role of IP3R type 2 (IP3R2) using mice deficient in this Ca2+ release channel (IP3R2−/−). Using live acinar cell Ca2+ imaging we found that loss of IP3R2 reduced the amplitude of the apical Ca2+ signal and caused a delay in its initiation. This was associated with a reduction in carbachol-stimulated amylase release and an accumulation of zymogen granules (ZGs). Specifically, there was a 2-fold increase in the number of ZGs (P<0.05) and an expansion of the ZG pool area within the cell. There was also a 1.6- and 2.6-fold increase in cellular amylase and trypsinogen, respectively. However, the mice did not have evidence of pancreatic injury at baseline, other than an elevated serum amylase level. Further, pancreatitis outcomes using a mild caerulein hyperstimulation model were similar between IP3R2−/− and wild type mice. In summary, IP3R2 modulates apical acinar cell Ca2+ signals and pancreatic enzyme secretion. IP3R-deficient acinar cells accumulate ZGs, but the mice do not succumb to pancreatic damage or worse pancreatitis outcomes.

Abstract 279: Inositol Triphosphate Receptor Activation Alters the Electrical Properties of Human Ventricular Myocytes

The recognition that ryanodine receptor (RyR) dysfunction is associated to arrhythmogenic diseases raises the possibility that alternative Ca2+ release channels alter the electrical properties of ventricular myocytes. The aim of this study was to determine whether inositol triphosphate receptor (IP3R)-mediated Ca2+ mobilization modulates Ca2+ cycling and electrical behavior in LV myocytes. Cells were obtained from human and mouse hearts and the expression and function of IP3Rs were evaluated. IP3Rs were identified in isolated myocytes by immunocytochemistry and Western blotting. In field-stimulated cells, IP3R activation via Gq-protein receptor agonists (ET-1, ATP) or enhancer of ligand affinity (thimerosal) increased diastolic Ca2+ and transient amplitude by 11% and 44%, respectively. Additionally, extra-systolic Ca2+ release and sustained Ca2+ elevations were detected. These effects were prevented by inhibition of IP3 production or by IP3R blockade. Importantly, myocytes obtained from mice infected in vivo with small hairpin RNA (shRNA) targeting IP3R type 2, failed to respond to Gq-protein receptor agonists. In patch-clamped human and mouse cells, changes in Ca2+ transient properties following IP3R activation were accompanied by a decrease in resting potential, action potential (AP) prolongation, and emergence of arrhythmic events. In mouse cardiomyocytes, assessment of excitation-contraction coupling gain and blockade of RyR channels under IP3R stimulation, excluded the contribution of RyRs to the effects induced by IP3R activation. In voltage-clamped cells, IP3R agonists promoted transient inward currents at the membrane potential of -70 mV and increased a nickel-sensitive current in the range of potentials corresponding to forward mode operation of the Na-Ca exchanger. To establish whether enhanced Ca2+ load was responsible for the altered electrical properties under IP3R activation, conditions buffering cytosolic Ca2+ levels were employed. Under these circumstances, IPR stimulation failed to prolong the AP and to induce arrhythmias. In conclusions, our observations demonstrate that Ca2+ mobilization via IP3Rs directly alters the electrical properties of human and rodent myocytes.

On the origin of rhythmic calcium transients in the ICC-MP of the mouse small intestine

Interstitial cells of Cajal associated with the myenteric plexus (ICC-MP) are pacemaker cells of the small intestine, producing the characteristic omnipresent electrical slow waves, which orchestrate peristaltic motor activity and are associated with rhythmic intracellular calcium oscillations. Our objective was to elucidate the origins of the calcium transients. We hypothesized that calcium oscillations in the ICC-MP are primarily regulated by the sarcoplasmic reticulum (SR) calcium release system. With the use of calcium imaging, study of the effect of T-type calcium channel blocker mibefradil revealed that T-type channels did not play a major role in generating the calcium transients. 2-Aminoethoxydiphenyl borate, an inositol 1,4,5 trisphosphate receptor (IP(3)R) inhibitor, and U73122, a phospholipase C inhibitor, both drastically decreased the frequency of calcium oscillations, suggesting a major role of IP(3) and IP(3)-induced calcium release from the SR. Immunohistochemistry proved the expression of IP(3)R type I (IP(3)R-I), but not type II (IP(3)R-II) and type III (IP(3)R-III) in ICC-MP, indicating the involvement of the IP(3)R-I subtype in calcium release from the SR. Cyclopiazonic acid, a SR/endoplasmic reticulum calcium ATPase pump inhibitor, strongly reduced or abolished calcium oscillations. The Na-Ca exchanger (NCX) in reverse mode is likely involved in refilling the SR because the NCX inhibitor KB-R7943 markedly reduced the frequency of calcium oscillations. Immunohistochemistry revealed 100% colocalization of NCX and c-Kit in ICC-MP. Testing a mitochondrial NCX inhibitor, we were unable to show an essential role for mitochondria in regulating calcium oscillations in the ICC-MP. In summary, ongoing IP(3) synthesis and IP(3)-induced calcium release from the SR, via the IP(3)R-I, are the major drivers of the calcium transients associated with ICC pacemaker activity. This suggests that a biochemical clock intrinsic to ICC determines the pacemaker frequency, which is likely directly linked to kinetics of the IP(3)-activated SR calcium channel and IP(3) metabolism.

Origin of Rhythmic Calcium Transients in the ICC-MP of the Mouse Small Intestine

Lowie B-J, Wang X-Y, White EJ, Huizinga JD. On the origin of rhythmic calcium transients in the ICC-MP of the mouse small intestine. Am J Physiol Gastrointest Liver Physiol 301: G835–G845, 2011. First published August 11, 2011; doi:10.1152/ajpgi.00077.2011.—Interstitial cells of Cajal associated with the myenteric plexus (ICC-MP) are pacemaker cells of the small intestine, producing the characteristic omnipresent electrical slow waves, which orchestrate peristaltic motor activity and are associated with rhythmic intracellular calcium oscillations. Our objective was to elucidate the origins of the calcium transients. We hypothesized that calcium oscillations in the ICC-MP are primarily regulated by the sarcoplasmic reticulum (SR) calcium release system. With the use of calcium imaging, study of the effect of T-type calcium channel blocker mibefradil revealed that T-type channels did not play a major role in generating the calcium transients. 2-Aminoethoxydiphenyl borate, an inositol 1,4,5 trisphosphate receptor (IP3R) inhibitor, and U73122, a phospholipase C inhibitor, both drastically decreased the frequency of calcium oscillations, suggesting a major role of IP3 and IP3-induced calcium release from the SR. Immunohistochemistry proved the expression of IP3R type I (IP3R-I), but not type II (IP3R-II) and type III (IP3R-III) in ICC-MP, indicating the involvement of the IP3R-I subtype in calcium release from the SR. Cyclopiazonic acid, a SR/endoplasmic reticulum calcium ATPase pump inhibitor, strongly reduced or abolished calcium oscillations. The Na-Ca exchanger (NCX) in reverse mode is likely involved in refilling the SR because the NCX inhibitor KB-R7943 markedly reduced the frequency of calcium oscillations. Immunohistochemistry revealed 100% colocalization of NCX and c-Kit in ICC-MP. Testing a mitochondrial NCX inhibitor, we were unable to show an essential role for mitochondria in regulating calcium oscillations in the ICCMP. In summary, ongoing IP3 synthesis and IP3-induced calcium release from the SR, via the IP3R-I, are the major drivers of the calcium transients associated with ICC pacemaker activity. This suggests that a biochemical clock intrinsic to ICC determines the pacemaker frequency, which is likely directly linked to kinetics of the IP3-activated SR calcium channel and IP3 metabolism.

Inositol 1,4,5-trisphosphate receptors are essential for the development of the second heart field

Congenital heart defects (CHDs) occur in 0.5–1% of live births, yet the underlying genetic etiology remains mostly unknown. Recently, a new source of myocardial cells, namely the second heart field (SHF), was discovered in the splanchnic mesoderm. Abnormal development of the SHF leads to a spectrum of outflow tract defects, such as persistent truncus arteriosus and tetralogy of Fallot. Intracellular Ca 2+ signaling is known to be essential for many aspects of heart biology including heart development, but its role in the SHF is uncertain. Here, we analyzed mice deficient for genes encoding inositol 1,4,5-trisphosphate receptors (IP 3Rs), which are intracellular Ca 2+ release channels on the endo/sarcoplasmic reticulum that mediate Ca 2+ mobilization. Mouse embryos that are double mutant for IP 3R type 1 and type 3 ( IP 3 R1 −/− IP 3 R3 −/− ) show hypoplasia of the outflow tract and the right ventricle, reduced expression of specific molecular markers and enhanced apoptosis of mesodermal cells in the SHF. Gene expression analyses suggest that IP 3R-mediated Ca 2+ signaling may involve, at least in part, the Mef2C–Smyd1 pathway, a transcriptional cascade essential for the SHF. These data reveal that IP 3R type 1 and type 3 may play a redundant role in the development of the SHF.

Differential Genomic and Proteomic Signatures In Cord Blood (CB) Vs Peripheral Blood (PB) CD56+Dim NK Cells: Over Expression of CD34 In CB Vs PB CD56+Dim NK Cells

AbstractAbstract 2781 Background.Umbilical cord blood (UCB) is a viable alternative source of allogeneic hematopoietic stem cells for the treatment of both malignant and non-malignant disease (Cairo et al BBMT 2008). UCB transplantation (UCBT) is known to be associated with decrease severe acute graft-versus-host disease (GvHD) compared to unrelated bone marrow (BM) and peripheral blood (PB) transplantation; however, it is associated with delayed hematopoietic and immune reconstitution (Szabolcs/Cairo et al Seminars in Hematology 2010). NK cells play important roles in both innate and adaptive immunity and are characterized as a CD56+ cell population. NK cell recovery is prompt by 2 months after hematopoietic stem cell transplantation (HSCT), while T-cell (after at least 9 mo HSCT) and B-cell (after 3 to 4 mo HSCT) reconstitutions are gradual and delayed. CD56+dim cells are primarily cytotoxic and make up 90% of PB NK populations (Shereck/Cairo PBC 2007). We previously demonstrated the ability to ex-vivo expand CB MNC into various phenotypes of CD56+dim and CD56+bright NK cells (totally 60%) and NKT cells (40%) with profound in vitro and in vivo cytotoxicity against hematological malignancies (Ayello/Cairo BBMT 2006 & Exp. Hematology 2009). Proteomic studies from our group demonstrated differential protein expression including ↑NKG2A, ↓IP3R type 3, ↓MAPKAPK5, and ↑NOTCH 2 in CB vs PB CD56+dim (Shereck/Cairo, ASH 2007; Shereck/Day/Cairo, ASBMT 2009). Objective.In these studies, we sought to determine the similarity or differences in genetic signatures in CB vs APB CD56+dim NK cells. Methods.CB MNCs were isolated on a ficoll gradient and NK CD56+16+dim cells isolated using a 2-step magnetic activated cell separation (MACS) process via a standard kit (Miltenyi Biotec). Enrichment was at least 94%. Isolated RNA from CB and PB CD56+dim cells were subjected to microarray studies (Affymetrix, U133A_2) as we have previously described (Jiang/Cairo et al J Immunol 2004). Data were analyzed by Agilent GeneSpring and Ingenuity pathway analyses. Welch test were used to perform statistical analysis and fold change of < 1.5 and values of p<0.05 were considered to be significant. Two-color ECL Plex fluorescence Western blotting (WB) was preformed to validate the proteomic data. Protein samples were separated using SDS-PAGE followed by transblotting. WB membranes were then incubated with target and control (GAPDH) primary antibodies. After rinse and wash, the membranes were further incubated with CY5 and CY3 conjugated secondary antibodies. The membranes were scanned with TYPHOON by green (532 laser and 580 filter) and red (633 laser and 670 filter) setting for CY3 and CY5, respectively, and then observed and quantified using ImageQuant. Results.CB vs PB CD56+dim cells significantly altered expressed 796 genes, in which 486 genes were over expressed, at the genomic level including: pro-apoptotic genes: CASP10 (3.1F), TNFSF11 (4.7F), CDC2 (3.0F), BCL2L1 (4.3F), NOTCH2 (1.5F); and cell development: PBX1 (7.6F), IL1RN (5.1F), CD24 (5.3F), CD34 (3.5F), CD55 (2.1F), CCL13 (2.2F). Conversely, there was significant under expression of NF1 (5.1F), MAP2K3 (1.7F), PIK3CD (2.1F), BAX (2.9F), and JUN (2.2F). Our WB results indicate that NOTCH2 (2.4F) and PBX1 (2.2F) proteins are increased in CB vs PB CD56+dim NK cells, consistent with our proteomic results. Conclusion.These results suggest that CB vs PB CD56+dim NK are more prone to undergo programmed cell death (apoptosis) secondary to over expression of numerous pro-apoptotic genes, and may be earlier in development (pro-NK) with over expression of the CD34 gene. Furthermore, decrease CB vs PB NK cytotoxicity maybe in part secondary to increase programmed cell death in particularly increase NOTCH2 at the genomic and proteomic levels. (The first two authors contribute equally.) Disclosures:No relevant conflicts of interest to declare.

Neuronal calcium sensor-1 regulation of calcium channels, secretion, and neuronal outgrowth.

Calcium (Ca(2+)) is an important intracellular messenger underlying cell physiology. Ca(2+) channels are the main entry route for Ca(2+) into excitable cells, and regulate processes such as neurotransmitter release and neuronal outgrowth. Neuronal Calcium Sensor-1 (NCS-1) is a member of the Calmodulin superfamily of EF-hand Ca(2+) sensing proteins residing in the subfamily of NCS proteins. NCS-1 was originally discovered in Drosophila as an overexpression mutant (Frequenin), having an increased frequency of Ca(2+)-evoked neurotransmission. NCS-1 is N-terminally myristoylated, can bind intracellular membranes, and has a Ca(2+) affinity of 0.3 μM. Over 10 years ago it was discovered that NCS-1 overexpression enhances Ca(2+)-evoked secretion in bovine adrenal chromaffin cells. The mechanism was unclear, but there was no apparent direct effect on the exocytotic machinery. It was revealed, again in chromaffin cells, that NCS-1 regulates voltage-gated Ca(2+) channels (Cavs) in G-Protein Coupled Receptor (GPCR) signaling pathways. This work in chromaffin cells highlighted NCS-1 as an important modulator of neurotransmission. NCS-1 has since been shown to regulate and/or directly interact with many proteins including Cavs (P/Q, N, and L), TRPC1/5 channels, GPCRs, IP3R, and PI4 kinase type IIIβ. NCS-1 also affects neuronal outgrowth having roles in learning and memory affecting both short- and long-term synaptic plasticity. It is not known if NCS-1 affects neurotransmission and synaptic plasticity via its effect on PIP2 levels, and/or via a direct interaction with Ca(2+) channels or their signaling complexes. This review gives a historical account of NCS-1 function, examining contributions from chromaffin cells, PC12 cells and other models, to describe how NCS-1's regulation of Ca(2+) channels allows it to exert its physiological effects.

Regulation of Inositol 1,4,5 Trisphosphate Receptors by InsP3 Receptor-Associated cGMP Kinase Substrate (IRAG)

Various factors interact with IP3R, regulating Ca2+ release and thus serve to define the spatial and temporal characteristics of the cytosolic Ca2+ signal. IP3R-associated cGMP kinase substrate (IRAG) has been reported to bind IP3R type-1 and inhibit Ca2+ mobilization in smooth muscle cells. No information is however available as to whether IRAG interacts or has functional effects on other IP3R family members. In this study, we examined whether IRAG binds to and regulates Ca2+ release via IP3R type-2 or type-3. cDNA encoding IP3R type-1, IRAG-GFP, and protein kinase G1β (PKG1β) were transiently transfected into COS cells. Following immunoprecipitation from cell lysates with an anti-GFP antibody, IP3R type-1 was detected by immunoblotting. In contrast, an IRAG-GFP construct (IRAGΔE12-GFP) in which 40 amino acids required for binding with IP3R was deleted, failed to interact with IP3R type-1, but was still capable of binding to PKG1β, an additional cognate binding partner of IRAG. Similarly, IP3R type-2 or IP3R type-3 could be shown to interact with IRAG-GFP but not IRAGΔE12-GFP in COS cells. Next, we investigated if IRAG regulates IP3-induced Ca-release using DT40-3KO cell lines stably expressing mammalian IP3R type-2 or type-3 in isolation. In DT40-3KO cells stably expressing IP3R type-2, and transiently expressing Muscarinic M3 receptor, IRAG-GFP and PKG1β, cell permeable PKG activators reduced the muscarinic agonist carbachol (CCh)-induced Ca2+-release. Ca2+ oscillations induced by low concentrations of CCh or by stimulating the endogenous B cell receptor were similarly attenuated. No inhibitory effect was evident in cells expressing IRAGΔE12-GFP or in the absence of IRAG-GFP. Similar results were obtained with DT40-3KO cells stably expressing IP3R type-3. These results indicate that Ca2+ release through all Inositol 1,4,5 trisphosphate receptors are inhibited by an interaction with IRAG and PKG1β.