The inositol 1,4,5-trisphosphate receptor (InsP 3 R) is an intracellular Ca 2؉ release channel that mediates the rise in cytoplasmic calcium in response to receptor-activated production of InsP 3 . The InsP 3 R-mediated signaling pathway appears to be ubiquitous and is involved in many cellular processes including cell division, smooth muscle contraction, and neuronal signaling. Different regions of the heart also express InsP 3 receptors. We report here that acutely dissociated ventricular myocytes from ferret and rat hearts express significant levels of InsP 3 R as indicated by immunoblotting with a receptor consensus antibody. InsP 3 binding experiments (K D ‫؍‬ 23.6 nM and B max ‫؍‬ 0.46 pmol/mg) suggest the myocytes contain the high affinity type 2 InsP 3 receptor. Exhaustive mRNA screening by polymerase chain reaction, RNase protection, and subsequent DNA sequencing positively identify the InsP 3 R as type 2. The type 2 receptor from ferret heart was then incorporated into planar lipid bilayers and formed Ca 2؉ -selective, InsP 3 -activated, heparin-blocked ion channels. We conclude that the predominant InsP 3 receptor isoform expressed in cardiac myocytes is type 2 and that it forms a functional InsP 3 -gated Ca 2؉ channel when reconstituted in planar lipid bilayers.Inositol 1,4,5-trisphosphate (InsP 3 ) 1 is a well known second messenger mediating the regulated release of intracellular calcium and is produced through the action of phospholipase C. Phospholipase C is activated in response to the stimulation of cell surface receptors coupled to heterotrimeric G-proteins and tyrosine kinases resulting in the hydrolysis of phosphatidylinositol 4,5-bisphosphate to liberate InsP 3 and diacylglycerol. InsP 3 is a readily diffusible compound that binds to specific (InsP 3 R) receptors localized to the endoplasmic reticulum and results in the release of calcium from intracellular stores (see reviews Refs.

The inositol 1,4,5-trisphosphate receptor (InsP 3 R) is a tetrameric assembly of highly conserved subunits that contain multiple membrane-spanning sequences in the C-terminal region of the protein. In studies aimed at investigating the oligomerization and transmembrane topology of the type-1 InsP 3 R, a series of membranespanning region truncation and deletion plasmids were constructed. These plasmids were transiently transfected in COS-1 cells, and the resulting expression products were analyzed for the ability to assemble into tetrameric structures. The topology of the membranespanning region truncations and the full-length receptor was determined by immunocytochemical analysis of transfected COS-1 cells using complete or selective permeabilization strategies. Our results are the first to experimentally define the presence of six membrane-spanning regions. These results are consistent with the current model for the organization of the InsP 3 R in the endoplasmic reticulum and show that the truncation mutants are properly targeted and oriented in the endoplasmic reticulum membrane, thus making them amenable reagents to study receptor subunit oligomerization. Fractionation of soluble and membrane protein components revealed that the first two membrane-spanning regions were necessary for membrane targeting of the receptor. Sedimentation and immunoprecipitation experiments show that assembly of the receptor subunits was an additive process as the number of membrane-spanning regions increased. Immunoprecipitations from cells co-expressing the full-length receptor and carboxyl-terminal truncations reveal that constructs expressing the first two or more membranespanning domains were capable of co-assembling with the full-length receptor. Inclusion of the fifth membrane-spanning segment significantly enhanced the degree of oligomerization. Furthermore, a deletion construct containing only membrane-spanning regions 5 and 6 oligomerized to a similar extent as that of the wild type protein. Membrane-spanning region deletion constructions that terminate with the receptor's 145 carboxyl-terminal amino acids were found to have enhanced assembly characteristics and implicate the carboxyl terminus as a determinant in oligomerization. Our results reveal a process of receptor assembly involving several distinct yet additive components and define the fifth and sixth membrane spanning regions as the key determinants in receptor oligomerization.Inositol 1,4,5-trisphosphate (InsP 3 ) 1 is a well known second messenger that plays a pivotal role in the regulated release of intracellular calcium. Plasma membrane receptor-coupled activation of G-protein or tyrosine kinase-induced hydrolysis of phosphotidylinositol 4,5-bisphosphate results in the production of InsP 3 and subsequent efflux of Ca 2ϩ from endoplasmic reticulum stores. InsP 3 -mediated calcium release has been implicated in numerous, very diverse cellular processes including the initiation/propagation of Ca 2ϩ waves, cell growth, secretion, fertilization, and development (1...

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Background: Coronary artery disease (CAD) is a major cause of out-of-hospital cardiac arrest (OHCA). The role of emergency coronary angiography (CAG) and percutaneous coronary intervention (PCI) following cardiac arrest in patients without ST-segment elevation myocardial infarction (STEMI) remains unclear. Aims: We aim to assess whether emergency CAG and PCI, when indicated, will improve survival with good neurological outcome in post-OHCA patients without STEMI who remain comatose. Methods: COUPE is a prospective, multicentre and randomized controlled clinical trial. A total of 166 survivors of OHCA without STEMI will be included. Potentially non-cardiac aetiology of the cardiac arrest will be ruled out prior to randomization. Randomization will be 1:1 for emergency (within 2 h) or deferred (performed before discharge) CAG. Both groups will receive routine care in the intensive cardiac care unit, including therapeutic hypothermia. The primary efficacy endpoint is a composite of in-hospital survival free of severe dependence, which will be evaluated using the Cerebral Performance Category Scale. The safety endpoint will be a composite of major adverse cardiac events including death, reinfarction, bleeding and ventricular arrhythmias. Conclusions: This study will assess the efficacy of an emergency CAG versus a deferred one in OHCA patients without STEMI in terms of survival and neurological impairment.