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.

Both d- and l-Glucose Polyphosphates Mimic d-myo-Inositol 1,4,5-Trisphosphate: New Synthetic Agonists and Partial Agonists at the Ins(1,4,5)P3 Receptor.

Chiral sugar derivatives are potential cyclitol surrogates of the Ca2+-mobilizing intracellular messenger d-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]. Six novel polyphosphorylated analogues derived from both d- and l-glucose were synthesized. Binding to Ins(1,4,5)P3 receptors [Ins(1,4,5)P3R] and the ability to release Ca2+ from intracellular stores via type 1 Ins(1,4,5)P3Rs were investigated. ß-d-Glucopyranosyl 1,3,4-tris-phosphate, with similar phosphate regiochemistry and stereochemistry to Ins(1,4,5)P3, and α-d-glucopyranosyl 1,3,4-tris-phosphate are full agonists, being equipotent and 23-fold less potent than Ins(1,4,5)P3, respectively, in Ca2+-release assays and similar to Ins(1,4,5)P3 and 15-fold weaker in binding assays. They can be viewed as truncated analogues of adenophostin A and refine understanding of structure-activity relationships for this Ins(1,4,5)P3R agonist. l-Glucose-derived ligands, methyl α-l-glucopyranoside 2,3,6-trisphosphate and methyl α-l-glucopyranoside 2,4,6-trisphosphate, are also active, while their corresponding d-enantiomers, methyl α-d-glucopyranoside 2,3,6-trisphosphate and methyl α-d-glucopyranoside 2,4,6-trisphosphate, are inactive. Interestingly, both l-glucose-derived ligands are partial agonists: they are among the least efficacious agonists of Ins(1,4,5)P3R yet identified, providing new leads for antagonist development.

[Medicinal Chemical Studies Based on the Theoretical Design of Bioactive Compounds].

I have engaged in medicinal chemical studies based on the theoretical design of bioactive compounds. First, I present a three-dimensional structural diversity-oriented conformational restriction strategy for developing bioactive compounds based on the characteristic steric and stereoelectronic features of cyclopropane. Using this strategy, various biologically active small molecule compounds, such as receptor agonists/antagonists and enzyme inhibitors, were effectively developed. The strategy was also applied to develop versatile peptidomimetics and membrane-permeable cyclic peptides. Next, studies on Ca2+-mobilizing second messengers, cyclic ADP-ribose (cADPR) and myo-inositol trisphosphates (IP3), are described. In these studies, stable equivalents of cADPR were developed, since biological studies of cADPR have been limited due to its instability. Various potent IP3 receptor ligands, which were designed using the d-glucose structure as a bioisostere of the myo-inositol moiety of IP3, have been identified. Organic chemistry studies have also been extensively performed, because excellent organic chemistry is essential for promoting high-level medicinal chemical studies. For examples, new methods for the synthesis of chiral cyclopropanes, new radical reactions with silicon tethers, and kinetic anomeric effect-dependent stereoselective glycosidations have been developed.

Competitive Fluorescent Ligand Assay for Inositol 1,4,5-Trisphosphate.

We present a novel method, termed competitive fluorescent ligand assay for inositol 1,4,5-trisphosphate (CFLA-IP3), to measure inositol 1,4,5-trisphosphate (IP3). This method is based on fluorescence resonance energy transfer (FRET) between two fluorescent molecules, a fluorescent IP3-binding protein and its fluorescent ligand. Binding of these fluorescent molecules generates a FRET signal, and the IP3-dependent decrease in the FRET signal due to displacement of the fluorescent ligand is detected by fluorescence microscopy.

Experimental and theoretical study of IBC domain from human IP3R2; molecular cloning, bacterial expression and protein purification.

IP3 is a ubiquitous second messenger in eukaryotic cells that triggers Ca2+-release from intracellular stores. IP3 binds to intracellular IP3-receptor (IP3R) and induces conformational change within the ligand-binding domain which regulates Ca2+-release; hence, both IP3 and IP3R are key components of the signal transduction mechanism. Here we present cDNA cloning of IP3-binding core (IBC) domain encoding only residues 224-604 of human IP3R type 2 that binds to IP3 with high affinity. RNA extraction, RT-PCR, PCR and cloning were carried out, and then the cloned DNA was checked by sequencing. Thereafter, expression vector pET-28a harboring the correct gene was transformed into different E. coli (DE3) strains and investigated its protein expression under various conditions. Finally, the IBC expression was induced at 20 °C for 20 h into BL21 strain at LB medium with 4 mM lactose and 0.5 mM IPTG, and then confirmed by western blotting. After protein purification, structural study was recorded in absence and presence of its ligand. Far-CD and intrinsic fluorescence spectra analysis of the purified protein with and without IP3 ligand showed change in secondary and tertiary IBC structure. Moreover, bioinformatics study demonstrated that the ligand binding site residues R269, K508 and R511 are conserved.

Structural features of human inositol phosphate multikinase rationalize its inositol phosphate kinase and phosphoinositide 3-kinase activities.

Human inositol phosphate multikinase (HsIPMK) critically contributes to intracellular signaling through its inositol-1,4,5-trisphosphate (Ins(1,4,5)P3) 3-kinase and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) 3-kinase activities. This catalytic profile is not conserved; orthologs from Arabidopsis thaliana and Saccharomyces cerevisiae are predominantly Ins(1,4,5)P3 6-kinases, and the plant enzyme cannot phosphorylate PtdIns(4,5)P2 Therefore, crystallographic analysis of the yeast and plant enzymes, without bound inositol phosphates, do not structurally rationalize HsIPMK activities. Here, we present 1.6-Å resolution crystal structures of HsIPMK in complex with either Ins(1,4,5)P3 or PtdIns(4,5)P2 The Ins(1,4,5)P3 headgroup of PtdIns(4,5)P2 binds in precisely the same orientation as free Ins(1,4,5)P3 itself, indicative of evolutionary optimization of 3-kinase activities against both substrates. We report on nucleotide binding between the separate N- and C-lobes of HsIPMK. The N-lobe exhibits a remarkable degree of conservation with protein kinase A (root mean square deviation = 1.8 Å), indicating common ancestry. We also describe structural features unique to HsIPMK. First, we observed a constrained, horseshoe-shaped substrate pocket, formed from an α-helix, a 310 helix, and a recently evolved tri-proline loop. We further found HsIPMK activities rely on a preponderance of Gln residues, in contrast to the larger Lys and Arg residues in yeast and plant orthologs. These conclusions are supported by analyzing 14 single-site HsIPMK mutants, some of which differentially affect 3-kinase and 6-kinase activities. Overall, we structurally rationalize phosphorylation of Ins(1,4,5)P3 and PtdIns(4,5)P2 by HsIPMK.

IP3 receptor signaling and endothelial barrier function.

The endothelium, a monolayer of endothelial cells lining vessel walls, maintains tissue-fluid homeostasis by restricting the passage of the plasma proteins and blood cells into the interstitium. The ion Ca2+, a ubiquitous secondary messenger, initiates signal transduction events in endothelial cells that is critical to control of vascular tone and endothelial permeability. The ion Ca2+ is stored inside the intracellular organelles and released into the cytosol in response to environmental cues. The inositol 1,4,5-trisphosphate (IP3) messenger facilitates Ca2+ release through IP3 receptors which are Ca2+-selective intracellular channels located within the membrane of the endoplasmic reticulum. Binding of IP3 to the IP3Rs initiates assembly of IP3R clusters, a key event responsible for amplification of Ca2+ signals in endothelial cells. This review discusses emerging concepts related to architecture and dynamics of IP3R clusters, and their specific role in propagation of Ca2+ signals in endothelial cells.

Circular oligomerization is an intrinsic property of synaptotagmin.

Previously, we showed that synaptotagmin1 (Syt1) forms Ca2+-sensitive ring-like oligomers on membranes containing acidic lipids and proposed a potential role in regulating neurotransmitter release (Zanetti et al., 2016). Here, we report that Syt1 assembles into similar ring-like oligomers in solution when triggered by naturally occurring polyphosphates (PIP2 and ATP) and magnesium ions (Mg2+). These soluble Syt1 rings were observed by electron microscopy and independently demonstrated and quantified using fluorescence correlation spectroscopy. Oligomerization is triggered when polyphosphates bind to the polylysine patch in C2B domain and is stabilized by Mg2+, which neutralizes the Ca2+-binding aspartic acids that likely contribute to the C2B interface in the oligomer. Overall, our data show that ring-like polymerization is an intrinsic property of Syt1 with reasonable affinity that can be triggered by the vesicle docking C2B-PIP2 interaction and raise the possibility that Syt1 rings could pre-form on the synaptic vesicle to facilitate docking.

Bacterial PhyA protein-tyrosine phosphatase-like myo-inositol phosphatases in complex with the Ins(1,3,4,5)P4 and Ins(1,4,5)P3 second messengers.

myo-Inositol phosphates (IPs) are important bioactive molecules that have multiple activities within eukaryotic cells, including well-known roles as second messengers and cofactors that help regulate diverse biochemical processes such as transcription and hormone receptor activity. Despite the typical absence of IPs in prokaryotes, many of these organisms express IPases (or phytases) that dephosphorylate IPs. Functionally, these enzymes participate in phosphate-scavenging pathways and in plant pathogenesis. Here, we determined the X-ray crystallographic structures of two catalytically inactive mutants of protein-tyrosine phosphatase-like myo-inositol phosphatases (PTPLPs) from the non-pathogenic bacteria Selenomonas ruminantium (PhyAsr) and Mitsuokella multacida (PhyAmm) in complex with the known eukaryotic second messengers Ins(1,3,4,5)P4 and Ins(1,4,5)P3 Both enzymes bound these less-phosphorylated IPs in a catalytically competent manner, suggesting that IP hydrolysis has a role in plant pathogenesis. The less-phosphorylated IP binding differed in both the myo-inositol ring position and orientation when compared with a previously determined complex structure in the presence of myo-inositol-1,2,3,4,5,6-hexakisphosphate (InsP6 or phytate). Further, we have demonstrated that PhyAsr and PhyAmm have different specificities for Ins(1,2,4,5,6)P5, have identified structural features that account for this difference, and have shown that the absence of these features results in a broad specificity toward Ins(1,2,4,5,6)P5 These features are main-chain conformational differences in loops adjacent to the active site that include the extended loop prior to the penultimate helix, the extended Ω-loop, and a ß-hairpin turn of the Phy-specific domain.

IP3-mediated gating mechanism of the IP3 receptor revealed by mutagenesis and X-ray crystallography.

The inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) is an IP3-gated ion channel that releases calcium ions (Ca2+) from the endoplasmic reticulum. The IP3-binding sites in the large cytosolic domain are distant from the Ca2+ conducting pore, and the allosteric mechanism of how IP3 opens the Ca2+ channel remains elusive. Here, we identify a long-range gating mechanism uncovered by channel mutagenesis and X-ray crystallography of the large cytosolic domain of mouse type 1 IP3R in the absence and presence of IP3 Analyses of two distinct space group crystals uncovered an IP3-dependent global translocation of the curvature α-helical domain interfacing with the cytosolic and channel domains. Mutagenesis of the IP3R channel revealed an essential role of a leaflet structure in the α-helical domain. These results suggest that the curvature α-helical domain relays IP3-controlled global conformational dynamics to the channel through the leaflet, conferring long-range allosteric coupling from IP3 binding to the Ca2+ channel.

Oocyte activation and latent HIV-1 reactivation: AMPK as a common mechanism of action linking the beginnings of life and the potential eradication of HIV-1.

In all mammalian species studied to date, the initiation of oocyte activation is orchestrated through alterations in intracellular calcium (Ca(2+)) signaling. Upon sperm binding to the oocyte plasma membrane, a sperm-associated phospholipase C (PLC) isoform, PLC zeta (PLCζ), is released into the oocyte cytoplasm. PLCζ hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) to produce diacylglycerol (DAG), which activates protein kinase C (PKC), and inositol 1,4,5-trisphosphate (IP3), which induces the release of Ca(2+) from endoplasmic reticulum (ER) Ca(2+) stores. Subsequent Ca(2+) oscillations are generated that drive oocyte activation to completion. Ca(2+) ionophores such as ionomycin have been successfully used to induce artificial human oocyte activation, facilitating fertilization during intra-cytoplasmic sperm injection (ICSI) procedures. Early studies have also demonstrated that the PKC activator phorbol 12-myristate 13-acetate (PMA) acts synergistically with Ca(2+) ionophores to induce parthenogenetic activation of mouse oocytes. Interestingly, the Ca(2+)-induced signaling cascade characterizing sperm or chemically-induced oocyte activation, i.e. the "shock and live" approach, bears a striking resemblance to the reactivation of latently infected HIV-1 viral reservoirs via the so called "shock and kill" approach, a method currently being pursued to eradicate HIV-1 from infected individuals. PMA and ionomycin combined, used as positive controls in HIV-1 latency reversal studies, have been shown to be extremely efficient in reactivating latent HIV-1 in CD4(+) memory T cells by inducing T cell activation. Similar to oocyte activation, T cell activation by PMA and ionomycin induces an increase in intracellular Ca(2+) concentrations and activation of DAG, PKC, and downstream Ca(2+)-dependent signaling pathways necessary for proviral transcription. Interestingly, AMPK, a master regulator of cell metabolism that is activated thorough the induction of cellular stress (e.g. increase in Ca(2+) concentration, reactive oxygen species generation, increase in AMP/ATP ratio) is essential for oocyte maturation, T cell activation, and mitochondrial function. In addition to the AMPK kinase LKB1, CaMKK2, a Ca(2+)/calmodulin-dependent kinase that also activates AMPK, is present in and activated on T cell activation and is also present in mouse oocytes and persists until the zygote and two-cell stages. It is our hypothesis that AMPK activation represents a central node linking T cell activation-induced latent HIV-1 reactivation and both physiological and artificial oocyte activation. We further propose the novel observation that various compounds that have been shown to reactivate latent HIV-1 (e.g. PMA, ionomycin, metformin, bryostatin, resveratrol, etc.) or activate oocytes (PMA, ionomycin, ethanol, puromycin, etc.) either alone or in combination likely do so via stress-induced activation of AMPK.

Calcium Wave Propagation Triggered by Local Mechanical Stimulation as a Method for Studying Gap Junctions and Hemichannels.

Intercellular communication is essential for the coordination and synchronization of cellular processes. Gap junction channels play an important role to communicate between cells and organs, including the brain, lung, liver, lens, retina, and heart. Gap junctions enable a direct route for ions like calcium and potassium, and low molecular weight compounds, such as inositol 1,4,5-trisphosphate, cyclic adenosine monophosphate, and various kinds of metabolites to pass between cells. Intercellular calcium wave propagation evoked by a local mechanical stimulus is one of the gap junction assays to study intercellular communication. In experimental settings, an intercellular calcium wave can be elicited by applying a mechanical stimulus to a single cell. Here, we describe the use of monolayers of primary bovine corneal endothelial cells as a model to study intercellular communication. Calcium wave propagation was assayed by imaging fluorescent calcium in bovine corneal endothelial cells loaded with a fluorescent calcium dye using a confocal microscope. Spatial changes in intercellular calcium concentration following mechanical stimulation were measured in the mechanical stimulated cell and in the neighboring cells. The active area (i.e., total surface area of responsive cells) of a calcium wave can be measured and used for studying the function and regulation of gap junction channels as well as hemichannels in a variety of cell systems.

Role of Conserved Disulfide Bridges and Aromatic Residues in Extracellular Loop 2 of Chemokine Receptor CCR8 for Chemokine and Small Molecule Binding.

Chemokine receptors play important roles in the immune system and are linked to several human diseases. The initial contact of chemokines with their receptors depends on highly specified extracellular receptor features. Here we investigate the importance of conserved extracellular disulfide bridges and aromatic residues in extracellular loop 2 (ECL-2) for ligand binding and activation in the chemokine receptor CCR8. We used inositol 1,4,5-trisphosphate accumulation and radioligand binding experiments to determine the impact of receptor mutagenesis on both chemokine and small molecule agonist and antagonist binding and action in CCR8. We find that the seven-transmembrane (TM) receptor conserved disulfide bridge (7TM bridge) linking transmembrane helix III (TMIII) and ECL-2 is crucial for chemokine and small molecule action, whereas the chemokine receptor conserved disulfide bridge between the N terminus and TMVII is needed only for chemokines. Furthermore, we find that two distinct aromatic residues in ECL-2, Tyr(184) (Cys + 1) and Tyr(187) (Cys + 4), are crucial for binding of the CC chemokines CCL1 (agonist) and MC148 (antagonist), respectively, but not for small molecule binding. Finally, using in silico modeling, we predict an aromatic cluster of interaction partners for Tyr(187) in TMIV (Phe(171)) and TMV (Trp(194)). We show in vitro that these residues are crucial for the binding and action of MC148, thus supporting their participation in an aromatic cluster with Tyr(187) This aromatic cluster appears to be present in a large number of CC chemokine receptors and thereby could play a more general role to be exploited in future drug development targeting these receptors.

IP3 receptors: Take four IP3 to open.

Inositol 1,4,5-trisphosphate receptors (IP3Rs) and ryanodine receptors are the channels responsible for Ca(2+)release from the endoplasmic and sarcoplasmic reticulum. Research inScience Signalingby Alzayadyet al show that all four IP3-binding sites within the tetrameric IP3R must bind IP3before the channel can open, which has important consequences for the distribution of both IP3and IP3R activity within cells.

Defining the stoichiometry of inositol 1,4,5-trisphosphate binding required to initiate Ca2+ release.

Inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) are tetrameric intracellular Ca(2+)-release channels with each subunit containing a binding site for IP3in the amino terminus. We provide evidence that four IP3molecules are required to activate the channel under diverse conditions. Comparing the concentration-response relationship for binding and Ca(2+)release suggested that IP3Rs are maximally occupied by IP3before substantial Ca(2+)release occurs. We showed that ligand binding-deficient subunits acted in a dominant-negative manner when coexpressed with wild-type monomers in the chicken immune cell line DT40-3KO, which lacks all three genes encoding IP3R subunits, and confirmed the same effect in an IP3R-null human cell line (HEK-3KO) generated by CRISPR/Cas9 technology. Using dimeric and tetrameric concatenated IP3Rs with increasing numbers of binding-deficient subunits, we addressed the obligate ligand stoichiometry. The concatenated IP3Rs with four ligand-binding sites exhibited Ca(2+)release and electrophysiological properties of native IP3Rs. However, IP3failed to activate IP3Rs assembled from concatenated dimers consisting of one binding-competent and one binding-deficient mutant subunit. Similarly, IP3Rs containing two monomers of IP3R2short, an IP3binding-deficient splice variant, were nonfunctional. Concatenated tetramers containing only three binding-competent ligand-binding sites were nonfunctional under a wide range of activating conditions. These data provide definitive evidence that IP3-induced Ca(2+)release only occurs when each IP3R monomer within the tetramer is occupied by IP3, thereby ensuring fidelity of Ca(2+)release.

Comparative Anterior Pituitary miRNA and mRNA Expression Profiles of Bama Minipigs and Landrace Pigs Reveal Potential Molecular Network Involved in Animal Postnatal Growth.

The anterior pituitary is the most important endocrine organ modulating animal postnatal growth, mainly by controlling growth hormone (GH) gene transcription, synthesis, and secretion. As an ideal model for animal postnatal growth studies, the Bama minipig is characterized as having a lower growth performance and fewer individual differences compared with larger pig breeds. In this study, anterior pituitaries from Bama minipig and Landrace pig were used for miRNA and mRNA expression profile analysis using miRNA microarrays and mRNA-seq. Consequently, a total of 222 miRNAs and 12,909 transcripts were detected, and both miRNAs and mRNAs in the two breeds showed high correlation (r > 0.97). Additionally, 41 differentially expressed miRNAs and 2,254 transcripts were identified. Pathways analysis indicated that 32 pathways significantly differed in the two breeds. Importantly, two GH-regulation-signalling pathways, cAMP and inositol 1, 4, 5-triphosphate (IP3), and multiple GH-secretion-related transcripts were significantly down-regulated in Bama minipigs. Moreover, TargetScan and RNAHybrid algorithms were used for predicting differentially expressed miRNAs (DE miRNAs) and differentially expressed mRNAs (DE mRNAs) interaction. By examining their fold-changes, interestingly, most DE miRNA-DE mRNA target pairs (63.68-71.33%) presented negatively correlated expression pattern. A possible network among miRNAs, mRNAs, and GH-regulation pathways was also proposed. Among them, two miRNA-mRNA interactions (Y-47 targets FSHB; ssc-miR-133a-3p targets GNAI3) were validated by dual-luciferase assay. These data will be helpful in understanding the possible molecular mechanisms involved in animal postnatal growth.

Differential Regulation of Multiple Steps in Inositol 1,4,5-Trisphosphate Signaling by Protein Kinase C Shapes Hormone-stimulated Ca2+ Oscillations.

How Ca(2+) oscillations are generated and fine-tuned to yield versatile downstream responses remains to be elucidated. In hepatocytes, G protein-coupled receptor-linked Ca(2+) oscillations report signal strength via frequency, whereas Ca(2+) spike amplitude and wave velocity remain constant. IP3 uncaging also triggers oscillatory Ca(2+) release, but, in contrast to hormones, Ca(2+) spike amplitude, width, and wave velocity were dependent on [IP3] and were not perturbed by phospholipase C (PLC) inhibition. These data indicate that oscillations elicited by IP3 uncaging are driven by the biphasic regulation of the IP3 receptor by Ca(2+), and, unlike hormone-dependent responses, do not require PLC. Removal of extracellular Ca(2+) did not perturb Ca(2+) oscillations elicited by IP3 uncaging, indicating that reloading of endoplasmic reticulum stores via plasma membrane Ca(2+) influx does not entrain the signal. Activation and inhibition of PKC attenuated hormone-induced Ca(2+) oscillations but had no effect on Ca(2+) increases induced by uncaging IP3. Importantly, PKC activation and inhibition differentially affected Ca(2+) spike frequencies and kinetics. PKC activation amplifies negative feedback loops at the level of G protein-coupled receptor PLC activity and/or IP3 metabolism to attenuate IP3 levels and suppress the generation of Ca(2+) oscillations. Inhibition of PKC relieves negative feedback regulation of IP3 accumulation and, thereby, shifts Ca(2+) oscillations toward sustained responses or dramatically prolonged spikes. PKC down-regulation attenuates phenylephrine-induced Ca(2+) wave velocity, whereas responses to IP3 uncaging are enhanced. The ability to assess Ca(2+) responses in the absence of PLC activity indicates that IP3 receptor modulation by PKC regulates Ca(2+) release and wave velocity.

Using concatenated subunits to investigate the functional consequences of heterotetrameric inositol 1,4,5-trisphosphate receptors.

Inositol 1,4,5-trisphosphate receptors (IP3Rs) are a family of ubiquitous, ER localized, tetrameric Ca2+ release channels. There are three subtypes of the IP3Rs (R1, R2, R3), encoded by three distinct genes, that share ∼60-70% sequence identity. The diversity of Ca2+ signals generated by IP3Rs is thought to be largely the result of differential tissue expression, intracellular localization and subtype-specific regulation of the three subtypes by various cellular factors, most significantly InsP3, Ca2+ and ATP. However, largely unexplored is the notion of additional signal diversity arising from the assembly of both homo and heterotetrameric InsP3Rs. In the present article, we review the biochemical and functional evidence supporting the existence of homo and heterotetrameric populations of InsP3Rs. In addition, we consider a strategy that utilizes genetically concatenated InsP3Rs to study the functional characteristics of heterotetramers with unequivocally defined composition. This approach reveals that the overall properties of IP3R are not necessarily simply a blend of the constituent monomers but that specific subtypes appear to dominate the overall characteristics of the tetramer. It is envisioned that the ability to generate tetramers with defined wild type and mutant subunits will be useful in probing fundamental questions relating to IP3R structure and function.

Intracellular Ca(2+) release from endoplasmic reticulum regulates slow wave currents and pacemaker activity of interstitial cells of Cajal.

Interstitial cells of Cajal (ICC) provide pacemaker activity in gastrointestinal muscles that underlies segmental and peristaltic contractions. ICC generate electrical slow waves that are due to large-amplitude inward currents resulting from anoctamin 1 (ANO1) channels, which are Ca(2+)-activated Cl(-) channels. We investigated the hypothesis that the Ca(2+) responsible for the stochastic activation of ANO1 channels during spontaneous transient inward currents (STICs) and synchronized activation of ANO1 channels during slow wave currents comes from intracellular Ca(2+) stores. ICC, obtained from the small intestine of Kit(+/copGFP) mice, were studied under voltage and current clamp to determine the effects of blocking Ca(2+) uptake into stores and release of Ca(2+) via inositol 1,4,5-trisphosphate (IP3)-dependent and ryanodine-sensitive channels. Cyclocpiazonic acid, thapsigargin, 2-APB, and xestospongin C inhibited STICs and slow wave currents. Ryanodine and tetracaine also inhibited STICs and slow wave currents. Store-active compounds had no direct effects on ANO1 channels expressed in human embryonic kidney-293 cells. Under current clamp, store-active drugs caused significant depolarization of ICC and reduced spontaneous transient depolarizations (STDs). After block of ryanodine receptors with ryanodine and tetracaine, repolarization did not restore STDs. ANO1 expressed in ICC has limited access to cytoplasmic Ca(2+) concentration, suggesting that pacemaker activity depends on Ca(2+) dynamics in restricted microdomains. Our data from studies of isolated ICC differ somewhat from studies on intact muscles and suggest that release of Ca(2+) from both IP3 and ryanodine receptors is important in generating pacemaker activity in ICC.

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

The two major calcium (Ca²âº) 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 Ca²âº 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.