IP3 Binding Research Articles

Agonist-Induced Ca2+ Signaling in HEK-293-Derived Cells Expressing a Single IP3 Receptor Isoform.

In mammals, three genes encode IP3 receptors (IP3Rs), which are involved in agonist-induced Ca2+ signaling in cells of apparently all types. Using the CRISPR/Cas9 approach for disruption of two out of three IP3R genes in HEK-293 cells, we generated three monoclonal cell lines, IP3R1-HEK, IP3R2-HEK, and IP3R3-HEK, with the single functional isoform, IP3R1, IP3R2, and IP3R3, respectively. All engineered cells responded to ACh with Ca2+ transients in an "all-or-nothing" manner, suggesting that each IP3R isotype was capable of mediating CICR. The sensitivity of cells to ACh strongly correlated with the affinity of IP3 binding to an IP3R isoform they expressed. Based on a mathematical model of intracellular Ca2+ signals induced by thapsigargin, a SERCA inhibitor, we developed an approach for estimating relative Ca2+ permeability of Ca2+ store and showed that all three IP3R isoforms contributed to Ca2+ leakage from ER. The relative Ca2+ permeabilities of Ca2+ stores in IP3R1-HEK, IP3R2-HEK, and IP3R3-HEK cells were evaluated as 1:1.75:0.45. Using the genetically encoded sensor R-CEPIA1er for monitoring Ca2+ signals in ER, engineered cells were ranged by resting levels of stored Ca2+ as IP3R3-HEK ≥ IP3R1-HEK > IP3R2-HEK. The developed cell lines could be helpful for further assaying activity, regulation, and pharmacology of individual IP3R isoforms.

Targeting calcium signaling by inositol trisphosphate receptors: A novel mechanism for the anti-asthmatic effects of Houttuynia cordata

Asthma is a chronic inflammatory disease characterized by airway hypersensitivity and remodeling. The current treatments provide only short-term benefits and may have undesirable side effects; thus, alternative or supplementary therapy is needed. Because intracellular calcium (Ca2+) signaling plays an essential role in regulating the contractility and remodeling of airway smooth muscle cells, the targeting of Ca2+ signaling is a potential therapeutic strategy for asthma. Houttuynia cordata is a traditional Chinese herb that is used to treat asthma due to its anti-allergic and anti-inflammatory properties. We hypothesized that H. cordata might modulate intracellular Ca2+ signaling and could help relieve asthmatic airway remodeling. We found that the mRNA and protein levels of inositol trisphosphate receptors (IP3Rs) were elevated in interleukin-stimulated primary human bronchial smooth muscle cells and a house dust mite–sensitized model of asthma. The upregulation of IP3R expression enhanced intracellular Ca2+ release upon stimulation and contributed to airway remodeling in asthma. Intriguingly, pretreatment with H. cordata essential oil rectified the disruption of Ca2+ signaling, mitigated asthma development, and prevented airway narrowing. Furthermore, our analysis suggested that houttuynin/2-undecanone could be the bioactive component in H. cordata essential oil because we found similar IP3R suppression in response to the commercially available derivative sodium houttuyfonate. An in silico analysis showed that houttuynin, which downregulates IP3R expression, binds to the IP3 binding domain of IP3R and may mediate a direct inhibitory effect. In summary, our findings suggest that H. cordata is a potential alternative treatment choice that may reduce asthma severity by targeting the dysregulation of Ca2+ signaling.

Conformational motions and ligand-binding underlying gating and regulation in IP3R channel

Inositol-1,4,5-trisphosphate receptors (IP3Rs) are activated by IP3 and Ca2+ and their gating is regulated by various intracellular messengers that finely tune the channel activity. Here, using single particle cryo-EM analysis we determined 3D structures of the nanodisc-reconstituted IP3R1 channel in two ligand-bound states. These structures provide unprecedented details governing binding of IP3, Ca2+ and ATP, revealing conformational changes that couple ligand-binding to channel opening. Using a deep-learning approach and 3D variability analysis we extracted molecular motions of the key protein domains from cryo-EM density data. We find that IP3 binding relies upon intrinsic flexibility of the ARM2 domain in the tetrameric channel. Our results highlight a key role of dynamic side chains in regulating gating behavior of IP3R channels. This work represents a stepping-stone to developing mechanistic understanding of conformational pathways underlying ligand-binding, activation and regulation of the channel.

Functional determination of calcium-binding sites required for the activation of inositol 1,4,5-trisphosphate receptors

Inositol 1,4,5-trisphosphate receptors (IP3Rs) initiate a diverse array of physiological responses by carefully orchestrating intracellular calcium (Ca2+) signals in response to various external cues. Notably, IP3R channel activity is determined by several obligatory factors, including IP3, Ca2+, and ATP. The critical basic amino acid residues in the N-terminal IP3-binding core (IBC) region that facilitate IP3 binding are well characterized. In contrast, the residues conferring regulation by Ca2+ have yet to be ascertained. Using comparative structural analysis of Ca2+-binding sites identified in two main families of intracellular Ca2+-release channels, ryanodine receptors (RyRs) and IP3Rs, we identified putative acidic residues coordinating Ca2+ in the cytosolic calcium sensor region in IP3Rs. We determined the consequences of substituting putative Ca2+ binding, acidic residues in IP3R family members. We show that the agonist-induced Ca2+ release, single-channel open probability (P0), and Ca2+ sensitivities are markedly altered when the negative charge on the conserved acidic side chain residues is neutralized. Remarkably, neutralizing the negatively charged side chain on two of the residues individually in the putative Ca2+-binding pocket shifted the Ca2+ required to activate IP3R to higher concentrations, indicating that these residues likely are a component of the Ca2+ activation site in IP3R. Taken together, our findings indicate that Ca2+ binding to a well-conserved activation site is a common underlying mechanism resulting in increased channel activity shared by IP3Rs and RyRs.

Quantal Ca2+ release mediated by very few IP3 receptors that rapidly inactivate allows graded responses to IP3.

SummaryInositol 1,4,5-trisphosphate receptors (IP3Rs) are intracellular Ca2+ channels that link extracellular stimuli to Ca2+ signals. Ca2+ release from intracellular stores is “quantal”: low IP3 concentrations rapidly release a fraction of the stores. Ca2+ release then slows or terminates without compromising responses to further IP3 additions. The mechanisms are unresolved. Here, we synthesize a high-affinity partial agonist of IP3Rs and use it to demonstrate that quantal responses do not require heterogenous Ca2+ stores. IP3Rs respond incrementally to IP3 and close after the initial response to low IP3 concentrations. Comparing functional responses with IP3 binding shows that only a tiny fraction of a cell’s IP3Rs mediate incremental Ca2+ release; inactivation does not therefore affect most IP3Rs. We conclude, and test by simulations, that Ca2+ signals evoked by IP3 pulses arise from rapid activation and then inactivation of very few IP3Rs. This allows IP3Rs to behave as increment detectors mediating graded Ca2+ release.

Regulation of Store-Operated Ca <sup>2+</sup> Entry by IP <sub>3</sub> Receptors Independent of Their Ability to Release Ca <sup>2+</sup>

Loss of ER Ca2+ activates store-operated Ca2+ entry (SOCE) by causing STIM1 to unfurl domains that activate Orai1 channels in the plasma membrane at membrane contact sites (MCS). In human neurons, SOCE evoked by thapsigargin to deplete ER Ca2+ is attenuated by loss of IP3Rs, and restored by expression of IP3Rs even when they cannot releaseCa2+, but only if they can bind IP3. In cells expressing pore-dead IP3Rs, IP3 enhances SOCE evoked by partial store depletion without enhancing Ca2+ release. Proximity ligation assays establish that IP3Rs enhance association of STIM1 with Orai1 in cells with empty stores; this requires an IP3-binding site, but not a pore. Over-expressing STIM1, or extended synaptotagmin-1 to exaggerate MCS, circumvents the need for IP3Rs. Thus, IP3 binding to IP3Rs stimulates SOCE by both mediating ER Ca2+ release and promoting STIM1-Orai1 interactions. Convergent regulation by IP3Rs may tune SOCE to respond selectively to IP3.

Bisphenol A stabilizes Nrf2 via Ca2+ influx by direct activation of the IP3 receptor.

Bisphenol A (BPA) is an endocrine-disrupting chemical used in polycarbonate and epoxy resins. Previously, we found that BPA stabilized the protein levels of nuclear factor erythroid 2-related factor 2 (Nrf2) by inducing Ca2+ efflux into the cytosol, followed by nitric oxide synthase activation, resulting in the enhanced nitrosylation of Keap1, which is a negative regulator of Nrf2. However, the mechanisms behind the stimulation of Ca2+ efflux by BPA remain unknown. In the present study, we found that BPA stimulated Ca2+ efflux into the cytosol from the ER, but not from outside of cells through the plasma membrane in Hep3B cells. Ca2+ efflux and Nrf2 stabilization by BPA were inhibited by an inhibitor of the inositol 1,4,5-trisphosphate (IP3) receptor, 2-aminoethoxydiphenylborane, in the endoplasmic reticulum. IP3 is produced by activation of phospholipase C (PLC) from a membrane lipid, phosphatidylinositol 4,5-bisphosphate (PIP2). The induction of Nrf2 by BPA was not inhibited by a PLC inhibitor, U-73122, suggesting that BPA does not induce the production of IP3 via PLC activation. We found that BPA bound directly to the IP3 binding core domain of the IP3 receptor, and BPA competed with IP3 on this site. In addition, overexpression of this domain of the IP3 receptor in Hep3B cells inhibited the stabilization of Nrf2 by BPA. These results clarified that the IP3 receptor is a new target of BPA, and that BPA induces Ca2+ efflux from the endoplasmic reticulum via activation of the IP3 receptor.

Biological Regulatory Network (BRN) Analysis and Molecular Docking Simulations to Probe the Modulation of IP3R Mediated Ca2+ Signaling in Cancer.

Inositol trisphosphate receptor (IP3R) mediated Ca+2 signaling is essential in determining the cell fate by regulating numerous cellular processes, including cell division and cell death. Despite extensive studies about the characterization of IP3R in cancer, the underlying molecular mechanism initiating the cell proliferation and apoptosis remained enigmatic. Moreover, in cancer, the modulation of IP3R in downstream signaling pathways, which control oncogenesis and cancer progression, is not well characterized. Here, we constructed a biological regulatory network (BRN), and describe the remodeling of IP3R mediated Ca2+ signaling as a central key that controls the cellular processes in cancer. Moreover, we summarize how the inhibition of IP3R affects the deregulated cell proliferation and cell death in cancer cells and results in the initiation of pro-survival responses in resistance of cell death in normal cells. Further, we also investigated the role of stereo-specificity of IP3 molecule and its analogs in binding with the IP3 receptor. Molecular docking simulations showed that the hydroxyl group at R6 position along with the phosphate group at R5 position in ‘R’ conformation is more favorable for IP3 interactions. Additionally, Arg-266 and Arg-510 showed π–π and hydrogen bond interactions and Ser-278 forms hydrogen bond interactions with the IP3 binding site. Thus, they are identified as crucial for the binding of antagonists.

Effects of Linker Flexibility and Conformational Changes of IP3 Receptor on Split Luciferase Complementation Assay.

Background: IP3-induced Ca2+ release, mediated by IP3R, is one of the most momentous cellular signaling mechanisms that regulate in a wide variety of essential cellular functions. Involvement of disrupted IP3 signaling pathways in numerous pathophysiology conditions is implicated to find the best methods for its measurement. Hence, several different biosensors have developed to monitor temporal changes of IP3 by using the IP3-binding domain of IP3 receptors.Objectives: Based on a previous study, we developed and characterized a series of bioluminescent biosensors using the human type-II IP3 receptor ligand binding domain (residues 1–604), named LAIRE (luminescent analyzer for IP3 receptor element) to study the effect of flexible and rigid linkers on the luminescence intensity of split luciferase. The effect of a mutation in IP3 binding residues and suppressor domain in the IP3 binding domain on luciferase complementary assay is also investigated.Materials and Methods: In the present study, first IP3-binding domain (residues 1-604) of IP3-receptor type 2 (LAIRE) was fused between complementary non-functional fragments of firefly luciferase and then the rigid linker sequence (LLRAIEAQQHLL), selected by ProDA database, introduced between Nluc and ligand binding domain and compared with that of the flexible linker ((GGGGS)2) in LAIRE chimera. The IP3-insensitive mutant of the biosensor was constructed using the Stratagene QuikChange® procedure. In order to the analysis of the dynamical movements of selected structures in the large-scale, coarse-graining method of molecular dynamics simulation (1µs) was applied.Results: As expected, the flexible linker brings two inactive fragments of luciferase together relative to the rigid linker and leads to complementation of luciferase activity, which is detected using luciferin. However, this conformational flexibility in linker increases background to noise ratio and attenuates fold induction.Conclusions: It seems that the ligand binding properties of IP3 binding core make it more suitable for the design of biosensor than the ligand binding domain.

Cryo-EM structure of human type-3 inositol triphosphate receptor reveals the presence of a self-binding peptide that acts as an antagonist

Calcium-mediated signaling through inositol 1,4,5-triphosphate receptors (IP3Rs) is essential for the regulation of numerous physiological processes, including fertilization, muscle contraction, apoptosis, secretion, and synaptic plasticity. Deregulation of IP3Rs leads to pathological calcium signaling and is implicated in many common diseases, including cancer and neurodegenerative, autoimmune, and metabolic diseases. Revealing the mechanism of activation and inhibition of this ion channel will be critical to an improved understanding of the biological processes that are controlled by IP3Rs. Here, we report structural findings of the human type-3 IP3R (IP3R-3) obtained by cryo-EM (at an overall resolution of 3.8 Å), revealing an unanticipated regulatory mechanism where a loop distantly located in the primary sequence occupies the IP3-binding site and competitively inhibits IP3 binding. We propose that this inhibitory mechanism must differ qualitatively among IP3R subtypes because of their diverse loop sequences, potentially serving as a key molecular determinant of subtype-specific calcium signaling in IP3Rs. In summary, our structural characterization of human IP3R-3 provides critical insights into the mechanistic function of IP3Rs and into subtype-specific regulation of these important calcium-regulatory channels.

ORP4L couples IP3 to ITPR1 in control of endoplasmic reticulum calcium release

Oxysterol-binding protein-related protein (ORP) 4L acts as a scaffold protein assembling CD3-ε, G-αq/11, and PLC-β3 into a complex at the plasma membrane that mediates inositol (1,4,5)-trisphosphate (IP3)-induced endoplasmic reticulum (ER) Ca2+ release and oxidative phosphorylation in T-cell acute lymphoblastic leukemia cells. Here, we offer new evidence that ORP4L interacts with the carboxyl terminus of the IP3 receptor type 1 (ITPR1) in Jurkat T cells. ORP4L enables IP3 binding to ITPR1; a truncated construct that lacks the ITPR1-binding region retains the ability to increase IP3 production but fails to mediate IP3 and ITPR1 binding. In association with this ability of ORP4L, it enhances Ca2+ release from the ER and subsequent cytosolic and mitochondrial parallel Ca2+ spike oscillations that stimulate mitochondrial energetics and thus maintains cell survival. These data support a novel model in which ORP4L is a cofactor of ITPR1, which increases ITPR1 sensitivity to IP3 and enables ER Ca2+ release.-Cao, X., Chen, J., Li, D., Xie, P., Xu, M., Lin, W., Li, S., Pan, G., Tang, Y., Xu, J., Olkkonen, V. M., Yan, D., Zhong, W. ORP4L couples IP3 to ITPR1 in control of endoplasmic reticulum calcium release.

Exploration of inositol 1,4,5-trisphosphate (IP3) regulated dynamics of N-terminal domain of IP3 receptor reveals early phase molecular events during receptor activation

Inositol 1, 4, 5-trisphosphate (IP3) binding at the N-terminus (NT) of IP3 receptor (IP3R) allosterically triggers the opening of a Ca2+-conducting pore located ~100 Å away from the IP3-binding core (IBC). However, the precise mechanism of IP3 binding and correlated domain dynamics in the NT that are central to the IP3R activation, remains unknown. Our all-atom molecular dynamics (MD) simulations recapitulate the characteristic twist motion of the suppressor domain (SD) and reveal correlated ‘clam closure’ dynamics of IBC with IP3-binding, complementing existing suggestions on IP3R activation mechanism. Our study further reveals the existence of inter-domain dynamic correlation in the NT and establishes the SD to be critical for the conformational dynamics of IBC. Also, a tripartite interaction involving Glu283-Arg54-Asp444 at the SD – IBC interface seemed critical for IP3R activation. Intriguingly, during the sub-microsecond long simulation, we observed Arg269 undergoing an SD-dependent flipping of hydrogen bonding between the first and fifth phosphate groups of IP3. This seems to play a major role in determining the IP3 binding affinity of IBC in the presence/absence of the SD. Our study thus provides atomistic details of early molecular events occurring within the NT during and following IP3 binding that lead to channel gating.

Allosteric response to ligand binding: Molecular dynamics study of the N-terminal domains in IP 3 receptor.

Inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) is a huge tetrameric intracellular Ca2+ channel that mediates cytoplasmic Ca2+ signaling. The structural basis of the gating in IP3R has been studied by X-ray crystallography and cryo-electron microscopy, focusing on the domain rearrangements triggered by IP3 binding. Here, we conducted molecular dynamics (MD) simulations of the three N-terminal domains of IP3R responsible for IP3 binding (IBC/SD; two domains of the IP3 binding core, IBCβ and IBCα, and suppressor domain, SD) as a model system to study the initial gating stage. The response upon removal of IP3 from the IP3-bound form of IBC/SD was traced in MD trajectories. The two IBC domains showed an immediate response of opening after removal of IP3, and SD showed a simultaneous opening motion indicating a tight dynamic coupling with IBC. However, when IBC remained in a more closed form, the dynamic coupling broke and SD exhibited a more amplified closing motion independently of IBC. This amplified SD motion was caused by the break of connection between SD and IBCβ at the hinge region, but was suppressed in the native tetrameric state. The analyses using Motion Tree and the linear response theory clarified that in the open form, SD and IBCα moved collectively relative to IBCβ with a response upon IP3 binding within the linear regime, whereas in the closed form, such collectiveness disappeared. These results suggest that the regulation of dynamics via the domain arrangement and multimerization is requisite for large-scale allosteric communication in IP3R gating machinery.

IP3 receptors - lessons from analyses ex cellula.

Inositol 1,4,5-trisphosphate receptors (IP3Rs) are widely expressed intracellular channels that release Ca2+ from the endoplasmic reticulum (ER). We review how studies of IP3Rs removed from their intracellular environment ('ex cellula'), alongside similar analyses of ryanodine receptors, have contributed to understanding IP3R behaviour. Analyses of permeabilized cells have demonstrated that the ER is the major intracellular Ca2+ store, and that IP3 stimulates Ca2+ release from this store. Radioligand binding confirmed that the 4,5-phosphates of IP3 are essential for activating IP3Rs, and facilitated IP3R purification and cloning, which paved the way for structural analyses. Reconstitution of IP3Rs into lipid bilayers and patch-clamp recording from the nuclear envelope have established that IP3Rs have a large conductance and select weakly between Ca2+ and other cations. Structural analyses are now revealing how IP3 binding to the N-terminus of the tetrameric IP3R opens the pore ∼7 nm away from the IP3-binding core (IBC). Communication between the IBC and pore passes through a nexus of interleaved domains contributed by structures associated with the pore and cytosolic domains, which together contribute to a Ca2+-binding site. These structural analyses provide evidence to support the suggestion that IP3 gates IP3Rs by first stimulating Ca2+ binding, which leads to pore opening and Ca2+ release.

Aberrant IP3 receptor activities revealed by comprehensive analysis of pathological mutations causing spinocerebellar ataxia 29

Spinocerebellar ataxia type 29 (SCA29) is autosomal dominant congenital ataxia characterized by early-onset motor delay, hypotonia, and gait ataxia. Recently, heterozygous missense mutations in an intracellular Ca2+ channel, inositol 1,4,5-trisphosphate (IP3) receptor type 1 (IP3R1), were identified as a cause of SCA29. However, the functional impacts of these mutations remain largely unknown. Here, we determined the molecular mechanisms by which pathological mutations affect IP3R1 activity and Ca2+ dynamics. Ca2+ imaging using IP3R-null HeLa cells generated by genome editing revealed that all SCA29 mutations identified within or near the IP3-binding domain of IP3R1 completely abolished channel activity. Among these mutations, R241K, T267M, T267R, R269G, R269W, S277I, K279E, A280D, and E497K impaired IP3 binding to IP3R1, whereas the T579I and N587D mutations disrupted channel activity without affecting IP3 binding, suggesting that T579I and N587D compromise channel gating mechanisms. Carbonic anhydrase-related protein VIII (CA8) is an IP3R1-regulating protein abundantly expressed in cerebellar Purkinje cells and is a causative gene of congenital ataxia. The SCA29 mutation V1538M within the CA8-binding site of IP3R1 completely eliminated its interaction with CA8 and CA8-mediated IP3R1 inhibition. Furthermore, pathological mutations in CA8 decreased CA8-mediated suppression of IP3R1 by reducing protein stability and the interaction with IP3R1. These results demonstrated the mechanisms by which pathological mutations cause IP3R1 dysfunction, i.e., the disruption of IP3 binding, IP3-mediated gating, and regulation via the IP3R-modulatory protein. The resulting aberrant Ca2+ homeostasis may contribute to the pathogenesis of cerebellar ataxia.

All three IP3 receptor subtypes generate Ca2+ puffs, the universal building blocks of IP3-evoked Ca2+ signals.

ABSTRACTAll three subtypes of inositol 1,4,5-trisphosphate receptor (IP3R) are intracellular Ca2+ channels that are co-regulated by IP3 and Ca2+. This allows IP3Rs to evoke regenerative Ca2+ signals, the smallest of which are Ca2+ puffs that reflect the coordinated opening of a few clustered IP3Rs. We use total internal reflection microscopy (TIRF) microscopy to record Ca2+ signals in HEK cells expressing all three IP3R subtypes or a single native subtype. Ca2+ puffs are less frequent in cells expressing one IP3R subtype, commensurate with them expressing fewer IP3Rs than wild-type cells. However, all three IP3R subtypes generate broadly similar Ca2+ puffs with similar numbers of IP3Rs contributing to each. This suggests that IP3R clusters may be assembled by conserved mechanisms that generate similarly sized clusters across different IP3R expression levels. The Ca2+ puffs evoked by IP3R2 had slower kinetics and more prolonged durations, which may be due to IP3 binding with greater affinity to IP3R2. We conclude that Ca2+ puffs are the building blocks for the Ca2+ signals evoked by all IP3Rs.

Characterization of a phospholipase C‐like protein (TbPI‐PLC2) from Trypanosoma brucei

Trypanosoma brucei is the causative agent of African Trypanosomiasis, a deadly disease affecting humans and cattle. There are very few drugs to treat the disease and evidence of mounting resistance raises the need for new drug development. The inositol 1,4,5 triphosphate/diacylglycerol (IP3/DAG) pathway regulates important processes in many organisms. T. brucei has an active IP3 receptor, localized to the acidocalcisome, that is essential for infection in mice. In previous work (King‐Keller et al., Eukaryot. Cell 14:486–494, 2015) we characterized a phosphoinositide phospholipase C (TbPI‐PLC1, Tb927.11.5970) from T. brucei that contains a domain organization characteristic of PI‐PLCs, such as X and Y catalytic domains, an EF‐hand calcium‐binding motif, and a C2 domain, but lacks a pleckstrin homology (PH) domain. In addition, TbPI‐PLC1 contains an N‐terminal myristoylation consensus sequence only found in trypanosomatid PI‐PLCs. Here we report the presence of a second PI‐PLC‐like protein (TbPI‐PLC2, Tb927.6.2090) that is very similar to TbPI‐PLC1 but lacks the Y catalytic domain and the C2 domain and possesses instead a PDZ domain. Recombinant TbPI‐PLC2 hydrolyzes neither phosphatidylinositol (PI) nor phosphatidylinositol 4,5‐bisphosphate (PIP2), and does not modulate TbPI‐PLC1 activity. However, knockdown of TbPI‐PLC2 expression alone or together with downregulation of TbPI‐PLC1 expression by RNAi resulted in growth inhibition. This is in contrast with the lack of effect of downregulation of expression of TbPI‐PLC1 alone. TbPI‐PLC2 has a plasma membrane and intracellular localization and it might be involved in IP3 binding as has been reported for the phospholipase C‐related catalytically inactive protein 1 (PRIP‐1) of mammalian cells (Uji et al., Life Sci 72:443–453, 2002). The PDZ domain could be involved in this binding and this is being investigated.Support or Funding InformationNIH grant # AI077538This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Region-specific proteolysis differentially regulates type 1 inositol 1,4,5-trisphosphate receptor activity

The inositol 1,4,5 trisphosphate receptor (IP3R) is an intracellular Ca2+ release channel expressed predominately on the membranes of the endoplasmic reticulum. IP3R1 can be cleaved by caspase or calpain into at least two receptor fragments. However, the functional consequences of receptor fragmentation are poorly understood. Our previous work has demonstrated that IP3R1 channels, formed following either enzymatic fragmentation or expression of the corresponding complementary polypeptide chains, retain tetrameric architecture and are still activated by IP3 binding despite the loss of peptide continuity. In this study, we demonstrate that region-specific receptor fragmentation modifies channel regulation. Specifically, the agonist-evoked temporal Ca2+ release profile and protein kinase A modulation of Ca2+ release are markedly altered. Moreover, we also demonstrate that activation of fragmented IP3R1 can result in a distinct functional outcome. Our work suggests that proteolysis of IP3R1 may represent a novel form of modulation of IP3R1 channel function and increases the repertoire of Ca2+ signals achievable through this channel.

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.

Calcium waves in a grid of clustered channels with synchronous IP3 binding and unbinding.

Calcium signals in cells occur at multiple spatial scales and variable temporal duration. However, a physical explanation for transitions between long-lasting global oscillations and localized short-term elevations (puffs) of cytoplasmic Ca2+ is still lacking. Here we introduce a phenomenological, coarse-grained model for the calcium variable, which is represented by ordinary differential equations. Due to its small number of parameters, and its simplicity, this model allows us to numerically study the interplay of multi-scale calcium concentrations with stochastic ion channel gating dynamics even in larger systems. We apply this model to a single cluster of inositol trisphosphate (IP 3) receptor channels and find further evidence for the results presented in earlier work: a single cluster may be capable of producing different calcium release types, where long-lasting events are accompanied by unbinding of IP 3 from the receptor (Rückl et al., PLoS Comput. Biol. 11, e1003965 (2015)). Finally, we show the practicability of the model in a grid of 64 clusters which is computationally intractable with previous high-resolution models. Here long-lasting events can lead to synchronized oscillations and waves, while short events stay localized. The frequency of calcium releases as well as their coherence can thereby be regulated by the amplitude of IP 3 stimulation. Finally the model allows for a new explanation of oscillating [IP 3], which is not based on metabolic production and degradation of IP 3.