IP3R Channels Research Articles

Dominant negative variants in ITPR3 impair T cell Ca2+ dynamics causing combined immunodeficiency.

The importance of calcium (Ca2+) as a second messenger in T cell signaling is exemplified by genetic deficiencies of STIM1 and ORAI1, which abolish store-operated Ca2+ entry (SOCE) resulting in combined immunodeficiency (CID). We report five unrelated patients with de novo missense variants in ITPR3, encoding a subunit of the inositol 1,4,5-trisphosphate receptor (IP3R), which forms a Ca2+ channel in the endoplasmic reticulum (ER) membrane responsible for the release of ER Ca2+ required to trigger SOCE, and for Ca2+ transfer to other organelles. The patients presented with CID, abnormal T cell Ca2+ homeostasis, incompletely penetrant ectodermal dysplasia, and multisystem disease. Their predominant T cell immunodeficiency is characterized by significant T cell lymphopenia, defects in late stages of thymic T cell development, and impaired function of peripheral T cells, including inadequate NF-κB- and NFAT-mediated, proliferative, and metabolic responses to activation. Pathogenicity is not due to haploinsufficiency, rather ITPR3 protein variants interfere with IP3R channel function leading to depletion of ER Ca2+ stores and blunted SOCE in T cells.

NF2 regulates IP3R-mediated Ca2+ signal and apoptosis in meningiomas.

Meningiomas are the most common primary intracranial tumors and account for nearly 30% of all nervous system tumors. Approximately half of meningioma patients exhibit neurofibromin 2 (NF2) gene inactivation. Here, NF2 was shown to interact with the endoplasmic reticulum (ER) calcium (Ca2+) channel inositol 1,4,5-trisphosphate receptor 1 (IP3R1) in IOMM-Lee, a high-grade malignant meningioma cell line, and the F1 subdomain of NF2 plays a critical role in this interaction. Functional assays indicated that NF2 promotes the phosphorylation of IP3R (Ser 1756) and IP3R-mediated endoplasmic reticulum (ER) Ca2+ release by binding to IP3R1, which results in Ca2+-dependent apoptosis. Knockout of NF2 decreased Ca2+ release and promoted resistance to apoptosis, which was rescued by wild-type NF2 overexpression but not by F1 subdomain deletion truncation overexpression. The effects of NF2 defects on the development of tumors were further studied in mouse models. The decreased expression level of NF2 caused by NF2 gene knockout or mutation affects the activity of the IP3R channel, which reduces Ca2+-dependent apoptosis, thereby promoting the development of tumors. We elucidated the interaction patterns of NF2 and IP3R1, revealed the molecular mechanism through which NF2 regulates IP3R1-mediated Ca2+ release, and elucidated the new pathogenic mechanism of meningioma-related NF2 variants. Our study broadens the current understanding of the biological function of NF2 and provides ideas for drug screening of NF2-associated meningioma.

KRAP regulates mitochondrial Ca2+ uptake by licensing IP3 receptor activity and stabilizing ER-mitochondrial junctions.

Inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) are high-conductance channels that allow the regulated redistribution of Ca2+ from the endoplasmic reticulum (ER) to the cytosol and, at specialized membrane contact sites (MCSs), to other organelles. Only a subset of IP3Rs release Ca2+ to the cytosol in response to IP3. These 'licensed' IP3Rs are associated with Kras-induced actin-interacting protein (KRAP, also known as ITPRID2) beneath the plasma membrane. It is unclear whether KRAP regulates IP3Rs at MCSs. We show, using simultaneous measurements of Ca2+ concentration in the cytosol and mitochondrial matrix, that KRAP also licenses IP3Rs to release Ca2+ to mitochondria. Loss of KRAP abolishes cytosolic and mitochondrial Ca2+ signals evoked by stimulation of IP3Rs via endogenous receptors. KRAP is located at ER-mitochondrial membrane contact sites (ERMCSs) populated by IP3R clusters. Using a proximity ligation assay between IP3R and voltage-dependent anion channel 1 (VDAC1), we show that loss of KRAP reduces the number of ERMCSs. We conclude that KRAP regulates Ca2+ transfer from IP3Rs to mitochondria by both licensing IP3R activity and stabilizing ERMCSs.

Characterization of PDGF-Induced Subcellular Calcium Regulation through Calcium Channels in Airway Smooth Muscle Cells by FRET Biosensors

The homeostasis of cellular calcium is fundamental for many physiological processes, while the calcium levels remain inhomogeneous within cells. During the onset of asthma, epithelial and inflammatory cells secrete platelet-derived growth factor (PDGF), inducing the proliferation and migration of airway smooth muscle (ASM) to the epidermal layer, narrowing the airway. The regulation of ASM cells by PDGF is closely related to the conduction of calcium signals. In this work, we generated subcellular-targeted FRET biosensors to investigate calcium regulation in the different compartments of ASM cells. A PDGF-induced cytoplasmic calcium [Ca2+]C increase was attributed to both extracellular calcium influx and endoplasmic reticulum (ER) calcium [Ca2+]ER release, which was partially regulated by the PLC-IP3R pathway. Interestingly, the removal of the extracellular calcium influx led to inhibited ER calcium release, likely through inhibitory effects on the calcium-dependent activation of the ER ryanodine receptor. The inhibition of the L-type calcium channel on the plasma membrane or the SERCA pump on the ER resulted in both reduced [Ca2+]C and [Ca2+]ER from PDGF stimulation, while IP3R channel inhibition led to reduced [Ca2+]C only. The inhibited SERCA pump caused an immediate [Ca2+]C increase and [Ca2+]ER decrease, indicating active calcium exchange between the cytosol and ER storage in resting cells. PDGF-induced calcium at the outer mitochondrial membrane sub-region showed a similar regulatory response to cytosolic calcium, not influenced by the inhibition of the mitochondrial calcium uniporter channel. Therefore, our work identifies calcium flow pathways among the extracellular medium, cell cytosol, and ER via regulatory calcium channels. Specifically, extracellular calcium flow has an essential function in fully activating ER calcium release.

Host IP3R channels are dispensable for rotavirus Ca2+ signaling but critical for intercellular Ca2+ waves that prime uninfected cells for rapid virus spread.

Many viruses exploit host Ca2+ signaling to facilitate their replication; however, little is known about how Ca2+ signals from different host and viral channels contribute to the overall dysregulation of Ca2+ signaling or promote virus replication. Using cells lacking IP3R, a host ER Ca2+ channel, we delineated intracellular Ca2+ signals within virus-infected cells and intercellular Ca2+ waves (ICWs), which increased Ca2+ signaling in neighboring, uninfected cells. In infected cells, IP3R was dispensable for rotavirus-induced Ca2+ signaling and replication, suggesting the rotavirus NSP4 viroporin supplies these signals. However, IP3R-mediated ICWs increase rotavirus replication kinetics and spread, indicating that the Ca2+ signals from the ICWs may prime nearby uninfected cells to better support virus replication upon eventual infection. This "pre-emptive priming" of uninfected cells by exploiting host intercellular pathways in the vicinity of virus-infected cells represents a novel mechanism for viral reprogramming of the host to gain a replication advantage.

Structural titration reveals Ca2+-dependent conformational landscape of the IP3 receptor

Inositol 1,4,5-trisphosphate receptors (IP3Rs) are endoplasmic reticulum Ca2+ channels whose biphasic dependence on cytosolic Ca2+ gives rise to Ca2+ oscillations that regulate fertilization, cell division and cell death. Despite the critical roles of IP3R-mediated Ca2+ responses, the structural underpinnings of the biphasic Ca2+ dependence that underlies Ca2+ oscillations are incompletely understood. Here, we collect cryo-EM images of an IP3R with Ca2+ concentrations spanning five orders of magnitude. Unbiased image analysis reveals that Ca2+ binding does not explicitly induce conformational changes but rather biases a complex conformational landscape consisting of resting, preactivated, activated, and inhibited states. Using particle counts as a proxy for relative conformational free energy, we demonstrate that Ca2+ binding at a high-affinity site allows IP3Rs to activate by escaping a low-energy resting state through an ensemble of preactivated states. At high Ca2+ concentrations, IP3Rs preferentially enter an inhibited state stabilized by a second, low-affinity Ca2+ binding site. Together, these studies provide a mechanistic basis for the biphasic Ca2+-dependence of IP3R channel activity.

Arriving at the next level of complexity in IP3R and SOCE signaling

The endoplasmic reticulum (ER) has long been recognized as the master regulator of cellular Ca2+ signaling. In this context, IP3R channels may be envisioned as this conductor's baton, which enables virtuous orchestration of cellular Ca2+ signaling tunes. IP3Rs serve the generation of spatiotemporally defined Ca2+ changes and are key for the ER´s function as an autonomous Ca2+ signaling unit, which is able to govern its own refilling from the extracellular Ca2+ pool. As yet, IP3R signaling has been primarily attributed to its precisely-tunable Ca2+ channel function and IP3-mediated control over Ca2+ levels within signaling domains. A recent report from the Hasan laboratory [1] provides evidence for an as yet overlooked function of IP3R1 in terms of supporting STIM/Orai-mediated SOCE in neurons. IP3R1 is demonstrated to remarkably facilitate productive STIM-Orai interactions and SOCE by a process that is triggered by IP3 but independent of the receptors’ function as an ER Ca2+ channel.

High-sensitivity calcium biosensor on the mitochondrial surface reveals that IP3R channels participate in the reticular Ca2+ leak towards mitochondria.

Genetically encoded biosensors based on fluorescent proteins (FPs) are widely used to monitor dynamics and sub-cellular spatial distribution of calcium ion (Ca2+) fluxes and their role in intracellular signaling pathways. The development of different mutations in the Ca2+-sensitive elements of the cameleon probes has allowed sensitive range of Ca2+ measurements in almost all cellular compartments. Region of the endoplasmic reticulum (ER) tethered to mitochondria, named as the mitochondrial-associated membranes (MAMs), has received an extended attention since the last 5 years. Indeed, as MAMs are essential for calcium homeostasis and mitochondrial function, molecular tools have been developed to assess quantitatively Ca2+ levels in the MAMs. However, sensitivity of the first generation Ca2+ biosensors on the surface of the outer-mitochondrial membrane (OMM) do not allow to measure μM or sub-μM changes in Ca2+ concentration which prevents to measure the native activity (unstimulated exogenously) of endogenous channels. In this study, we assembled a new ratiometric highly sensitive Ca2+ biosensor expressed on the surface of the outer-mitochondrial membrane (OMM). It allows the detection of smaller differences than the previous biosensor in or at proximity of the MAMs. Noteworthy, we demonstrated that IP3-receptors have an endogenous activity which participate to the Ca2+ leak channel on the surface of the OMM during hypoxia or when SERCA activity is blocked.

β-carotene targets IP3R/GRP75/VDAC1-MCU axis to renovate LPS-induced mitochondrial oxidative damage by regulating STIM1

Endoplasmic reticulum (ER) and mitochondria are the main sites for the storage and regulation of Ca2+ homeostasis. An imbalance of Ca2+ homeostasis can cause ER stress and mitochondrial dysfunction, thereby inducing apoptosis. The store-operated calcium entry (SOCE) is the main channel for extracellular calcium influx. Mitochondria-associated endoplasmic reticulum (MAM) is an important agent for Ca2+ transfer from the ER to the mitochondria. Therefore, regulation of SOCE and MAMs has potential therapeutic value for disease prevention and treatment. In this study, bovine mammary epithelial cells (BMECs) and mice were used as models to explore the mechanisms of β-carotene to relieve ER stress and mitochondrial dysfunction. BAPTA-AM, EGTA (Ca2+ inhibitor), and BTP2 (SOCE channel inhibitor) alleviated ER stress and mitochondrial oxidative damage induced by increased intracellular Ca2+ levels after lipopolysaccharide (LPS) stimulation. Furthermore, inhibition of ER stress by 4-PBA (ER stress inhibitor), 2-APB (IP3R inhibitor), and ruthenium red (mitochondrial calcium uniporter (MCU) inhibitor) restored mitochondrial function by reducing mitochondrial ROS. Our data also confirm that β-carotene targeted STIM1 and IP3R channels to repair LPS-induced ER stress and mitochondrial disorders. Consistent with the in vitro study, in vito experiments in mice further showed that β-carotene attenuated LPS-induced ER stress and mitochondrial oxidative damage by inhibiting the expression of STIM1 and ORAI1, and reducing the level of Ca2+ in mouse mammary glands. Therefore, ER stress-mitochondrial oxidative damage mediated by the STIM1-ER-IP3R/GRP75/VDAC1-MCU axis plays an vital role in the development of mastitis. Our results provided novel ideas and therapeutic targets for the prevention and treatment of mastitis.

MDFI regulates fast-to-slow muscle fiber type transformation via the calcium signaling pathway

Muscle fiber is the basic unit of skeletal muscle with strong self-adaptability, and its type is closely related to meat quality. Myod family inhibitor (Mdfi) has the function of regulating myogenic regulatory factors during cell differentiation, but how Mdfi regulates muscle fiber type transformation in myoblasts is still unclear. In the present study, we constructed overexpressing and interfering with Mdfi C2C12 cell models by lipofection. The immunofluorescence, quantitative real-time PCR (qPCR), and western blot results show that the elevated MDFI promoted mitochondrial biogenesis, aerobic metabolism and the calcium level by activating CaMKK2 and AMPK phosphorylation and then stimulated the conversion of C2C12 cells from fast glycolytic to slow oxidative type. In addition, after inhibiting IP3R and RYR channels, the higher MDFI reversed the blockage of calcium release from the endoplasmic reticulum by calcium channel receptor inhibitors and increased intracellular calcium levels. Therefore, we propose that the higher MDFI promotes muscle fiber types conversion through the calcium signaling pathway. These findings further broaden our understanding of the regulatory mechanism of MDFI in muscle fiber type transformation. Furthermore, our results suggest potential therapeutic targets for skeletal muscle and metabolic-related diseases.

Orai, RyR, and IP3R channels cooperatively regulate calcium signaling in brain mid-capillary pericytes

Pericytes are multifunctional cells of the vasculature that are vital to brain homeostasis, yet many of their fundamental physiological properties, such as Ca2+ signaling pathways, remain unexplored. We performed pharmacological and ion substitution experiments to investigate the mechanisms underlying pericyte Ca2+ signaling in acute cortical brain slices of PDGFRβ-Cre::GCaMP6f mice. We report that mid-capillary pericyte Ca2+ signalling differs from ensheathing type pericytes in that it is largely independent of L- and T-type voltage-gated calcium channels. Instead, Ca2+ signals in mid-capillary pericytes were inhibited by multiple Orai channel blockers, which also inhibited Ca2+ entry triggered by endoplasmic reticulum (ER) store depletion. An investigation into store release pathways indicated that Ca2+ transients in mid-capillary pericytes occur through a combination of IP3R and RyR activation, and that Orai store-operated calcium entry (SOCE) is required to sustain and amplify intracellular Ca2+ increases evoked by the GqGPCR agonist endothelin-1. These results suggest that Ca2+ influx via Orai channels reciprocally regulates IP3R and RyR release pathways in the ER, which together generate spontaneous Ca2+ transients and amplify Gq-coupled Ca2+ elevations in mid-capillary pericytes. Thus, SOCE is a major regulator of pericyte Ca2+ and a target for manipulating their function in health and disease.

High-sensitivity calcium biosensor on the mitochondrial surface reveals that IP3R channels participate to the reticular Ca2+leak towards mitochondria

High-sensitivity calcium biosensor on the mitochondrial surface reveals that IP3R channels participate to the reticular Ca2+leak towards mitochondria

Missense mutations in inositol 1,4,5-trisphosphate receptor type 3 result in leaky Ca2+ channels and activation of store-operated Ca2+ entry.

Mutations in all subtypes of the inositol 1,4,5-trisphosphate receptor Ca2+ release channel are associated with human diseases. In this report, we investigated the functionality of three neuropathy-associated missense mutations in IP3R3 (V615M, T1424M, and R2524C). The mutants only exhibited function when highly over-expressed compared to endogenous hIP3R3. All variants resulted in elevated basal cytosolic Ca2+ levels, decreased endoplasmic reticulum Ca2+ store content, and constitutive store-operated Ca2+ entry in the absence of any stimuli, consistent with a leaky IP3R channel pore. These variants differed in channel function; when stably over-expressed the R2524C mutant was essentially dead, V615M was poorly functional, and T1424M exhibited activity greater than that of the corresponding wild-type following threshold stimulation. These results demonstrate that a common feature of these mutations is decreased IP3R3 function. In addition, these mutations exhibit a novel phenotype manifested as a constitutively open channel, which inappropriately gates SOCE in the absence of stimulation.

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.

KRAP is required for diffuse and punctate IP3-mediated Ca2+ liberation and determines the number of functional IP3R channels within clusters

KRas-induced actin-interacting protein (KRAP) has been identified as crucial for the appropriate localization and functioning of the inositol trisphosphate receptors (IP3Rs) that mediate Ca2+ release from the endoplasmic reticulum. Here, we used siRNA knockdown of KRAP expression in HeLa and HEK293 cells to examine the roles of KRAP in the generation of IP3-mediated local Ca2+ puffs and global, cell-wide Ca2+ signals. High resolution Ca2+ imaging revealed that the mean amplitude of puffs was strongly reduced by KRAP knockdown, whereas the Ca2+ flux during openings of individual IP3R channels was little affected. In both control and KRAP knockdown cells the numbers of functional channels in the clusters underlying puff sites were stochastically distributed following a Poisson relationship, but the mean number of functional channels per site was reduced by about two thirds by KRAP knockdown. We conclude that KRAP is required for activity of IP3R channels at puff sites and stochastically ‘licenses’ the function of individual channels on a one-to-one basis, rather than determining the functioning of the puff site as a whole. In addition to puff activity (‘punctate’ Ca2+ release), global, cell-wide Ca2+ signals evoked by higher levels of IP3 are further composed from a discrete ‘diffuse’ mode of Ca2+ release. By applying fluctuation analysis to isolate the punctate component during global Ca2+ signals, we find that KRAP knockdown suppresses to similar extents punctate and diffuse Ca2+ release in wild-type cells and in HEK293 cells exclusively expressing type 1 and type 3 IP3Rs. Thus, KRAP appears essential for the functioning of the IP3Rs involved in diffuse Ca2+ release as well as the clustered IP3Rs that generate local Ca2+ puffs.

The Isoxazole Derivative of Usnic Acid Induces an ER Stress Response in Breast Cancer Cells That Leads to Paraptosis-like Cell Death.

Derivatives of usnic acid (UA), a secondary metabolite from lichens, were synthesized to improve its anticancer activity and selectivity. Recently we reported the synthesis and activity of an UA isoxazole derivative, named 2b, against cancer cells of different origins. Herein, the molecular mechanisms underlying its activity and efficacy in vivo were tested. The viability of breast cancer or normal cells has been tested using an MTT assay. Cell and organelle morphology was analyzed using light, electron and fluorescence microscopy. Gene expression was evaluated by RNAseq and protein levels were evaluated by Western blotting. In vivo anticancer activity was evaluated in a mice xenograft model. We found that 2b induced massive vacuolization which originated from the endoplasmic reticulum (ER). ER stress markers were upregulated both at the mRNA and protein levels. ER stress was caused by the release of Ca2+ ions from the ER by IP3R channels which was mediated, at least partly, by phospholipase C (PLC)-synthetized 1,4,5-inositol triphosphate (IP3). ER stress led to cell death with features of apoptosis and paraptosis. When applied to nude mice with xenografted breast cancer cells, 2b stopped tumour growth. In mice treated with 2b, vacuolization was observed in tumour cells, but not in other organs. This study shows that the antiproliferative activity of 2b relates to the induction of ER stress in cancer, not in healthy, cells and it leads to breast cancer cell death in vitro and in vivo.

Endoplasmic Reticulum-Based Calcium Dysfunctions in Synucleinopathies.

Neuronal calcium dyshomeostasis has been associated to Parkinson's disease (PD) development based on epidemiological studies on users of calcium channel antagonists and clinical trials are currently conducted exploring the hypothesis of increased calcium influx into neuronal cytosol as basic premise. We reported in 2018 an opposite hypothesis based on the demonstration that α-synuclein aggregates stimulate the endoplasmic reticulum (ER) calcium pump SERCA and demonstrated in cell models the existence of an α-synuclein-aggregate dependent neuronal state wherein cytosolic calcium is decreased due to an increased pumping of calcium into the ER. Inhibiting the SERCA pump protected both neurons and an α-synuclein transgenic C. elegans model. This models two cellular states that could contribute to development of PD. First the prolonged state with reduced cytosolic calcium that could deregulate multiple signaling pathways. Second the disease ER state with increased calcium concentration. We will discuss our hypothesis in the light of recent papers. First, a mechanistic study describing how variation in the Inositol-1,4,5-triphosphate (IP3) kinase B (ITPKB) may explain GWAS studies identifying the ITPKB gene as a protective factor toward PD. Here it was demonstrated that how increased ITPKB activity reduces influx of ER calcium to mitochondria via contact between IP3-receptors and the mitochondrial calcium uniporter complex in ER-mitochondria contact, known as mitochondria-associated membranes (MAMs). Secondly, it was demonstrated that astrocytes derived from PD patients contain α-synuclein accumulations. A recent study has demonstrated how human astrocytes derived from a few PD patients carrying the LRRK2-2019S mutation express more α-synuclein than control astrocytes, release more calcium from ER upon ryanodine receptor (RyR) stimulation, show changes in ER calcium channels and exhibit a decreased maximal and spare respiration indicating altered mitochondrial function in PD astrocytes. Here, we summarize the previous findings focusing the effect of α-synuclein to SERCA, RyR, IP3R, MCU subunits and other MAM-related channels. We also consider how the SOCE-related events could contribute to the development of PD.

Cryo-EM structure of type 1 IP3R channel in a lipid bilayer

Type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) is the predominant Ca2+-release channel in neurons. IP3R1 mediates Ca2+ release from the endoplasmic reticulum into the cytosol and thereby is involved in many physiological processes. Here, we present the cryo-EM structures of full-length rat IP3R1 reconstituted in lipid nanodisc and detergent solubilized in the presence of phosphatidylcholine determined in ligand-free, closed states by single-particle electron cryo-microscopy. Notably, both structures exhibit the well-established IP3R1 protein fold and reveal a nearly complete representation of lipids with similar locations of ordered lipids bound to the transmembrane domains. The lipid-bound structures show improved features that enabled us to unambiguously build atomic models of IP3R1 including two membrane associated helices that were not previously resolved in the TM region. Our findings suggest conserved locations of protein-bound lipids among homotetrameric ion channels that are critical for their structural and functional integrity despite the diversity of structural mechanisms for their gating.

Upregulated SOCC and IP3R calcium channels and subsequent elevated cytoplasmic calcium signaling promote nonalcoholic fatty liver disease by inhibiting autophagy.

Nonalcoholic fatty liver disease (NAFLD) is related to elevated cytoplasmic calcium signaling in hepatocytes, which may be mediated by store-operated calcium channel (SOCC) and inositol triphosphate receptor (IP3R). However, the regulatory effect of calcium signaling on lipid accumulation and degeneration in hepatocytes and the underlying molecular mechanism remain unknown. Autophagy inhibition promotes lipid accumulation and steatosis in hepatocytes. However, the association between elevated calcium signaling and autophagy inhibition in hepatocytes and its effect on hepatocyte fatty lesions remain unclear. Here, we established a mouse hepatocyte fatty gradient model using oleic acid. SOCC and IP3R channel opening and cytoplasmic calcium levels gradually increased with the hepatocyte pimelosis degree, whereas autophagy gradually decreased. We also established an optimal oleic acid (OOA) hepatocyte model, observing significantly increased SOCC and IP3R channel opening and calcium influx alongside significantly decreased autophagy and aggravated cellular fatty lesion. Calcium channel blockers (CCBs) and calcium channel gene silencing reagents (CCGSRs), respectively, reversed these effects, indicating that elevated cytoplasmic calcium signaling promotes NAFLD occurrence and the development by inhibiting hepatocyte autophagy. In the OOA model, upregulated extracellular regulated protein kinases 1/2 (ERK1/2), which can be regulated by SOCC and IP3R proteins transient receptor potential canonical 1 (TRPC1)/IP3R with elevated cytoplasmic calcium signaling, over-inhibited forkhead/winged helix O (FOXO) signaling and over-activated mammalian target of rapamycin complex 1 (mTORC1) signaling. Over-inhibited FOXO signaling significantly downregulated autophagy-related gene 12, which inhibits autophagosome maturation, while over-activated mTORC1 signaling over-inactivated Unc-51 like autophagy activating kinase 1, which inhibits preautophagosome formation. CCBs and CCGSRs recovered autophagy by significantly downregulating ERK1/2 to block abnormal changes in FOXO and mTORC1 signaling. Our findings indicate that upregulated SOCC and IP3R channels and subsequent elevated cytoplasmic calcium signaling in hepatocyte fatty lesions inhibits hepatocyte autophagy through (TRPC1/IP3R)/ERK/(FOXO/mTORC1) signaling pathways, causes lipid accumulation and degeneration in hepatocytes, and promotes NAFLD occurrence and development.