Phosphoinositide Research Articles

Getting attached to membranes - How plant signalling networks employ PtdIns(4,5)P2.

In eukaryotes, a small subset of membrane lipids, the phosphoinositides (PIs), exert regulatory effects on membrane-associated processes with profound impact on the organism, and PIs are relevant also for the physiology and development of plants. The PI, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) has emerged as an important regulatory player in plants, and in recent years this lipid has received substantial attention. This Update Review focuses on our current understanding of how PtdIns(4,5)P2 exerts its regulatory functions, how biosynthesis and degradation of this important regulatory lipid is controlled, and how PtdIns(4,5)P2 is linked to upstream and downstream elements within plant signalling networks.

GhFAD3-4 Promotes Fiber Cell Elongation and Cell Wall Thickness by Increasing PI and IP3 Accumulation in Cotton.

The omega-3 fatty acid desaturase enzyme gene FAD3 is responsible for converting linoleic acid to linolenic acid in plant fatty acid synthesis. Despite limited knowledge of its role in cotton growth, our study focused on GhFAD3-4, a gene within the FAD3 family, which was found to promote fiber elongation and cell wall thickness in cotton. GhFAD3-4 was predominantly expressed in elongating fibers, and its suppression led to shorter fibers with reduced cell wall thickness and phosphoinositide (PI) and inositol triphosphate (IP3) levels. Transcriptome analysis of GhFAD3-4 knock-out mutants revealed significant impacts on genes involved in the phosphoinositol signaling pathway. Experimental evidence demonstrated that GhFAD3-4 positively regulated the expression of the GhBoGH3B and GhPIS genes, influencing cotton fiber development through the inositol signaling pathway. The application of PI and IP6 externally increased fiber length in GhFAD3-4 knock-out plants, while inhibiting PI led to a reduced fiber length in GhFAD3-4 overexpressing plants. These findings suggest that GhFAD3-4 plays a crucial role in enhancing fiber development by promoting PI and IP3 biosynthesis, offering the potential for breeding cotton varieties with superior fiber quality.

Characterization of the First Secreted Sorting Nexin Identified in the Leishmania Protists.

Proteins of the sorting nexin (SNX) family present a modular structural architecture with a phox homology (PX) phosphoinositide (PI)-binding domain and additional PX structural domains, conferring to them a wide variety of vital eukaryotic cell's functions, from signal transduction to membrane deformation and cargo binding. Although SNXs are well studied in human and yeasts, they are poorly investigated in protists. Herein, is presented the characterization of the first SNX identified in Leishmania protozoan parasites encoded by the LdBPK_352470 gene. In silico secondary and tertiary structure prediction revealed a PX domain on the N-terminal half and a Bin/amphiphysin/Rvs (BAR) domain on the C-terminal half of this protein, with these features classifying it in the SNX-BAR subfamily of SNXs. We named the LdBPK_352470.1 gene product LdSNXi, as it is the first SNX identified in Leishmania (L.) donovani. Its expression was confirmed in L. donovani promastigotes under different cell cycle phases, and it was shown to be secreted in the extracellular medium. Using an in vitro lipid binding assay, it was demonstrated that recombinant (r) LdSNXi (rGST-LdSNXi) tagged with glutathione-S-transferase (GST) binds to the PtdIns3P and PtdIns4P PIs. Using a specific a-LdSNXi antibody and immunofluorescence confocal microscopy, the intracellular localization of endogenous LdSNXi was analyzed in L. donovani promastigotes and axenic amastigotes. Additionally, rLdSNXi tagged with enhanced green fluorescent protein (rLdSNXi-EGFP) was heterologously expressed in transfected HeLa cells and its localization was examined. All observed localizations suggest functions compatible with the postulated SNX identity of LdSNXi. Sequence, structure, and evolutionary analysis revealed high homology between LdSNXi and the human SNX2, while the investigation of protein-protein interactions based on STRING (v.11.5) predicted putative molecular partners of LdSNXi in Leishmania.

Knockdown of INPP5K compromises the differentiation of N2A cells.

Inositol polyphosphate 5-phosphatase K (INPP5K), also known as SKIP (skeletal muscle and kidney-enriched inositol phosphatase), is a cytoplasmic enzyme with 5-phosphatase activity toward phosphoinositides (PIs). Mutations in INPP5K are associated with autosomal recessive congenital muscular dystrophy with cataracts and intellectual disability (MDCCAID). Notably, muscular dystrophy is characterized by the hypoglycosylation of dystroglycan. Thus, far, the underlying mechanisms are only partially understood. In this study, we show that INPP5K expression increases during brain development. Knockdown of INPP5K in the neuroblastoma-derived cell line N2A impaired their neuronal-like differentiation and interfered with protein glycosylation.

Marked alteration of phosphoinositide signaling-associated molecules in postmortem prefrontal cortex with bipolar disorder.

The etiology of bipolar disorder (BD) remains unknown; however, lipid abnormalities in BD have received increasing attention in recent years. In this study, we examined the expression levels of enzyme proteins associated with the metabolic pathway of phosphoinositides (PIs) and their downstream effectors, protein kinase B (Akt1) and glycogen synthase kinase 3β (GSK3β), which have been assumed to be the targets of mood stabilizers such as lithium, in the postmortem brains of patients with BD. The protein expression levels of phosphatidylinositol 4-phosphate 5-kinase type-1 gamma (PIP5K1C), phosphatidylinositol 4-kinase alpha (PIK4CA), phosphatase and tensin homolog deleted from chromosome 10 (PTEN), Akt1, and GSK3β were measured using enzyme-linked immunosorbent assays and multiplex fluorescent bead-based immunoassays in the prefrontal cortex (PFC). Specifically, PTEN, Akt1, GSK3β, and PIP5K1C were measured in seven BD patients and 48 controls. Additionally, PIK4CA was analyzed in 10 cases and 34 controls. PTEN expression levels were markedly decreased in the PFCs of patients with BD, whereas those of Akt and GSK3β were prominently elevated. Moreover, patients medicated with lithium exhibited higher Akt1 expression levels and lower PTEN expression levels in comparison with the untreated group. Our results suggest that the expression levels of Akt1/GSK3β and its upstream regulator PTEN are considerably altered.

Aggregatibacter actinomycetemcomitans cytolethal distending toxin modulates host phagocytic function.

Cytolethal distending toxins (Cdt) are a family of toxins produced by several human pathogens which infect mucocutaneous tissue and induce inflammatory disease. Human macrophages exposed to Aggregatibacter actinomycetemcomitans (Aa) Cdt respond through canonical and non-canonical inflammasome activation to stimulate cytokine release. The inflammatory response is dependent on PI3K signaling blockade via the toxin's phosphatidylinositol-3,4,5-triphosphate (PIP3) phosphatase activity; converting PIP3 to phosphatidylinsoitol-3,4-diphosphate (PI3,4P2) thereby depleting PIP3 pools. Phosphoinositides, also play a critical role in phagosome trafficking, serving as binding domains for effector proteins during phagosome maturation and subsequent fusion with lysosomes. We now demonstrate that AaCdt manipulates the phosphoinositide (PI) pools of phagosome membranes and alters Rab5 association. Exposure of macrophages to AaCdt slowed phagosome maturation and decreased phago-lysosome formation, thereby compromising macrophage phagocytic function. Moreover, macrophages exposed to Cdt showed decreased bactericidal capacity leading to increase in Aggregatibacter actinomycetemcomitans survival. Thus, Cdt may contribute to increased susceptibility to bacterial infection. These studies uncover an underexplored aspect of Cdt function and provide new insight into the virulence potential of Cdt in mediating the pathogenesis of disease caused by Cdt-producing organisms such as Aa.

MGluR1/IP3/ERK signaling pathway regulates vestibular compensation in ON UBCs of the cerebellar flocculus.

To investigate the role of mGluR1α in cerebellar unipolar brush cells (UBC) in mediating vestibular compensation (VC), using mGluR1α agonist and antagonist to modulate ON UBC neurons, and explore the mGluR1/IP3/extracellular signal-regulated kinase (ERK) signaling pathway. First, AAV virus that knockdown ON UBC (mGluR1α) were injected into cerebellar UBC by stereotactic, and verified by immunofluorescence and western blot. The effect on VC was evaluated after unilateral labyrinthectomy (UL). Second, saline, (RS)-3,5-dihydroxyphenylglycine (DHPG), and LY367385 were injected into tubes implanted in rats at different time points after UL separately. The effect on ON UBC neuron activity was evaluated by immunofluorescence. Then, Phosphoinositide (PI) and p-ERK1/2 levels of mGluR1α were analyzed by ELISA after UL. The protein levels of p-ERK and total ERK were verified by western blot. In addition, the effect of mGluR1α activation or inhibition on VC-related behavior was observed. mGluR1α knockdown induced VC phenotypes. DHPG increased ON UBC activity, while LY367385 reduced ON UBC activity. DHPG group showed an increase in PI and p-ERK1/2 levels, while LY367385 group showed a decrease in PI and p-ERK1/2 levels in cerebellar UBC of rats. The western blot results of p-ERK and total ERK confirm and support the observations. DHPG alleviated VC-related behavior phenotypes, while LY367385 exacerbated vestibular decompensation-like behavior induced by UL. mGluR1α activity in cerebellar ON UBC is crucial for mediating VC through the mGluR1/IP3/ERK signaling pathway, which affects ON UBC neuron activity and contributes to the pathogenesis of VC.

Regulation of Phosphoinositide Signaling by Scaffolds at Cytoplasmic Membranes.

Cytoplasmic phosphoinositides (PI) are critical regulators of the membrane-cytosol interface that control a myriad of cellular functions despite their low abundance among phospholipids. The metabolic cycle that generates different PI species is crucial to their regulatory role, controlling membrane dynamics, vesicular trafficking, signal transduction, and other key cellular events. The synthesis of phosphatidylinositol (3,4,5)-triphosphate (PI3,4,5P3) in the cytoplamic PI3K/Akt pathway is central to the life and death of a cell. This review will focus on the emerging evidence that scaffold proteins regulate the PI3K/Akt pathway in distinct membrane structures in response to diverse stimuli, challenging the belief that the plasma membrane is the predominant site for PI3k/Akt signaling. In addition, we will discuss how PIs regulate the recruitment of specific scaffolding complexes to membrane structures to coordinate vesicle formation, fusion, and reformation during autophagy as well as a novel lysosome repair pathway.

Phosphoinositides in New Spaces.

Phosphoinositides (PIs) are phospholipids derived from phosphatidylinositol. PIs are regulated via reversible phosphorylation, which is directed by the opposing actions of PI kinases and phosphatases. PIs constitute a minor fraction of the total cellular lipid pool but play pleiotropic roles in multiple aspects of cell biology. Genetic mutations of PI regulatory enzymes have been identified in rare congenital developmental syndromes, including ciliopathies, and in numerous human diseases, such as cancer and metabolic and neurological disorders. Accordingly, PI regulatory enzymes have been targeted in the design of potential therapeutic interventions for human diseases. Recent advances place PIs as central regulators of membrane dynamics within functionally distinct subcellular compartments. This brief review focuses on the emerging role PIs play in regulating cell signaling within the primary cilium and in directing transfer of molecules at interorganelle membrane contact sites and identifies new roles for PIs in subcellular spaces.

In Silico Identification and Analysis of Proteins Containing the Phox Homology Phosphoinositide-Binding Domain in Kinetoplastea Protists: Evolutionary Conservation and Uniqueness of Phox-Homology-Domain-Containing Protein Architectures.

Kinetoplastea are free living and parasitic protists with unique features among Eukaryota. Pathogenic Kinetoplastea parasites (i.e., Trypanosoma and Leishmania spp.) undergo several developmental transitions essential for survival in their hosts. These transitions require membrane and cytoskeleton reorganizations that involve phosphoinositides (PIs). Phospholipids like PIs are key regulators of vital functions in all eukaryotes including signal transduction, protein transport and sorting, membrane trafficking, and cytoskeleton and membrane remodeling. A large repertoire of PI-metabolizing enzymes and PI-binding proteins/effectors carrying distinct PI-binding modules like the PX (phox homology) module could play significant roles in the life and virulence of pathogenic Kinetoplastea. The aim of this study was to retrieve the entire spectrum of Kinetoplastea protein sequences containing the PX module (PX-proteins), predict their structures, and identify in them evolutionary conserved and unique traits. Using a large array of bioinformatics tools, protein IDs from two searches (based on PFam's pHMM for PX domain (PF00787)) were combined, aligned, and utilized for the construction of a new Kinetoplastea_PX pHMM. This three-step search retrieved 170 PX-protein sequences. Structural domain configuration analysis identified PX, Pkinase, Lipocalin_5, and Vps5/BAR3-WASP domains and clustered them into five distinct subfamilies. Phylogenetic tree and domain architecture analysis showed that some domain architectures exist in proteomes of all Kinetoplastea spp., while others are genus-specific. Finally, amino acid conservation logos of the Kinetoplastea spp. and Homo sapiens PX domains revealed high evolutionary conservation in residues forming the critical structural motifs for PtdIns3P recognition. This study highlights the PX-Pkinase domain architecture as unique within Trypanosoma spp. and forms the basis for a targeted functional analysis of Kinetoplastea PX-proteins as putative targets for a rational design of anti-parasitic drugs.

De novo missense variants in phosphatidylinositol kinase PIP5KIγ underlie a neurodevelopmental syndrome associated with altered phosphoinositide signaling

Phosphoinositides (PIs) are membrane phospholipids produced through the local activity of PI kinases and phosphatases that selectively add or remove phosphate groups from the inositol head group. PIs control membrane composition and play key roles in many cellular processes including actin dynamics, endosomal trafficking, autophagy, and nuclear functions. Mutations in phosphatidylinositol 4,5 bisphosphate [PI(4,5)P2] phosphatases cause a broad spectrum of neurodevelopmental disorders such as Lowe and Joubert syndromes and congenital muscular dystrophy with cataracts and intellectual disability, which are thus associated with increased levels of PI(4,5)P2. Here, we describe a neurodevelopmental disorder associated with an increase in the production of PI(4,5)P2 and with PI-signaling dysfunction. We identified three de novo heterozygous missense variants in PIP5K1C, which encodes an isoform of the phosphatidylinositol 4-phosphate 5-kinase (PIP5KIγ), in nine unrelated children exhibiting intellectual disability, developmental delay, acquired microcephaly, seizures, visual abnormalities, and dysmorphic features. We provide evidence that the PIP5K1C variants result in an increase of the endosomal PI(4,5)P2 pool, giving rise to ectopic recruitment of filamentous actin at early endosomes (EEs) that in turn causes dysfunction in EE trafficking. In addition, we generated an invivo zebrafish model that recapitulates the disorder we describe with developmental defects affecting the forebrain, including the eyes, as well as craniofacial abnormalities, further demonstrating the pathogenic effect of the PIP5K1C variants.

A Plethora of Functions Condensed into Tiny Phospholipids: The Story of PI4P and PI(4,5)P2.

Phosphoinositides (PIs) are small, phosphorylated lipids that serve many functions in the cell. They regulate endo- and exocytosis, vesicular trafficking, actin reorganization, and cell mobility, and they act as signaling molecules. The most abundant PIs in the cell are phosphatidylinositol-4-monophosphate (PI4P) and phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2]. PI4P is mostly localized at the Golgi apparatus where it regulates the anterograde trafficking from the Golgi apparatus to the plasma membrane (PM), but it also localizes at the PM. On the other hand, the main localization site of PI(4,5)P2 is the PM where it regulates the formation of endocytic vesicles. The levels of PIs are regulated by many kinases and phosphatases. Four main kinases phosphorylate the precursor molecule phosphatidylinositol into PI4P, divided into two classes (PI4KIIα, PI4KIIβ, PI4KIIIα, and PI4KIIIβ), and three main kinases phosphorylate PI4P to form PI(4,5)P2 (PI4P5KIα, PI4P5KIβ, and PI4P5KIγ). In this review, we discuss the localization and function of the kinases that produce PI4P and PI(4,5)P2, as well as the localization and function of their product molecules with an overview of tools for the detection of these PIs.

Inactivating the lipid kinase activity of PI3KC2β is sufficient to rescue myotubular myopathy in mice.

Phosphoinositides (PIs) are membrane lipids that regulate signal transduction and vesicular trafficking. X-linked centronuclear myopathy (XLCNM), also called myotubular myopathy, results from loss-of-function mutations in the MTM1 gene, which encodes the myotubularin phosphatidylinositol 3-phosphate (PtdIns3P) lipid phosphatase. No therapy for this disease is currently available. Previous studies showed that loss of expression of the class II phosphoinositide 3-kinase (PI3K) PI3KC2β (PI3KC2B) protein improved the phenotypes of an XLCNM mouse model. PI3Ks are well known to have extensive scaffolding functions and the importance of the catalytic activity of this PI3K for rescue remains unclear. Here, using PI3KC2β kinase-dead mice, we show that the selective inactivation of PI3KC2β kinase activity is sufficient to fully prevent muscle atrophy and weakness, histopathology, and sarcomere and triad disorganization in Mtm1-knockout mice. This rescue correlates with normalization of PtdIns3P level and mTORC1 activity, a key regulator of protein synthesis and autophagy. Conversely, lack of PI3KC2β kinase activity did not rescue the histopathology of the BIN1 autosomal CNM mouse model. Overall, these findings support the development of specific PI3KC2β kinase inhibitors to cure myotubular myopathy.

PI(4,5)P2 regulates the gating of NaV1.4 channels.

Voltage-gated sodium (NaV) channels are densely expressed in most excitable cells and activate in response to depolarization, causing a rapid influx of Na+ ions that initiates the action potential. The voltage-dependent activation of NaV channels is followed almost instantaneously by fast inactivation, setting the refractory period of excitable tissues. The gating cycle of NaV channels is subject to tight regulation, with perturbations leading to a range of pathophysiological states. The gating properties of most ion channels are regulated by the membrane phospholipid, phosphatidylinositol (4,5) bisphosphate (PI(4,5)P2). However, it is not known whether PI(4,5)P2 modulates the activity of NaV channels. Here, we utilize optogenetics to activate specific, membrane-associated phosphoinositide (PI)-phosphatases that dephosphorylate PI(4,5)P2 while simultaneously recording NaV1.4 channel currents. We show that dephosphorylating PI(4,5)P2 left-shifts the voltage-dependent gating of NaV1.4 to more hyperpolarized membrane potentials, augments the late current that persists after fast inactivation, and speeds the rate at which channels recover from fast inactivation. These effects are opposed by exogenous diC8PI(4,5)P2. We provide evidence that PI(4,5)P2 is a negative regulator that tunes the gating behavior of NaV1.4 channels.

Exploring the Role of PI3P in Platelets: Insights from a Novel External PI3P Pool

Phosphoinositides (PIs) play a crucial role in regulating intracellular signaling, actin cytoskeleton rearrangements, and membrane trafficking by binding to specific domains of effector proteins. They are primarily found in the membrane leaflets facing the cytosol. Our study demonstrates the presence of a pool of phosphatidylinositol 3-monophosphate (PI3P) in the outer leaflet of the plasma membrane of resting human and mouse platelets. This pool of PI3P is accessible to exogenous recombinant myotubularin 3-phosphatase and ABH phospholipase. Mouse platelets with loss of function of class III PI 3-kinase and class II PI 3-kinase α have a decreased level of external PI3P, suggesting a contribution of these kinases to this pool of PI3P. After injection in mouse, or incubation ex vivo in human blood, PI3P-binding proteins decorated the platelet surface as well as α-granules. Upon activation, these platelets were able to secrete the PI3P-binding proteins. These data sheds light on a previously unknown external pool of PI3P in the platelet plasma membrane that recognizes PI3P-binding proteins, leading to their uptake towards α-granules. This study raises questions about the potential function of this external PI3P in the communication of platelets with the extracellular environment, and its possible role in eliminating proteins from the plasma.

Membrane lipid metabolism and scavenging of reactive oxygen species are essential for cold resistance of Solanum habrochaites (LA1777): Lipidomics and transcriptomes analysis

Low temperature significantly limits the production of tomato (Solanum lycopersicum) in greenhouse. Solanum habrochaites is a wild species of tomato closely related to Solanum lycopersicum and is more tolerant to cold than S. lycopersicum. However, the mechanism by which S. habrochaites resists low temperature is unknown. This study aimed to analyze the physiology, transcriptomics, and lipidomics of LA1777 (S. habrochaites variety) after exposure to cold stress for 3 days to assess its specific mechanism to cold tolerance. A cold-sensitive cultivated tomato variety AC was used as controls. Physiological analysis showed that the two tomato varieties were significantly affected by cold stress. However, LA1777 had more stable physiological characteristics and maintained relatively good photosynthetic capacity than the AC variety. Transcriptome analysis suggested that the stability of cell membrane structure and function might promote cold tolerance of LA1777. Furthermore, 243 differentially expressed genes (DEGs) involved in lipid metabolism were identified. Particularly, the upregulation of temperature-induced lipocalin genes (TILs) and fatty acid desaturase genes (FADs) promoted cold tolerance by inhibiting the excessive accumulation of reactive oxygen species (ROS) and increasing membrane lipid unsaturation, respectively. Meanwhile, lipidomics data indicated that monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) levels were significantly reduced in both plants under cold stress. MGDG levels decreased by 28% and 54% in LA1777 and AC, respectively, while DGDG decreased by 7% and 29%, respectively. Further results showed that high phosphoinositide (PI) content may increase cold tolerance in LA1777 by maintaining phosphatidylinositol signaling system. Also, results showed that adenosine triphosphate (ATP) produced via β-oxidation of fatty acids in mitochondria can maintain a higher metabolic level of wild tomato under cold stress. A metabolic model of the mechanism of cold tolerance in tomato was also constructed. This study provides a basis for further analysis of the molecular mechanism of cold resistance in tomato, which is crucial for genetic improvement.

Atlas of phosphoinositide signatures in the retina identifies heterogeneity between cell types.

Phosphoinositides (PIPs) are a family of minor acidic phospholipids in the cell membrane. Phosphoinositide (PI) kinases and phosphatases can rapidly convert one PIP product into another resulting in the generation of seven distinct PIPs. The retina is a heterogeneous tissue composed of several cell types. In the mammalian genome, around 50 genes encode PI kinases and PI phosphatases; however, there are no studies describing the distribution of these enzymes in the various retinal cell types. Using translating ribosome affinity purification, we have identified the in vivo distribution of PI-converting enzymes from the rod, cone, retinal pigment epithelium (RPE), Müller glia, and retinal ganglion cells, generating a physiological atlas for PI-converting enzyme expression in the retina. The retinal neurons, rods, cones, and RGCs, are characterized by the enrichment of PI-converting enzymes, whereas the Müller glia and RPE are characterized by the depletion of these enzymes. We also found distinct differences between the expression of PI kinases and PI phosphatases in each retinal cell type. Since mutations in PI-converting enzymes are linked to human diseases including retinal diseases, the results of this study will provide a guide for what cell types are likely to be affected by retinal degenerative diseases brought on by changes in PI metabolism.

Comprehensive classification of proteins based on structures that engage lipids by COMPOSEL

Structures can now be predicted for any protein using programs like AlphaFold and Rosetta, which rely on a foundation of experimentally determined structures of architecturally diverse proteins. The accuracy of such artificial intelligence and machine learning (AI/ML) approaches benefits from the specification of restraints which assist in navigating the universe of folds to converge on models most representative of a given protein's physiological structure. This is especially pertinent for membrane proteins, with structures and functions that depend on their presence in lipid bilayers. Structures of proteins in their membrane environments could conceivably be predicted from AI/ML approaches with user-specificized parameters that describe each element of the architecture of a membrane protein accompanied by its lipid environment. We propose the Classification Of Membrane Proteins based On Structures Engaging Lipids (COMPOSEL), which builds on existing nomenclature types for monotopic, bitopic, polytopic and peripheral membrane proteins as well as lipids. Functional and regulatory elements are also defined in the scripts, as shown with membrane fusing synaptotagmins, multidomain PDZD8 and Protrudin proteins that recognize phosphoinositide (PI) lipids, the intrinsically disordered MARCKS protein, caveolins, the β barrel assembly machine (BAM), an adhesion G-protein coupled receptor (aGPCR) and two lipid modifying enzymes - diacylglycerol kinase DGKε and fatty aldehyde dehydrogenase FALDH. This demonstrates how COMPOSEL communicates lipid interactivity as well as signaling mechanisms and binding of metabolites, drug molecules, polypeptides or nucleic acids to describe the operations of any protein. Moreover COMPOSEL can be scaled to express how genomes encode membrane structures and how our organs are infiltrated by pathogens such as SARS-CoV-2.

Optogenetic regulation of PIP2 alters the voltage-dependence of activation and fast inactivation of skeletal muscle Nav1.4 channels.

Since the late 1970's phosphatidylinositol-4,5-bisphosphate PI(4,5)P2 has garnered much attention for its role as a substrate that initiates the production of both inositol-1,4,5-triphosphate (IP3) and 1,2-diacylglycerol (DAG). These secondary messengers regulate the cellular function of many ion channels including voltage-gated sodium channels (NaV). In addition to this, PI(4,5)P2 can recruit and modulate the activity of many ion channels. However, to date, it is yet to be elucidated whether PI(4,5)P2 modulates the activity of NaV channels. NaV channels are critical in the initiation and propagation of action potentials in excitable cells. The voltage-dependent activation and instantaneous fast inactivation gating are tightly regulated as they set the refractory period in excitable cells. Changes in this regulation lead to various sodium channelopathies. With simultaneous use of both patch-clamp and optogenetic techniques, here, we report the effect of dephosphorylation of PI(4,5)P2 by specific phosphoinositide (PI)- phosphatases on Nav1.4 gating. In this study, we utilized the 4’ phosphatase Sac2 (CRY2-Sac2) or the 5’ phosphatase CRY2-5’PtaseOCRL (CRY2-5’POCRL) to show that dephosphorylating PI(4,5)P2 left-shifts the voltage-dependent gating of NaV1.4, the skeletal muscle channel, to more hyperpolarized membrane potentials. This data provides evidence that PI(4,5)P2 is important for the gating of NaV1.4 channels.

Loss of INPP5K attenuates IP3-induced Ca2+ responses in the glioblastoma cell line U-251 MG cells

INPP5K (inositol polyphosphate 5-phosphatase K) is an endoplasmic reticulum (ER)-resident enzyme that acts as a phosphoinositide (PI) 5-phosphatase, capable of dephosphorylating various PIs including PI 4,5-bisphosphate (PI(4,5)P2), a key phosphoinositide found in the plasma membrane. Given its ER localization and substrate specificity, INPP5K may play a role in ER-plasma membrane contact sites. Furthermore, PI(4,5)P2 serves as a substrate for phospholipase C, an enzyme activated downstream of extracellular agonists acting on Gq-coupled receptors or tyrosine-kinase receptors, leading to IP3 production and subsequent release of Ca2+ from the ER, the primary intracellular Ca2+ storage organelle. In this study, we investigated the impact of INPP5K on ER Ca2+ dynamics using a previously established INPP5K-knockdown U-251 MG glioblastoma cell model. We here describe that loss of INPP5K impairs agonist-induced, IP3 receptor (IP3R)-mediated Ca2+ mobilization in intact cells, while the ER Ca2+ content and store-operated Ca2+ influx remain unaffected. To further elucidate the underlying mechanisms, we examined Ca2+ release in permeabilized cells stimulated with exogenous IP3. Interestingly, the absence of INPP5K also disrupted IP3-induced Ca2+ release events. These results suggest that INPP5K may directly influence IP3R activity through mechanisms yet to be resolved. The findings from this study point towards role of INPP5K in modulating ER calcium dynamics, particularly in relation to IP3-mediated signaling pathways. However, further work is needed to establish the general nature of our findings and to unravel the exact molecular mechanisms underlying the interplay between INNP5K function and Ca2+ signaling.