Restoration of PITPNA in Type 2 diabetic human islets reverses pancreatic beta-cell dysfunction.

Defects in insulin processing and granule maturation are linked to pancreatic beta-cell failure during type 2 diabetes (T2D). Phosphatidylinositol transfer protein alpha (PITPNA) stimulates activity of phosphatidylinositol (PtdIns) 4-OH kinase to produce sufficient PtdIns-4-phosphate (PtdIns-4-P) in the trans-Golgi network to promote insulin granule maturation. PITPNA in beta-cells of T2D human subjects is markedly reduced suggesting its depletion accompanies beta-cell dysfunction. Conditional deletion of Pitpna in the beta-cells of Ins-Cre, Pitpnaflox/flox mice leads to hyperglycemia resulting from decreasing glucose-stimulated insulin secretion (GSIS) and reducing pancreatic beta-cell mass. Furthermore, PITPNA silencing in human islets confirms its role in PtdIns-4-P synthesis and leads to impaired insulin granule maturation and docking, GSIS, and proinsulin processing with evidence of ER stress. Restoration of PITPNA in islets of T2D human subjects reverses these beta-cell defects and identify PITPNA as a critical target linked to beta-cell failure in T2D.

ER Stress-Induced Sphingosine-1-Phosphate Lyase Phosphorylation Potentiates the Mitochondrial Unfolded Protein Response.

The unfolded protein response (UPR) is an elaborate signaling network that evolved to maintain proteostasis in the endoplasmic reticulum (ER) and mitochondria (mt). These organelles are functionally and physically associated, and consequently, their stress responses are often intertwined. It is unclear how these two adaptive stress responses are coordinated during ER stress. The inositol-requiring enzyme-1 (IRE1), a central ER stress sensor and proximal regulator of the UPRER, harbors dual kinase and endoribonuclease (RNase) activities. IRE1 RNase activity initiates the transcriptional layer of the UPRER, but IRE1's kinase substrate(s) and their functions are largely unknown. Here, we discovered that sphingosine 1-phosphate (S1P) lyase (SPL), the enzyme that degrades S1P, is a substrate for the mammalian IRE1 kinase. Our data show that IRE1-dependent SPL phosphorylation inhibits SPL's enzymatic activity, resulting in increased intracellular S1P levels. S1P has previously been shown to induce the activation of mitochondrial UPR (UPRmt) in nematodes. We determined that IRE1 kinase-dependent S1P induction during ER stress potentiates UPRmt signaling in mammalian cells. Phosphorylation of eukaryotic translation initiation factor 2α (eif2α) is recognized as a critical molecular event for UPRmt activation in mammalian cells. Our data further demonstrate that inhibition of the IRE1-SPL axis abrogates the activation of two eif2α kinases, namely double-stranded RNA-activated protein kinase (PKR) and PKR-like ER kinase upon ER stress. These findings show that the IRE1-SPL axis plays a central role in coordinating the adaptive responses of ER and mitochondria to ER stress in mammalian cells.

Monitoring the cellular metabolism of a membrane-permeant photo-caged phosphatidylinositol 3,4,5-trisphosphate derivative.

To deliver charged lipid derivatives to the cell interior, bioactivatable and photo-activatable protecting groups are frequently used. The intracellular metabolism of the protecting groups, as well as the lipid itself, are key factors that determine biological activity. Here we followed the cellular metabolism of cell-permeant photo-activatable ("caged") and non-caged membrane-permeant analogs of dioctanoyl phosphatidylinositol 3,4,5-trisphosphate (diC8PIP3) carrying biodegradable protecting groups by mass spectrometry. After successful cell entry, the photo-activatable group can be removed on demand by a light pulse. Hence, UV irradiation acts as a switch to expose the cellular metabolism to a bolus of active compound. To investigate lipid metabolites and to capture a more complete metabolome, we adapted standard extraction methods and employed multi-reaction monitoring mass spectrometry (MRM-MS). This required a previously developed permethylation method that stabilized metabolites and enhanced volatility of the phosphoinositide metabolites. The mass spectrometric analysis allowed for the monitoring of the intracellular removal of photo-activatable caging as well as biodegradable protecting groups from the membrane-permeant phosphoinositides along with cellular turnover, namely by dephosphorylation. We found that phosphate masking groups, namely acetoxymethyl esters, were rapidly removed by endogenous enzymes while butyrates masking hydroxy groups showed a longer lifetime, giving rise to trapped intermediates. We further identified key intermediate metabolites and demonstrated the beneficial effect of caging groups and their removal on the formation of favorable metabolites. Surprisingly, caging and protecting groups were found to influence each other's stability.

Inositol-requiring enzyme-1 regulates phosphoinositide signaling lipids and macrophage growth.

The ER-bound kinase/endoribonuclease (RNase), inositol-requiring enzyme-1 (IRE1), regulates the phylogenetically most conserved arm of the unfolded protein response (UPR). However, the complex biology and pathology regulated by mammalian IRE1 cannot be fully explained by IRE1's one known, specific RNA target, X box-binding protein-1 (XBP1) or the RNA substrates of IRE1-dependent RNA degradation (RIDD) activity. Investigating other specific substrates of IRE1 kinase and RNase activities may illuminate how it performs these diverse functions in mammalian cells. We report that macrophage IRE1 plays an unprecedented role in regulating phosphatidylinositide-derived signaling lipid metabolites and has profound impact on the downstream signaling mediated by the mammalian target of rapamycin (mTOR). This cross-talk between UPR and mTOR pathways occurs through the unconventional maturation of microRNA (miR) 2137 by IRE1's RNase activity. Furthermore, phosphatidylinositol (3,4,5) phosphate (PI(3,4,5)P3 ) 5-phosphatase-2 (INPPL1) is a direct target of miR-2137, which controls PI(3,4,5)P3 levels in macrophages. The modulation of cellular PI(3,4,5)P3 /PIP2 ratio and anabolic mTOR signaling by the IRE1-induced miR-2137 demonstrates how the ER can provide a critical input into cell growth decisions.

ACLY is the novel signaling target of PIP2/PIP3 and Lyn in acute myeloid leukemia.

A fundamental feature of tumor progression is reprogramming of metabolic pathways. ATP citrate lyase (ACLY) is a key metabolic enzyme that catalyzes the generation of Acetyl-CoA and is upregulated in cancer cells and required for their growth. The phosphoinositide 3-kinase (PI3K) and Src-family kinase (SFK) Lyn are constitutively activate in many cancers. We show here, for the first time, that both the substrate and product of PI3K, phosphatidylinositol-(4,5)-bisphosphate (PIP2) and phosphatidylinositol-(3,4,5)-trisphosphate (PIP3), respectively, bind to ACLY in Acute Myeloid Leukemia (AML) patient-derived, but not normal donor-derived cells. We demonstrate the binding of PIP2 to the CoA-binding domain of ACLY and identify the six tyrosine residues of ACLY that are phosphorylated by Lyn. Three of them (Y682, Y252, Y227) can be also phosphorylated by Src and they are located in catalytic, citrate binding and ATP binding domains, respectively. PI3K and Lyn inhibitors reduce the ACLY enzyme activity, ACLY-mediated Acetyl-CoA synthesis, phospholipid synthesis, histone acetylation and cell growth. Thus, PIP2/PIP3 binding and Src tyrosine kinases-mediated stimulation of ACLY links oncogenic pathways to Acetyl-CoA-dependent pro-growth and survival metabolic pathways in cancer cells. These results indicate a novel function for Lyn, as a regulator of Acetyl-CoA-mediated metabolic pathways.

Plasma membrane processes are differentially regulated by type I phosphatidylinositol phosphate 5-kinases and RASSF4.

Phosphoinositide lipids regulate many cellular processes and are synthesized by lipid kinases. Type I phosphatidylinositol phosphate 5-kinases (PIP5KIs) generate phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]. Several phosphoinositide-sensitive readouts revealed the nonequivalence of overexpressing PIP5KIß, PIP5KIγ or Ras association domain family 4 (RASSF4), believed to activate PIP5KIs. Mass spectrometry showed that each of these three proteins increased total cellular phosphatidylinositol bisphosphates (PtdInsP2) and trisphosphates (PtdInsP3) at the expense of phosphatidylinositol phosphate (PtdInsP) without changing lipid acyl chains. Analysis of KCNQ2/3 channels and PH domains confirmed an increase in plasma membrane PtdIns(4,5)P2 in response to PIP5KIß or PIP5KIγ overexpression, but RASSF4 required coexpression with PIP5KIγ to increase plasma membrane PtdIns(4,5)P2 Effects on the several steps of store-operated calcium entry (SOCE) were not explained by plasma membrane phosphoinositide increases alone. PIP5KIß and RASSF4 increased STIM1 proximity to the plasma membrane, accelerated STIM1 mobilization and speeded onset of SOCE; however, PIP5KIγ reduced STIM1 recruitment but did not change induced Ca2+ entry. These differences imply actions through different segregated pools of phosphoinositides and specific protein-protein interactions and targeting.This article has an associated First Person interview with the first author of the paper.

Quantification of phosphoinositides reveals strong enrichment of PIP2 in HIV-1 compared to producer cell membranes.

Human immunodeficiency virus type 1 (HIV-1) acquires its lipid envelope during budding from the plasma membrane of the host cell. Various studies indicated that HIV-1 membranes differ from producer cell plasma membranes, suggesting budding from specialized membrane microdomains. The phosphoinositide PI(4,5)P2 has been of particular interest since PI(4,5)P2 is needed to recruit the viral structural polyprotein Gag to the plasma membrane and thus facilitates viral morphogenesis. While there is evidence for an enrichment of PIP2 in HIV-1, fully quantitative analysis of all phosphoinositides remains technically challenging and therefore has not been reported, yet. Here, we present a comprehensive analysis of the lipid content of HIV-1 and of plasma membranes from infected and non-infected producer cells, resulting in a total of 478 quantified lipid compounds, including molecular species distribution of 25 different lipid classes. Quantitative analyses of phosphoinositides revealed strong enrichment of PIP2, but also of PIP3, in the viral compared to the producer cell plasma membrane. We calculated an average of ca. 8,000 PIP2 molecules per HIV-1 particle, three times more than Gag. We speculate that the high density of PIP2 at the HIV-1 assembly site is mediated by transient interactions with viral Gag polyproteins, facilitating PIP2 concentration in this microdomain. These results are consistent with our previous observation that PIP2 is not only required for recruiting, but also for stably maintaining Gag at the plasma membrane. We believe that this quantitative analysis of the molecular anatomy of the HIV-1 lipid envelope may serve as standard reference for future investigations.

Fatty-acyl chain profiles of cellular phosphoinositides.

Phosphoinositides are rapidly turning-over phospholipids that play key roles in intracellular signaling and modulation of membrane effectors. Through technical refinements we have improved sensitivity in the analysis of the phosphoinositide PI, PIP, and PIP2 pools from living cells using mass spectrometry. This has permitted further resolution in phosphoinositide lipidomics from cell cultures and small samples of tissue. The technique includes butanol extraction, derivatization of the lipids, post-column infusion of sodium to stabilize formation of sodiated adducts, and electrospray ionization mass spectrometry in multiple reaction monitoring mode, achieving a detection limit of 20pg. We describe the spectrum of fatty-acyl chains in the cellular phosphoinositides. Consistent with previous work in other mammalian primary cells, the 38:4 fatty-acyl chains dominate in the phosphoinositides of the pineal gland and of superior cervical ganglia, and many additional fatty acid combinations are found at low abundance. However, Chinese hamster ovary cells and human embryonic kidney cells (tsA201) in culture have different fatty-acyl chain profiles that change with growth state. Their 38:4 lipids lose their dominance as cultures approach confluence. The method has good time resolution and follows well the depletion in <20s of both PIP2 and PIP that results from strong activation of Gq-coupled receptors. The receptor-activated phospholipase C exhibits no substrate selectivity among the various fatty-acyl chain combinations.

Osmoregulatory inositol transporter SMIT1 modulates electrical activity by adjusting PI(4,5)P2 levels.

Myo-inositol is an important cellular osmolyte in autoregulation of cell volume and fluid balance, particularly for mammalian brain and kidney cells. We find it also regulates excitability. Myo-inositol is the precursor of phosphoinositides, key signaling lipids including phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. However, whether myo-inositol accumulation during osmoregulation affects signaling and excitability has not been fully explored. We found that overexpression of the Na(+)/myo-inositol cotransporter (SMIT1) and myo-inositol supplementation enlarged intracellular PI(4,5)P2 pools, modulated several PI(4,5)P2-dependent ion channels including KCNQ2/3 channels, and attenuated the action potential firing of superior cervical ganglion neurons. Further experiments using the rapamycin-recruitable phosphatase Sac1 to hydrolyze PI(4)P and the P4M probe to visualize PI(4)P suggested that PI(4)P levels increased after myo-inositol supplementation with SMIT1 expression. Elevated relative levels of PIP and PIP2 were directly confirmed using mass spectrometry. Inositol trisphosphate production and release of calcium from intracellular stores also were augmented after myo-inositol supplementation. Finally, we found that treatment with a hypertonic solution mimicked the effect we observed with SMIT1 overexpression, whereas silencing tonicity-responsive enhancer binding protein prevented these effects. These results show that ion channel function and cellular excitability are under regulation by several "physiological" manipulations that alter the PI(4,5)P2 setpoint. We demonstrate a previously unrecognized linkage between extracellular osmotic changes and the electrical properties of excitable cells.

Dynamic formation of ER-PM junctions presents a lipid phosphatase to regulate phosphoinositides.

Endoplasmic reticulum-plasma membrane (ER-PM) contact sites play an integral role in cellular processes such as excitation-contraction coupling and store-operated calcium entry (SOCE). Another ER-PM assembly is one tethered by the extended synaptotagmins (E-Syt). We have discovered that at steady state, E-Syt2 positions the ER and Sac1, an integral ER membrane lipid phosphatase, in discrete ER-PM junctions. Here, Sac1 participates in phosphoinositide homeostasis by limiting PM phosphatidylinositol 4-phosphate (PI(4)P), the precursor of PI(4,5)P2 Activation of G protein-coupled receptors that deplete PM PI(4,5)P2disrupts E-Syt2-mediated ER-PM junctions, reducing Sac1's access to the PM and permitting PM PI(4)P and PI(4,5)P2to recover. Conversely, depletion of ER luminal calcium and subsequent activation of SOCE increases the amount of Sac1 in contact with the PM, depleting PM PI(4)P. Thus, the dynamic presence of Sac1 at ER-PM contact sites allows it to act as a cellular sensor and controller of PM phosphoinositides, thereby influencing many PM processes.

Dynamics of Phosphoinositide-Dependent Signaling in Sympathetic Neurons.

In neurons, loss of plasma membrane phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] leads to a decrease in exocytosis and changes in electrical excitability. Restoration of PI(4,5)P2 levels after phospholipase C activation is therefore essential for a return to basal neuronal activity. However, the dynamics of phosphoinositide metabolism have not been analyzed in neurons. We measured dynamic changes of PI(4,5)P2, phosphatidylinositol 4-phosphate, diacylglycerol, inositol 1,4,5-trisphosphate, and Ca(2+) upon muscarinic stimulation in sympathetic neurons from adult male Sprague-Dawley rats with electrophysiological and optical approaches. We used this kinetic information to develop a quantitative description of neuronal phosphoinositide metabolism. The measurements and analysis show and explain faster synthesis of PI(4,5)P2 in sympathetic neurons than in electrically nonexcitable tsA201 cells. They can be used to understand dynamic effects of receptor-mediated phospholipase C activation on excitability and other PI(4,5)P2-dependent processes in neurons. SIGNIFICANCE STATEMENT: Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is a minor phospholipid in the cytoplasmic leaflet of the plasma membrane. Depletion of PI(4,5)P2 via phospholipase C-mediated hydrolysis leads to a decrease in exocytosis and alters electrical excitability in neurons. Restoration of PI(4,5)P2 is essential for a return to basal neuronal activity. However, the dynamics of phosphoinositide metabolism have not been analyzed in neurons. We studied the dynamics of phosphoinositide metabolism in sympathetic neurons upon muscarinic stimulation and used the kinetic information to develop a quantitative description of neuronal phosphoinositide metabolism. The measurements and analysis show a several-fold faster synthesis of PI(4,5)P2 in sympathetic neurons than in an electrically nonexcitable cell line, and provide a framework for future studies of PI(4,5)P2-dependent processes in neurons.

PIP3 induces the recycling of receptor tyrosine kinases.

Down-regulation of receptor tyrosine kinases such as the epidermal growth factor receptor (EGFR) is achieved by endocytosis of the receptor followed by degradation or recycling. We demonstrated that in the absence of ligand, increased phosphatidylinositol 3,4,5-trisphosphate (PIP3) concentrations induced clathrin- and dynamin-mediated endocytosis of EGFR but not that of transferrin or G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptors. Endocytosis of the receptor in response to binding of EGF resulted in a decrease in the abundance of the EGFR, but PIP3-induced internalization decreased receptor ubiquitination and phosphorylation and resulted in recycling of the receptor to the plasma membrane. An RNA interference (RNAi) screen directed against lipid-binding domain-containing proteins identified polarity complex proteins, including PARD3 (partitioning defective 3), as essential for PIP3-induced receptor tyrosine kinase recycling. Thus, PIP3 and polarity complex proteins regulate receptor tyrosine kinase trafficking, which may enhance cellular responsiveness to growth factors.

Control of TMEM16A by INO-4995 and other inositolphosphates.

BACKGROUND AND PURPOSE: Ca(2+)-dependent Cl(-) secretion (CaCC) in airways and other tissues is due to activation of the Cl(-) channel TMEM16A (anoctamin 1). Earlier studies suggested that Ca(2+) -activated Cl(-) channels are regulated by membrane lipid inositol phosphates, and that 1-O-octyl-2-O-butyryl-myo-inositol 3,4,5,6-tetrakisphosphate octakis(propionoxymethyl) ester (INO-4995) augments CaCC. Here we examined whether TMEM16A is the target for INO-4995 and if the channel is regulated by inositol phosphates. EXPERIMENTAL APPROACH: The effects of INO-4995 on CaCC were examined in overexpressing HEK293, colonic and primary airway epithelial cells as well as Xenopus oocytes. We used patch clamping, double electrode voltage clamp and Ussing chamber techniques. KEY RESULTS: We found that INO-4995 directly activates a TMEM16A whole cell conductance of 6.1 ± 0.9 nS pF(-1) in overexpressing cells. The tetrakisphosphates Ins(3,4,5,6)P(4) or Ins(1,3,4,5)P(4) and enzymes controlling levels of InsP(4) or PIP(2) and PIP(3) had no effects on the magnitude or kinetics of TMEM16A currents. In contrast in Xenopus oocytes, human airways and colonic cells, which all express TMEM16A endogenously, Cl(-) currents were not acutely activated by INO-4995. However incubation with INO-4995 augmented 1.6- to 4-fold TMEM16A-dependent Cl(-) currents activated by ionomycin or ATP, while intracellular Ca(2+) signals were not affected. The potentiating effect of INO-4995 on transient ATP-activated TMEM16A-currents in cystic fibrosis (CF) airways was twice of that observed in non-CF airways. CONCLUSIONS AND IMPLICATIONS: These data indicate that TMEM16A is the target for INO-4995, although the mode of action appears different for overexpressed and endogenous channels. INO-4995 may be useful for the treatment of CF lung disease.

INO-4995 therapeutic efficacy is enhanced with repeat dosing in cystic fibrosis knockout mice and human epithelia.

Progressive lung damage in cystic fibrosis (CF) has been linked to inadequate airway mucosal hydration. We previously demonstrated that an inositol tetrakisphosphate analog, 1-O-octyl-2-O-butyryl-myo-inositol 3,4,5,6-tetrakisphosphate octakis(propionoxymethyl)ester (INO-4995), regulates airway secretory and absorptive processes, affecting mucosal hydration by prolonged (24 h) inhibition of Na(+) and fluid absorption in CF human nasal epithelia (CFHNE). The objectives of this study were to further assess clinical potential of INO-4995 in CF through ascertaining in vivo activity in mice with CF, determining the effects of repeated administration on potency and determining cytoplasmic half-life. Uptake and metabolism of [(3)H]INO-4995 was monitored with HPLC to calculate intracellular half-life. INO-4995 was administered in vitro repeatedly over 4 to 8 days to CFHNE. Fluid absorption was assessed by blue dextran exclusion, and basal short-circuit current was measured in Ussing chambers. INO-4995 (1-100 microg/kg) was dosed intranasally either as a single dose or once per day over 4 days to gut-corrected CF mice. [(3)H]INO-4995 was rapidly taken up by epithelial cultures and converted to the active drug, which had a half-life of 40 hours. Repeated daily application of INO-4995 to CFHNE lowered the effective concentration for inhibition of fluid absorption and amiloride-sensitive short-circuit current in cultured CFHNE, and reduced nasal potential difference to nearly control levels in gut-corrected CF mice. Ca(2+)-activated Cl(-) channel activity was also boosted in cultures. Mouse nasal levels fell from abnormal levels to within 2 muA of normal with repeated exposure to 0.8 microg/kg over 4 days. These data support further development of INO-4995 for the treatment of CF.

Inositol polyphosphate derivative inhibits Na+ transport and improves fluid dynamics in cystic fibrosis airway epithelia.

Amiloride-sensitive, epithelial Na(+) channel (ENaC)-mediated, active absorption of Na(+) is elevated in the airway epithelium of cystic fibrosis (CF) patients, resulting in excess fluid removal from the airway lumen. This excess fluid/volume absorption corresponds to CF transmembrane regulator-linked defects in ENaC regulation, resulting in the reduced mucociliary clearance found in CF airways. Herein we show that INO-4995, a synthetic analog of the intracellular signaling molecule, D-myo-inositol 3,4,5,6-tetrakisphosphate, inhibits Na(+) and fluid absorption across CF airway epithelia, thus alleviating this critical pathology. This conclusion was based on electrophysiological studies, fluid absorption, and (22)Na(+) flux measurements in CF airway epithelia, contrasted with normal epithelia, and on electrophysiological studies in Madin-Darby canine kidney cells and 3T3 cells overexpressing ENaC. The effects of INO-4995 were long-lasting, dose-dependent, and more pronounced in epithelia from CF patients vs. controls. These findings support preclinical development of INO-4995 for CF treatment and demonstrate for the first time the therapeutic potential of inositol polyphosphate derivatives.

Lindane stimulates neutrophils by selectively activating phospholipase C and phosphoinositide-kinase activity.

The organochlorine insecticide lindane is a known activator of neutrophil responses. In this work we delineated the biochemical pathways by which lindane stimulates neutrophil oxidant production. Plasma membrane GTPase activity was not stimulated by lindane, ruling out a role for lindane-induced activation of G-proteins or G-protein coupled receptors, whereas inhibition of phospholipase C inhibited lindane-induced oxidant production. Together these data pointed to phospholipase C as the direct target of lindane activation. Type I phosphoinositide 3-kinase was not significantly activated by lindane and an inhibitor of phosphoinositide 3-kinases inhibited oxidant production by only 40%. Thus, Type I phosphoinositide 3-kinase played a minor role, if any, in lindane-induced oxidant production. Lindane stimulated an increase in phosphatidylinositol phosphate suggesting a Type II or III phosphotidylinositol 3-kinase or phosphatidylinositol 4-kinase activity was also stimulated.

Antagonists of myo-inositol 3,4,5,6-tetrakisphosphate allow repeated epithelial chloride secretion.

Cystic fibrosis (CF) patients suffer from a defect in hydration of mucosal membranes due to mutations in the cystic fibrosis transmembrane regulator (CFTR), an apical chloride channel in mucosal epithelia. Disease expression in CF knockout mice is organ specific, varying with the level of expression of calcium activated Cl(-) channels (CLCA). Therefore, restoring transepithelial Cl(-) secretion by augmenting alternate Cl(-) channels, such as CLCA, could be beneficial. However, CLCA-mediated Cl(-) secretion is transient, due in part to the inhibitory effects of myo-inositol 3,4,5,6-tetrakisphosphate [Ins(3,4,5,6)P(4)]. This suggests that antagonists of Ins(3,4,5,6)P(4) could be useful in treatment of CF. We have, therefore, synthesized a series of membrane-permeant Ins(3,4,5,6)P(4) derivatives, carrying alkyl substituents on the hydroxyl groups and screened them for effects on Cl(-) secretion in a human colonic epithelial cell line, T(84). While membrane-permeant Ins(3,4,5,6)P(4) derivatives had no direct effects on carbachol-stimulated Cl(-) secretion, Ins(3,4,5,6)P(4) derivatives, but not enantiomeric Ins(1,4,5,6)P(4) derivatives, reversed the inhibitory effect of Ins(3,4,5,6)P(4) on subsequent thapsigargin activation of Cl(-) secretion. The extent of the antagonistic effect of the Ins(3,4,5,6)P(4) derivatives varied with the position of the alkyl substituents. Derivatives with a cyclohexylidene ketal or a butyl-chain at the 1-position reversed the Ins(3,4,5,6)P(4)-mediated inhibition of Cl(-) secretion by up to 96 and 85%, respectively, whereas butylation of the 1- and 2-position generated a reversal effect of only 65%. Derivatives carrying the butyl chain only at the 2-position showed no antagonistic effect. These data: (1) Support the hypothesis that Ins(3,4,5,6)P(4) stereospecifically inhibits Ca(2+) activated Cl(-) secretion and that Ins(3,4,5,6)P(4) mediates most, if not all of the cholinergic-mediated inhibition of chloride secretion in T(84) cells; (2) Demonstrate Ins(3,4,5,6)P(4)-mediated inhibition can be completely reversed with rationally designed membrane-permeant Ins(3,4,5,6)P(4) antagonists; (3) Demonstrate that a SAR for membrane-permeant Ins(3,4,5,6) P(4) antagonists can be generated and screened in a physiologically relevant cell-based assay; (4) Indicate that Ins(3,4,5,6)P(4) derivatives could serve as a starting point for the development of therapeutics to treat cystic fibrosis.