Insights into the inhibitory mechanism of triazole-based small molecules on phosphatidylinositol-4,5-bisphosphate binding pleckstrin homology domain

BackgroundPhosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] is an important regulator of several cellular processes and a precursor for other second messengers which are involved in cell signaling pathways. Signaling proteins preferably interact with PI(4,5)P₂ through its pleckstrin homology (PH) domain. Efforts are underway to design small molecule-based antagonist, which can specifically inhibit the PI(4,5)P₂/PH-domain interaction to establish an alternate strategy for the development of drug(s) for phosphoinositide signaling pathways.MethodsSurface plasmon resonance, molecular docking, circular dichroism, competitive Förster resonance energy transfer, isothermal titration calorimetric analyses and liposome pull down assay were used.ResultsIn this study, we employed 1,2,3-triazol-4-yl methanol containing small molecule (CIPs) as antagonists for PI(4,5)P₂/PH-domain interaction and determined their inhibitory effect by using competitive-surface plasmon resonance analysis (IC₅₀ ranges from 53 to 159 nM for PI(4,5)P₂/PLCδ1-PH domain binding assay). We also used phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P₃], phosphatidylinositol 3,4-bisphosphate [PI(3,4)P₂], PI(4,5)P₂ specific PH-domains to determine binding selectivity of the compounds. Various physicochemical analyses showed that the compounds have weak affect on fluidity of the model membrane but, strongly interact with the phospholipase C δ1 (PLCδ1)-PH domains. The 1,2,3-triazol-4-yl methanol moiety and nitro group of the compounds are essential for their exothermic interaction with the PH-domains. Potent compound can efficiently displace PLCδ1-PH domain from plasma membrane to cytosol in A549 cells.ConclusionsOverall, our studies demonstrate that these compounds interact with the PIP-binding PH-domains and inhibit their membrane recruitment.General significanceThese results suggest specific but differential binding of these compounds to the PLCδ1-PH domain and emphasize the role of their structural differences in binding parameters. These triazole-based compounds could be directly used/further developed as potential inhibitor for PH domain-dependent enzyme activity.     

PLC-mediated PI(4,5)P2 hydrolysis regulates activation and inactivation of TRPC6/7 channels

Transient receptor potential classical (or canonical) (TRPC)3, TRPC6, and TRPC7 are a subfamily of TRPC channels activated by diacylglycerol (DAG) produced through the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) by phospholipase C (PLC). PI(4,5)P₂ depletion by a heterologously expressed phosphatase inhibits TRPC3, TRPC6, and TRPC7 activity independently of DAG; however, the physiological role of PI(4,5)P₂ reduction on channel activity remains unclear. We used Förster resonance energy transfer (FRET) to measure PI(4,5)P₂ or DAG dynamics concurrently with TRPC6 or TRPC7 currents after agonist stimulation of receptors that couple to G_q and thereby activate PLC. Measurements made at different levels of receptor activation revealed a correlation between the kinetics of PI(4,5)P₂ reduction and those of receptor-operated TRPC6 and TRPC7 current activation and inactivation. In contrast, DAG production correlated with channel activation but not inactivation; moreover, the time course of channel inactivation was unchanged in protein kinase C–insensitive mutants. These results suggest that inactivation of receptor-operated TRPC currents is primarily mediated by the dissociation of PI(4,5)P₂. We determined the functional dissociation constant of PI(4,5)P₂ to TRPC channels using FRET of the PLCδ Pleckstrin homology domain (PHd), which binds PI(4,5)P₂, and used this constant to fit our experimental data to a model in which channel gating is controlled by PI(4,5)P₂ and DAG. This model predicted similar FRET dynamics of the PHd to measured FRET in either human embryonic kidney cells or smooth muscle cells, whereas a model lacking PI(4,5)P₂ regulation failed to reproduce the experimental data, confirming the inhibitory role of PI(4,5)P₂ depletion on TRPC currents. Our model also explains various PLC-dependent characteristics of channel activity, including limitation of maximum open probability, shortening of the peak time, and the bell-shaped response of total current. In conclusion, our studies demonstrate a fundamental role for PI(4,5)P₂ in regulating TRPC6 and TRPC7 activity triggered by PLC-coupled receptor stimulation.     

PTPRN2 and PLCβ1 promote metastatic breast cancer cell migration through PI(4,5)P2‐dependent actin remodeling

Altered abundance of phosphatidyl inositides (PIs) is a feature of cancer. Various PIs mark the identity of diverse membranes in normal and malignant cells. Phosphatidylinositol 4,5‐bisphosphate (PI(4,5)P₂) resides predominantly in the plasma membrane, where it regulates cellular processes by recruiting, activating, or inhibiting proteins at the plasma membrane. We find that PTPRN2 and PLCβ1 enzymatically reduce plasma membrane PI(4,5)P₂ levels in metastatic breast cancer cells through two independent mechanisms. These genes are upregulated in highly metastatic breast cancer cells, and their increased expression associates with human metastatic relapse. Reduction in plasma membrane PI(4,5)P₂ abundance by these enzymes releases the PI(4,5)P₂‐binding protein cofilin from its inactive membrane‐associated state into the cytoplasm where it mediates actin turnover dynamics, thereby enhancing cellular migration and metastatic capacity. Our findings reveal an enzymatic network that regulates metastatic cell migration through lipid‐dependent sequestration of an actin‐remodeling factor.     

Phosphatidylinositol 5-phosphate 4-kinase type II beta is required for vitamin D receptor-dependent E-cadherin expression in SW480 cells

Numerous epidemiological data indicate that vitamin D receptor (VDR) signaling induced by its ligand or active metabolite 1α,25-dihydroxyvitamin D₃ (1α,25(OH)₂D₃) has anti-cancer activity in several colon cancers. 1α,25(OH)₂D₃ induces the epithelial differentiation of SW480 colon cancer cells expressing VDR (SW480-ADH) by upregulating E-cadherin expression; however, its precise mechanism remains unknown. We found that phosphatidylinositol-5-phosphate 4-kinase type II beta (PIPKIIβ) but not PIPKIIα is required for VDR-mediated E-cadherin induction in SW480-ADH cells. The syntenin-2 postsynaptic density protein/disc large/zona occludens (PDZ) domain and pleckstrin homology domain of phospholipase C-delta1 (PLCδ1 PHD) possess high affinity for phosphatidylinositol-4,5-bisphosphate (PI(4,5)P₂) mainly localized to the nucleus and plasma membrane, respectively. The expression of syntenin-2 PDZ but not PLCδ1 PHD inhibited 1α,25(OH)₂D₃-induced E-cadherin upregulation, suggesting that nuclear PI(4,5)P₂ production mediates E-cadherin expression through PIPKIIβ in a VDR-dependent manner. PIPKIIβ is also involved in the suppression of the cell motility induced by 1α,25(OH)₂D₃. These results indicate that PIPKIIβ-mediated PI(4,5)P₂ signaling is important for E-cadherin upregulation and inhibition of cellular motility induced by VDR activation.     

Signal-dependent hydrolysis of phosphatidylinositol 4,5-bisphosphate without activation of phospholipase C: implications on gating of Drosophila TRPL (transient receptor potential-like) channel

In Drosophila, a phospholipase C (PLC)-mediated signaling cascade, couples photo-excitation of rhodopsin to the opening of the transient receptor potential (TRP) and TRP-like (TRPL) channels. A lipid product of PLC, diacylglycerol (DAG), and its metabolites, polyunsaturated fatty acids (PUFAs) may function as second messengers of channel activation. However, how can one separate between the increase in putative second messengers, change in pH, and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) depletion when exploring the TRPL gating mechanism? To answer this question we co-expressed the TRPL channels together with the muscarinic (M1) receptor, enabling the openings of TRPL channels via G-protein activation of PLC. To dissect PLC activation of TRPL into its molecular components, we used a powerful method that reduced plasma membrane-associated PI(4,5)P₂ in HEK cells within seconds without activating PLC. Upon the addition of a dimerizing drug, PI(4,5)P₂ was selectively hydrolyzed in the cell membrane without producing DAG, inositol trisphosphate, or calcium signals. We show that PI(4,5)P₂ is not an inhibitor of TRPL channel activation. PI(4,5)P₂ hydrolysis combined with either acidification or application of DAG analogs failed to activate the channels, whereas PUFA did activate the channels. Moreover, a reduction in PI(4,5)P₂ levels or inhibition of DAG lipase during PLC activity suppressed the PLC-activated TRPL current. This suggests that PI(4,5)P₂ is a crucial substrate for PLC-mediated activation of the channels, whereas PUFA may function as the channel activator. Together, this study defines a narrow range of possible mechanisms for TRPL gating.     

Differential inhibition of the TRPM8 ion channel by Gαq and Gα11

Cold temperature is encoded by the cold-sensitive ion channel TRPM8 in somatosensory neurons. It has been unclear how TRPM8 is modulated so that it can mediate distinct type of cold signaling. We have recently reported that activated Gα_q directly inhibits TRPM8 after activation of Gq-coupled receptors. Here, we further show that activation of the muscarinic receptor M1R, which is known to inhibit M currents through PLCβ-mediated hydrolysis of PtdIns(4,5)P₂, similarly inhibited TRPM8 potently, but inhibition was not prevented by the PLC inhibitor U73122. Interestingly, although Gα_q and Gα₁₁ are indistinguishable in activating PLCβ and hydrolysing PtdIns(4,5)P₂, activated Gα₁₁ inhibited TRPM8 to a lesser extent than activated Gα_q. The differential TRPM8 inhibition is determined by a specific residue E197 on Gα₁₁, because mutating this residue to the corresponding residue on Gα_q restored TRPM8 inhibition to a similar degree as mediated by Gα_q. These results reinforce the idea that activated Gα_q directly inhibits TRPM8 independently from PtdIns(4,5)P₂ hydrolysis-mediated inhibition of TRPM8.     

Kv12.1 channels are not sensitive to GqPCR-triggered activation of phospholipase Cβ

K_v12.1 K⁺ channels are expressed in several brain areas, but no physiological function could be attributed to these subunits so far. As genetically-modified animal models are not available, identification of native K_v12.1 currents must rely on characterization of distinct channel properties. Recently, it was shown in Xenopus laevis oocytes that K_v12.1 channels were modulated by membrane PI(4,5)P₂. However, it is not known whether these channels are also sensitive to physiologically-relevant PI(4,5)P₂ dynamics. We thus studied whether K_v12.1 channels were modulated by activation of phospholipase C β (PLCβ) and found that they were insensitive to receptor-triggered depletion of PI(4,5)P₂. Thus, K_v12.1 channels add to the growing list of K⁺ channels that are insensitive to PLCβ signaling, although modulated by PI(4,5)P₂ in Xenopus laevis oocytes.     

Different signaling pathway between sphingosine-1-phosphate and lysophosphatidic acid in Xenopus oocytes: Functional coupling of the sphingosine-1-phosphate receptor to PLC-xβ in Xenopus oocytes

In Xenopus oocytes, both sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) activate Ca²⁺-dependent oscillatory Cl⁻ currents by acting through membrane-bound receptors. External application of 50 μM S1P elicited a long-lasting oscillatory current that continued over 30 min from the beginning of oscillation, with 300 nA (n = 11) as a usual maximum peak of current, whereas 1-μM LPA treatment showed only transiently oscillating but more vigorous current responses, with 2,800 nA (n = 18) as a maximum peak amplitude. Both phospholipid-induced Ca²⁺-dependent Cl⁻ currents were observed in the absence of extracellular Ca²⁺, were blocked by intracellular injection of the Ca²⁺ chelator, EGTA, and could not be elicited by treatment with thapsigargin, an inhibitor of endoplasmic reticulum (ER) Ca²⁺ ATPase. Intracellular Ca²⁺ release appeared to be from inositol 1,4,5-trisphosphate (IP₃)-sensitive Ca²⁺ store, because Cl⁻ currents were blocked by heparin injection. Pretreatment with the aminosteroid, U-73122, an inhibitor of G protein-mediated phospholipase C (PLC) activation, to oocytes inhibited the current responses evoked both by S1P and LPA. However, when they were injected with 10 ng of antisense oligonucleotide (AS-ODN) against Xenopus phospholipase C (PLC-xβ), oocytes could not respond to S1P application, whereas they responded normally to LPA, indicating that the S1P signaling pathway goes through PLC-xβ, whereas LPA signaling goes through another unknown PLC. To determine the types of G proteins involved, we introduced AS-ODNs against four types of G-protein α subunits that were identified in Xenopus laevis; G_qα, G₁₁α, G₀α, and G_i1α. Among AS-ODNs against the Gαs tested, AS-G_qα and AS-G_i1α to S1P and AS-G_qα and AS-G₁₁α to LPA specifically reduced current responses, respectively, to about 20–30% of controls. These results demonstrate that LPA and S1P, although they have similar structural features, release intracellular Ca²⁺ from the IP₃-sensitive pool, use different components in their signal transduction pathways in Xenopus oocytes. J. Cell. Physiol. 176:412–423, 1998. © 1998 Wiley-Liss, Inc.     

Sprouty2 Regulates PI(4,5)P2/Ca Signaling and HIV-1 Gag Release

We reported recently that activation of the inositol 1,4,5-triphosphate receptor (IP3R) is required for efficient HIV-1 Gag trafficking and viral particle release. IP3R activation requires phospholipase C (PLC)-catalyzed hydrolysis of PI(4,5)P₂ to IP3 and diacylglycerol. We show that Sprouty2 (Spry2), which binds PI(4,5)P₂ and PLCγ, interfered with PI(4,5)P₂ in a manner similar to that of U73122, an inhibitor of PI(4,5)P₂ hydrolysis, suggesting that Spry2 negatively regulates IP3R by preventing formation of its activating ligand, IP3. Mutation to Asp of R252, a crucial determinant of PI(4,5)P₂ binding in the C-terminal domain of Spry2, prevented the interference, indicating that binding to the phospholipid is required. By contrast, deletion of the PLCγ binding region or mutation of a critical Tyr residue in the region did not prevent the interference but Spry2-PI(4,5)P₂ colocalization was not detected, suggesting that PLC binding is required for their stable association. Like U73122, Spry2 over-expression inhibited wild type Gag release as virus-like particles. Disrupting either binding determinant relieved the inhibition. IP3R-mediated Ca²⁺signaling, in turn, was found to influence Spry2 subcellular distribution and ERK, a Spry2 regulator. Our findings suggest that Spry2 influences IP3R function through control of PI(4,5)P₂ and IP3R influences Spry2 function by controlling its distribution and ERK activation.     

Molecular Determinants of Phosphatidylinositol 4,5-Bisphosphate (PI(4,5)P2) Binding to Transient Receptor Potential V1 (TRPV1) Channels

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) has been recognized as an important activator of certain transient receptor potential (TRP) channels. More specifically, TRPV1 is a pain receptor activated by a wide range of stimuli. However, whether or not PI(4,5)P₂ is a TRPV1 agonist remains open to debate. Utilizing a combined approach of mutagenesis and molecular modeling, we identified a PI(4,5)P₂ binding site located between the TRP box and the S4-S5 linker. At this site, PI(4,5)P₂ interacts with the amino acid residues Arg-575 and Arg-579 in the S4-S5 linker and with Lys-694 in the TRP box. We confirmed that PI(4,5)P₂ behaves as a channel agonist and found that Arg-575, Arg-579, and Lys-694 mutations to alanine reduce PI(4,5)P₂ binding affinity. Additionally, in silico mutations R575A, R579A, and K694A showed that the reduction in binding affinity results from the delocalization of PI(4,5)P₂ in the binding pocket. Molecular dynamics simulations indicate that PI(4,5)P₂ binding induces conformational rearrangements of the structure formed by S6 and the TRP domain, which cause an opening of the lower TRPV1 channel gate.     

Profilin binding to sub-micellar concentrations of phosphatidylinositol (4,5) bisphosphate and phosphatidylinositol (3,4,5) trisphosphate

Profilin is a small (12-15 kDa) actin binding protein which promotes filament turnover. Profilin is also involved in the signaling pathway linking receptors in the cell membrane to the microfilament system within the cell. Profilin is thought to play critical roles in this signaling pathway through its interaction with phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] and phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P₃] (P.J. Lu, W.R. Shieh, S.G. Rhee, H.L. Yin, C.S. Chen, Lipid products of phosphoinositide 3-kinase bind human profilin with high affinity, Biochemistry 35 (1996) 14027-14034). To date, profilin's interaction with polyphosphoinositides (PPI) has only been studied in micelles or small vesicles. Profilin binds with high affinity to small clusters of PI(4,5)P₂ molecules. In this work, we investigated the interactions of profilin with sub-micellar concentrations of PI(4,5)P₂ and PI(3,4,5)P₃. Fluorescence anisotropy was used to determine the relevant dissociation constants for binding of sub-micellar concentrations of fluorescently labeled PPI lipids to profilin and we show that these are significantly different from those determined for profilin interaction with micelles or small vesicles. We also show that profilin binds more tightly to sub-micellar concentrations of PI(3,4,5)P₃ (K_D = 720 μM) than to sub-micellar concentrations of PI(4,5)P₂ (K_D = 985 μM). Despite the low affinity for sub-micellar concentration of PI(4,5)P₂, profilin was shown to bind to giant unilamellar vesicles in presence of 0.5% mole fraction of PI(4,5)P₂ The implications of these findings are discussed.     

Gα16 interacts with Class IA phosphatidylinositol 3-kinases and inhibits Akt signaling

Phosphatidylinositol 3-kinase (PI3K) mediates receptor tyrosine kinase and G protein coupled receptor (GPCR) signaling by phosphorylating phosphoinositides to elicit various biological responses. Gα_q has previously been shown to inhibit class IA PI3K by interacting with the p110α subunit. However, it is not known if PI3Ks can associate with other Gα_q family members such as Gα₁₆. Here, we demonstrated that class IA PI3Ks, p85/p110α and p85/p110β, could form stable complexes with wild type Gα₁₆ and its constitutively active mutant (Gα₁₆QL) in HEK293 cells. In contrast, no interaction between Gα₁₆ and class IB PI3K was observed. The Gα₁₆/p110α signaling complex could be detected in hematopoietic cells that endogenously express Gα₁₆. Overexpression of class I PI3Ks did not inhibit Gα₁₆QL-induced IP₃ production and, unlike p63RhoGEF, class IA PI3Ks did not attenuate the binding of PLCβ₂ to Gα₁₆QL. On the contrary, the function of class IA PI3Ks was suppressed by Gα₁₆QL as revealed by diminished production of PIP₃ as well as inhibition of EGF-induced Akt phosphorylation. Taken together, these results suggest that Gα₁₆ can bind to class IA PI3Ks and inhibit the PI3K signaling pathway.     

The inositol 5-phosphatase SHIP2 regulates endocytic clathrin-coated pit dynamics

Phosphatidylinositol (PI) 4,5-bisphosphate (PI(4,5)P₂) and its phosphorylated product PI 3,4,5-triphosphate (PI(3,4,5)P₃) are two major phosphoinositides concentrated at the plasma membrane. Their levels, which are tightly controlled by kinases, phospholipases, and phosphatases, regulate a variety of cellular functions, including clathrin-mediated endocytosis and receptor signaling. In this study, we show that the inositol 5-phosphatase SHIP2, a negative regulator of PI(3,4,5)P₃-dependent signaling, also negatively regulates PI(4,5)P₂ levels and is concentrated at endocytic clathrin-coated pits (CCPs) via interactions with the scaffold protein intersectin. SHIP2 is recruited early at the pits and dissociates before fission. Both knockdown of SHIP2 expression and acute production of PI(3,4,5)P₃ shorten CCP lifetime by enhancing the rate of pit maturation, which is consistent with a positive role of both SHIP2 substrates, PI(4,5)P₂ and PI(3,4,5)P₃, on coat assembly. Because SHIP2 is a negative regulator of insulin signaling, our findings suggest the importance of the phosphoinositide metabolism at CCPs in the regulation of insulin signal output.     

Odorant-stimulated phosphoinositide signaling in mammalian olfactory receptor neurons

Recent evidence has revived interest in the idea that phosphoinositides (PIs) may play a role in signal transduction in mammalian olfactory receptor neurons (ORNs). To provide direct evidence that odorants indeed activate PI signaling in ORNs, we used adenoviral vectors carrying two different fluorescently tagged probes, the pleckstrin homology (PH) domains of phospholipase Cδ1 (PLCδ1) and the general receptor of phosphoinositides (GRP1), to monitor PI activity in the dendritic knobs of ORNs in vivo. Odorants mobilized PI(4,5)P₂/IP₃ and PI(3,4,5)P₃, the substrates and products of PLC and PI3K. We then measured odorant activation of PLC and PI3K in olfactory ciliary-enriched membranes in vitro using a phospholipid overlay assay and ELISAs. Odorants activated both PLC and PI3K in the olfactory cilia within 2 s of odorant stimulation. Odorant-dependent activation of PLC and PI3K in the olfactory epithelium could be blocked by enzyme-specific inhibitors. Odorants activated PLC and PI3K with partially overlapping specificity. These results provide direct evidence that odorants indeed activate PI signaling in mammalian ORNs in a manner that is consistent with the idea that PI signaling plays a role in olfactory transduction.     

Non-inositol 1,4,5-trisphosphate (IP3) receptor IP3-binding proteins

Conventionally, myo-D-inositol 1, 4,5-trisphosphate (IP₃) is thought to exert its second messenger effects through the gating of IP₃R Ca²⁺ release channels, located in Ca²⁺-storage organelles like the endoplasmic reticulum. However, there is considerable indirect evidence to support the concept that IP₃ might interact with other, non-IP₃R proteins within cells. To explore this possibility further, the Protein Data Bank was searched using the term “IP3”. This resulted in the retrieval of 203 protein structures, the majority of which were members of the IP₃R/ryanodine receptor superfamily of channels. Only 49 of these structures were complexed with IP₃. These were inspected for their ability to interact with the carbon-1 phosphate of IP₃, since this is the least accessible phosphate group of its precursor, phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂). This reduced the number of structures retrieved to 35, of which 9 were IP₃Rs. The remaining 26 structures represent a diverse range of proteins, including inositol-lipid metabolizing enzymes, signal transducers, PH domain containing proteins, cytoskeletal anchor proteins, the TRPV4 ion channel, a retroviral Gag protein and fibroblast growth factor 2. Such proteins may impact on IP₃ signalling and its effects on cell-biology. This represents an area open for exploration in the field of IP₃ signalling.     

Transient receptor potential melastatin 3 is a phosphoinositide-dependent ion channel

Phosphoinositides are emerging as general regulators of the functionally diverse transient receptor potential (TRP) ion channel family. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) has been reported to positively regulate many TRP channels, but in several cases phosphoinositide regulation is controversial. TRP melastatin 3 (TRPM3) is a heat-activated ion channel that is also stimulated by chemical agonists, such as pregnenolone sulfate. Here, we used a wide array of approaches to determine the effects of phosphoinositides on TRPM3. We found that channel activity in excised inside-out patches decreased over time (rundown), an attribute of PI(4,5)P₂-dependent ion channels. Channel activity could be restored by application of either synthetic dioctanoyl (diC₈) or natural arachidonyl stearyl (AASt) PI(4,5)P₂. The PI(4,5)P₂ precursor phosphatidylinositol 4-phosphate (PI(4)P) was less effective at restoring channel activity. TRPM3 currents were also restored by MgATP, an effect which was inhibited by two different phosphatidylinositol 4-kinase inhibitors, or by pretreatment with a phosphatidylinositol-specific phospholipase C (PI-PLC) enzyme, indicating that MgATP acted by generating phosphoinositides. In intact cells, reduction of PI(4,5)P₂ levels by chemically inducible phosphoinositide phosphatases or a voltage-sensitive 5′-phosphatase inhibited channel activity. Activation of PLC via muscarinic receptors also inhibited TRPM3 channel activity. Overall, our data indicate that TRPM3 is a phosphoinositide-dependent ion channel and that decreasing PI(4,5)P₂ abundance limits its activity. As all other members of the TRPM family have also been shown to require PI(4,5)P₂ for activity, our data establish PI(4,5)P₂ as a general positive cofactor of this ion channel subfamily.     

Acute and Chronic Ethanol Consumption Effects on the Immunolabeling of Gq/11α Subunit Protein and Phospholipase C Isozymes in the Rat Brain

Abstract: The goal of this investigation was to examine whether postreceptor sites [G_q/11 protein and phospholipase C (PLC) isozymes] of the phosphoinositide signal transduction system are involved in neuroadaptational mechanisms in the brain during chronic ethanol consumption. It was observed that acute ethanol treatment has no effect on the immunolabeling of PLC-β₁, -γ₁, and -δ₁ and the α subunit of G_q/11 protein in the rat cortex as determined by western blotting using specific monoclonal antibodies. On the other hand, chronic ethanol consumption (15 days) resulted in a significant decrease in the immunolabeling of PLC-β₁, whereas under identical conditions, the immunolabeling of PLC-γ₁ and -δ₁ isozymes was not significantly altered. The decreased immunolabeling of PLC-β₁ during chronic ethanol consumption was not altered by 24 h of withdrawal after 15 days of ethanol consumption. The immunolabeling of the α subunit of G_q/11 protein was significantly decreased after 15 days of ethanol consumption but had returned to normal levels after 24 h of ethanol withdrawal. Also, chronic ethanol treatment resulted in a significant decrease in phosphatidylinositol 4,5-bisphosphate-specific PLC activity, which remained the same after 24 h of ethanol withdrawal. These results suggest that decreased PLC activity during ethanol consumption and its withdrawal may be due to decreased protein levels of the G_q/11 protein-coupled PLC-β₁ isozyme but not the PLC-γ₁ or -δ₁ isozyme in the rat cortex. It is possible that changes in the protein levels of the G_q/11 protein-coupled PLC-β₁ isozyme and in PLC activity in the brain may be involved in the cellular adaptation to chronic ethanol exposure.     

Ca induces PI(4,5)P2 clusters on lipid bilayers at physiological PI(4,5)P2 and Ca concentrations

Calcium has been shown to induce clustering of PI(4,5)P₂ at high and non-physiological concentrations of both the divalent ion and the phosphatidylinositol, or on supported lipid monolayers. In lipid bilayers at physiological conditions, clusters are not detected through microscopic techniques. Here, we aimed to determine through spectroscopic methodologies if calcium plays a role in PI(4,5)P₂ lateral distribution on lipid bilayers under physiological conditions. Using several different approaches which included information on fluorescence quantum yield, polarization, spectra and diffusion properties of a fluorescent derivative of PI(4,5)P₂ (TopFluor(TF)-PI(4,5)P₂), we show that Ca^2 + promotes PI(4,5)P₂ clustering in lipid bilayers at physiological concentrations of both Ca^2 + and PI(4,5)P₂. Fluorescence depolarization data of TF-PI(4,5)P₂ in the presence of calcium suggests that under physiological concentrations of PI(4,5)P₂ and calcium, the average cluster size comprises ~ 15 PI(4,5)P₂ molecules. The presence of Ca^2 +-induced PI(4,5)P₂ clusters is supported by FCS data. Additionally, calcium mediated PI(4,5)P₂ clustering was more pronounced in liquid ordered (l_o) membranes, and the PI(4,5)P₂-Ca^2 + clusters presented an increased affinity for l_o domains. In this way, PI(4,5)P₂ could function as a lipid calcium sensor and the increased efficiency of calcium-mediated PI(4,5)P₂ clustering on l_o domains might provide targeted nucleation sites for PI(4,5)P₂ clusters upon calcium stimulus.     

Novel regulatory roles of PtdIns(4,5)P2 generating enzyme EhPIPKI in actin dynamics and phagocytosis of Entamoeba histolytica

Motility and phagocytosis are the two important processes that are intricately linked to survival and virulence potential of the protist parasite Entamoeba histolytica. These processes primarily rely on actin‐dependent pathways, and regulation of these pathways is critical for understanding the pathology of E. histolytica. Generally, phosphoinositides dynamics have not been explored in amoebic actin dynamics and particularly during phagocytosis in E. histolytica. We have explored the roles of PtdIns(4,5)P₂ as well as the enzyme that produces this metabolite, EhPIPKI during phagocytosis. Immunofluorescence and live cell images showed enrichment of EhPIPKI in different stages of phagocytosis from initiation till the cups progressed towards closure. However, the enzyme was absent after phagosomes are pinched off from the membrane. Overexpression of a dominant negative mutant revealed a reduction in the formation of phagocytic cups and inhibition in the rate of engulfment of erythrocytes. Moreover, EhPIPKI binds directly to F and G‐actin unlike PIPKs from other organisms. PtdIns(4,5)P₂, the product of the enzyme, also followed a similar distribution pattern during phagocytosis as determined by a GFP‐tagged PH‐domain from PLCδ, which specifically binds PtdIns(4,5)P₂ in trophozoites. In summary, EhPIPKI regulates initiation of phagocytosis by regulating actin dynamics.