Role Of PIP2 Research Articles

PIP2-Effector Protein MPRIP Regulates RNA Polymerase II Condensation and Transcription.

The specific post-translational modifications of the C-terminal domain (CTD) of the Rpb1 subunit of RNA polymerase II (RNAPII) correlate with different stages of transcription. The phosphorylation of the Ser5 residues of this domain associates with the initiation condensates, which are formed through liquid-liquid phase separation (LLPS). The subsequent Tyr1 phosphorylation of the CTD peaks at the promoter-proximal region and is involved in the pause-release of RNAPII. By implementing super-resolution microscopy techniques, we previously reported that the nuclear Phosphatidylinositol 4,5-bisphosphate (PIP2) associates with the Ser5-phosphorylated-RNAPII complex and facilitates the RNAPII transcription. In this study, we identified Myosin Phosphatase Rho-Interacting Protein (MPRIP) as a novel regulator of the RNAPII transcription that recruits Tyr1-phosphorylated CTD (Tyr1P-CTD) to nuclear PIP2-containing structures. The depletion of MPRIP increases the number of the initiation condensates, indicating a defect in the transcription. We hypothesize that MPRIP regulates the condensation and transcription through affecting the association of the RNAPII complex with nuclear PIP2-rich structures. The identification of Tyr1P-CTD as an interactor of PIP2 and MPRIP further points to a regulatory role in RNAPII pause-release, where the susceptibility of the transcriptional complex to leave the initiation condensate depends on its association with nuclear PIP2-rich structures. Moreover, the N-terminal domain of MPRIP, which is responsible for the interaction with the Tyr1P-CTD, contains an F-actin binding region that offers an explanation of how nuclear F-actin formations can affect the RNAPII transcription and condensation. Overall, our findings shed light on the role of PIP2 in RNAPII transcription through identifying the F-actin binding protein MPRIP as a transcription regulator and a determinant of the condensation of RNAPII.

Coarse-Grained Simulations Suggest Potential Competing Roles of Phosphoinositides and Amphipathic Helix Structures in Membrane Curvature Sensing of the AP180 N-Terminal Homology Domain.

The generation and sensing of membrane curvature by proteins has become of increasing interest to researchers with multiple mechanisms, from hydrophobic insertion to protein crowding, being identified. However, the role of charged lipids in the membrane curvature-sensing process is still far from understood. Many proteins involved in endocytosis bind phosphatidylinositol 4,5-bisphosphate (PIP2) lipids, allowing these proteins to accumulate at regions of local curvature. Here, using coarse-grained molecular dynamics simulations, we study the curvature-sensing behavior of the ANTH domain, a protein crucial for endocytosis. We selected three ANTH crystal structures containing either an intact, split, or truncated terminal amphipathic helix. On neutral membranes, the ANTH domain has innate curvature-sensing ability. In the presence of PIP2, however, only the domain with an intact helix senses curvature. Our work sheds light on the role of PIP2 and its modulation of membrane curvature sensing by proteins.

PIP2 regulation of TRPC5 channel activation and desensitization

Transient receptor potential canonical type 5 (TRPC5) ion channels are expressed in the brain and kidney and have been identified as promising therapeutic targets whose selective inhibition can protect against diseases driven by a leaky kidney filter, such as focal segmental glomerular sclerosis. TRPC5 channels are activated not only by elevated levels of extracellular Ca2+or lanthanide ions but also by G protein (Gq/11) stimulation. Phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis by phospholipase C enzymes leads to PKC-mediated phosphorylation of TRPC5 channels and their subsequent desensitization. However, the roles of PIP2 in activation and maintenance of TRPC5 channel activity via its hydrolysis product diacyl glycerol (DAG), as well as the mechanism of desensitization of TRPC5 activity by DAG-stimulated PKC activity, remain unclear. Here, we designed experiments to distinguish between the processes underlying channel activation and inhibition. Employing whole-cell patch-clamp, we used an optogenetic tool to dephosphorylate PIP2 and assess channel–PIP2 interactions influenced by activators, such as DAG, or inhibitors, such as PKC phosphorylation. Using total internal reflection microscopy, we assessed channel cell surface density. We show that PIP2 controls both the PKC-mediated inhibition and the DAG- and lanthanide-mediated activation of TRPC5 currents via control of gating rather than channel cell surface density. These mechanistic insights promise to aid in the development of more selective and precise inhibitors to block TRPC5 channel activity and illuminate new opportunities for targeted therapies for a group of chronic kidney diseases for which there is currently a great unmet need.

Phosphatidylinositol 4,5-bisphosphate (PIP2) and Ca2+ are both required to open the Cl− channel TMEM16A

Transmembrane member 16A (TMEM16A) is a widely expressed Ca2+-activated Cl- channel with various physiological functions ranging from mucosal secretion to regulating smooth muscle contraction. Understanding how TMEM16A controls these physiological processes and how its dysregulation may cause disease requires a detailed understanding of how cellular processes and second messengers alter TMEM16A channel gating. Here we assessed the regulation of TMEM16A gating by recording Ca2+-evoked Cl- currents conducted by endogenous TMEM16A channels expressed in Xenopus laevis oocytes, using the inside-out configuration of the patch clamp technique. During continuous application of Ca2+, we found that TMEM16A-conducted currents decay shortly after patch excision. Such current rundown is common among channels regulated by phosphatidylinositol 4,5-bisphosphate (PIP2). Thus, we sought to investigate a possible role of PIP2 in TMEM16A gating. Consistently, synthetic PIP2 rescued the current after rundown, and the application of PIP2 modulating agents altered the speed kinetics of TMEM16A current rundown. First, two PIP2 sequestering agents, neomycin and anti-PIP2, applied to the intracellular surface of excised patches sped up TMEM16A current rundown to nearly twice as fast. Conversely, rephosphorylation of phosphatidylinositol (PI) derivatives into PIP2 using Mg-ATP or inhibiting dephosphorylation of PIP2 using β-glycerophosphate slowed rundown by nearly 3-fold. Our results reveal that TMEM16A regulation is more complicated than it initially appeared; not only is Ca2+ necessary to signal TMEM16a opening, but PIP2 is also required. These findings improve our understanding of how the dysregulation of these pathways may lead to disease and suggest that targeting these pathways could have utility for potential therapies.

Phosphatidylinositol 4,5-bisphosphate drives Ca2+-independent membrane penetration by the tandem C2 domain proteins synaptotagmin-1 and Doc2β

Exocytosis mediates the release of neurotransmitters and hormones from neurons and neuroendocrine cells. Tandem C2 domain proteins in the synaptotagmin (syt) and double C2 domain (Doc2) families regulate exocytotic membrane fusion via direct interactions with Ca2+ and phospholipid bilayers. Syt1 is a fast-acting, low-affinity Ca2+ sensor that penetrates membranes upon binding Ca2+ to trigger synchronous vesicle fusion. The closely related Doc2β is a slow-acting, high-affinity Ca2+ sensor that triggers spontaneous and asynchronous vesicle fusion, but whether it also penetrates membranes is unknown. Both syt1 and Doc2β bind the dynamically regulated plasma membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2), but it is unclear whether PIP2 serves only as a membrane contact or enables specialized membrane-binding modes by these Ca2+ sensors. Furthermore, it has been shown that PIP2 uncaging can trigger rapid, syt1-dependent exocytosis in the absence of Ca2+ influx, suggesting that current models for the action of these Ca2+ sensors are incomplete. Here, using a series of steady-state and time-resolved fluorescence measurements, we show that Doc2β, like syt1, penetrates membranes in a Ca2+-dependent manner. Unexpectedly, we observed that PIP2 can drive membrane penetration by both syt1 and Doc2β in the absence of Ca2+, providing a plausible mechanism for Ca2+-independent, PIP2-dependent exocytosis. Quantitative measurements of penetration depth revealed that, in the presence of Ca2+, PIP2 drives Doc2β, but not syt1, substantially deeper into the membrane, defining a biophysical regulatory mechanism specific to this high-affinity Ca2+ sensor. Our results provide evidence of a novel role for PIP2 in regulating, and under some circumstances triggering, exocytosis.

Role of lipid inositol phosphatase SHIP-1 in Germinal Center B cell selection and function

Abstract Germinal Centers (GC) are sites within secondary lymphoid organs in which B cells undergo proliferation and differentiation. The PI3K signaling pathway is a central modulator of GC B cells (GCBC) selection and function. The PI3K pathway can be regulated by several enzymes including Src homology 2-containing inositol 5’-phosphatase (SHIP-1). SHIP-1 is known to be hyper-phosphorylated in GCBCs. Upon activation, SHIP-1 hydrolyzes phosphatidyl inositol triphosphate (PIP3) to generate phosphatidyl inositol 3,4 biphosphate (PIP2). PIP2 can bind to signaling proteins that are crucial to BCR signaling such as TAPP1/2 and Akt/PKB. To ascertain the role of SHIP-1 in survival and selection of GCBC we used a tamoxifen inducible Cre system to delete SHIP-1 from B cells during an ongoing GC response to NP-CGG. SHIP-1 deletion led to bi-modal expression of SHIP-1 in GCBC expressing intermediate (SHIP-1 int) and low levels of SHIP-1 (SHIP-1 lo) compared to WT controls (SHIP-1 hi). SHIP-1 int GCBC showed reduced phosphorylation of signaling proteins S6 and increased phosphorylation of Btk upon BCR crosslinking with anti-u antibody. SHIP-1 lo cells were un-responsive to BCR crosslinking and had the highest frequency of dead cells and Caspase 3 positive cells among all 3 groups. This data suggests that SHIP-1 expression and activity is crucial to GCBC signaling and survival. The role of PIP2 in regulating BCR signaling in GCBC remains to be elucidated.

PIP2 and Talin Join Forces to Activate Integrin.

Integrins are major players in cell adhesion and migration, and malfunctions in controlling their activity are associated with various diseases. Nevertheless, the details of integrin activation are not completely understood, and the role of lipids in the process is largely unknown. Herein, we show using atomistic molecular dynamics simulations that the interplay of phosphatidylinositol 4,5-bisphosphate (PIP2) and talin may directly alter the conformation of integrin αIIbβ3. Our results provide a new perspective on the role of PIP2 in integrin activation and indicate that the charged PIP2 lipid headgroup can perturb a clasp at the cytoplasmic face of the integrin heterodimer.

Phosphatidylinositol 4,5‐bisphosphate Activates the Cell Adhesion Molecule CD44 and Assembles a CD44‐Ezrin Heterotetramer Complex as Revealed by Small Angle neutron Scattering

The cell adhesion molecule CD44 regulates diverse cellular functions, including cell‐cell and cell‐matrix interaction, cell motility, migration, differentiation, and growth. In cells, CD44 co‐localizes with the membrane‐cytoskeleton adapter protein Ezrin, which links the CD44 assembled receptor signaling complexes to the cytoskeletal actin and organizes the spatial and temporal localization of signaling events. Here we report that the cytoplasmic tail of CD44 (CD44ct) is largely disordered and adopts an auto‐inhibited conformation, which prevents CD44ct from binding directly to activated Ezrin in solution. Binding to the signaling lipid phosphatidylinositol 4,5‐biphosphlate (PIP2) disrupts autoinhibition in CD44ct, and activates CD44ct to associate with Ezrin. Further, using contrast variation small angle neutron scattering, we show that PIP2 mediates the assembly of a specific hetero‐tetramer complex of CD44ct with Ezrin. This study reveals a novel auto‐regulation mechanism in the cytoplasmic tail of CD44 and the role of PIP2 in mediating the assembly of multimeric CD44ct‐Ezrin complexes. We hypothesize that polyvalent electrostatic interactions are responsible for the assembly of multimeric PIP2‐CD44‐Ezrin complexes.

Abstract 5: ApoAI Binds to Phosphatidylinositol 4,5 Bisphosphate (PIP2), Which is Exposed on the Cell Surface by Novel PIP2 Floppase Activity of ABCA1, and Promotes Cholesterol Efflux

Introduction: ApoAI-ABCA1 mediated nascent HDL assembly promotes cholesterol efflux and reverse cholesterol transport, but the mechanism of HDL assembly is unknown. Objective: To determine role of PIP2 in ApoAI-ABCA1 mediated nascent HDL assembly. Methods and Results: To determine which lipid species are directly bound by apoAI, a lipid-protein overlay assay was performed using PIPstrips and sphingostrips from Echelon Biosciences. ApoAI bound directly to phosphatidylinositol phosphates (PIPs) rather than PC, PS, PE, SM or cholesterol. This interaction was not solely electrostatic, as PIP3, the most negative charged PIP had less apoAI binding than various PIP2 species. ApoAI did not bind to other charged lipids such as sphingosine 1-P, PI(3)-P, PI(4)-P, and PI(5)-P. Surface plasmon resonance assays using immobilized apoAI or PIP2 were performed, showing that apoAI interacted reversibly with PI(4,5)P2 in a dose dependent manner at the nM concentration range. The central domain of apoAI (residues 44-185) was sufficient for PIP2 binding. In RAW264.7 macrophages, depletion of cellular PIPs by treatments with PI3K inhibitor or PTEN inhibitor decreased cholesterol efflux by 45±1.4% and 41±6.4% respectively (p<0.005). Degradation of cell surface PIP2 via treatment with PI specific PLC decreased cell surface binding of apoAI by 37±6% and cholesterol efflux by 48±8% (p<0.005). In mouse bone-marrow derived macrophages, cholesterol efflux to apoAI was increased ~47% by GRIP (PIP2 binding PH domain of PLCδ) and decreased 41±8% by PTEN inhibition (p<0.005). Liposomes made with POPC:POPS:FC:PIP2 (65:20:10:5 mole ratio) were solubilized by apoAI at 37 o C, pH5, but apoAI had little activity using similar liposomes without PIP2 . ABCA1 expression led to 2-fold higher cell surface PIP2, assayed by flow cytometry using a PIP2 specific antibody. RAW cells stably transfected with PIP2 binding domain 2X PH-PLCδ-eGFP showed enrichment of this PIP2 reporter on the plasma membrane, while expression of ABCA1 led to partial redistribution to cytoplasm, consistent with ABCA1 having PIP2 floppase activity. Conclusions: ApoAI has novel PIP2 binding activity, ABCA1 is a novel PIP2 floppase, and cell surface PIP2 is required for optimal cholesterol acceptor activity of apoAI.

Phosphatidylinositol 4,5-bisphosphate depletion fails to affect neurosteroid modulation of GABAA receptor function

Neurosteroids and likely other lipid modulators access transmembrane sites on the GABAA receptor (GABAAR) by partitioning into and diffusing through the plasma membrane. Therefore, specific components of the plasma membrane may affect the potency or efficacy of neurosteroid-like modulators. Here, we tested a possible role for phosphatidylinositol 4,5-bisphosphate (PIP2), a phospholipid that governs activity of many channels and transporters, in modulation or function of GABAARs. In these studies, we sought to deplete plasma-membrane PIP2 and probe for a change in the strength of potentiation by submaximal concentrations of the neurosteroid allopregnanolone (3α5αP) and other anesthetics, including propofol, pentobarbital, and ethanol. We also tested for a change in the behavior of negative allosteric modulators pregnenolone sulfate and dipicrylamine. We used Xenopus oocytes expressing the ascidian voltage-sensitive phosphatase (Ci-VSP) to deplete PIP2. Voltage pulses to positive membrane potentials were used to deplete PIP2 in Ci-VSP-expressing cells. GABAARs composed of α1β2γ2L and α4β2δ subunits were challenged with GABA and 3α5αP or other modulators before and after PIP2 depletion. KV7.1 channels and NMDA receptors (NMDARs) were used as positive controls to verify PIP2 depletion. We found no evidence that PIP2 depletion affected modulation of GABAARs by positive or negative allosteric modulators. By contrast, Ci-VSP-induced PIP2 depletion depressed KV7.1 activation and NMDAR activity. We conclude that despite a role for PIP2 in modulation of a wide variety of ion channels, PIP2 does not affect modulation of GABAARs by neurosteroids or related compounds.

Membrane Remodeling by Curvature-Inducing Proteins

A host of membrane-binding proteins are responsible for dynamically remodeling cell membranes during vital cell processes, including endocytosis, exocytosis, cell migration, and morphogenesis. Experiments show that the protein exo70, a component of the exocyst, generates membrane protrusions on cell surfaces. We employ coarse-grained molecular dynamics simulations to validate confocal microscopy results which show that dimerization of exo70 is necessary to induce sufficient curvature for tubular invaginations in synthetic vesicles. These simulations provide mechanistic insight into the molecular details of membrane bending by exo70. Calculation of the local three-dimensional stress tensor reveals the shape and depth of the resulting curvature field and this parameter informs a mesoscale for membrane bending which includes the anisotropy of the induced curvature field. We also probe the role of binding to phosphatidylinositol (4,5)bisphosphate (PIP2), a key membrane constituent which binds many other curvature-inducing proteins including epsin and the Bin-Amphyphsin-Rvs (BAR) domains. PIP2 modulates a number of cellular signaling pathways, and anchors curvature-inducing proteins to the membrane via electrostatic interactions. Our simulations also validate experiments which show that a double mutation of positively charged residues in exo70 disrupts the binding to negatively charged PIP2, abolishing membrane binding and the resulting tubular invaginations. These modeling methods are extended to other curvature-inducing proteins, including the epsin N-terminal homology domain (ENTH), which senses and drives membrane curvature during clathrin-mediated endocytosis. Our simulations provide a link between detailed all-atom simulations, which capture the physics of membrane-protein binding and the role of PIP2, with mesoscopic models for membrane sculpting. These simulations provide molecular details for membrane remodeling by exo70 observed by confocal microscopy in synthetic vesicles, and fluorescence microscopy in live cells.

The Role of PIP2 in the Voltage-Dependent Activation of Kv7.1

Phosphatidylinositol 4,5-bisphosphate (PIP2) is an important regulator of ion channel activity. While PIP2 modulation of voltage-independent Kir channels has been extensively studied, the mechanism of how PIP2 potentiates the activity of voltage-dependent ion channels remains uknown. In this work, we study PIP2 activation of a voltage-dependent potassium channel (Kv7.1). Kv7.1 channels have a canonical Kv structure with a central pore-gate domain (PGD) surrounded by four peripheral voltage-sensing domains (VSDs). Kv channel activation is thought to involve two general steps. First, membrane depolarization is sensed by gating charges in the VSDs resulting in conformational changes within the VSDs from the resting state to the activated state. After all four VSDs have been activated, the second general step in Kv activation involves a concerted motion during which the PGD opens to allow ion permeation. In this study we ask if PIP2 regulates activation of Kv7.1 by potentiating the early VSD conformational changes, the concerted opening of the PGD, or both. Using the voltage-clamp fluorometry (VCF) technique to assay local conformation changes in the VSDs and the PGD simultaneously, we are able to show that although depletion of PIP2 eliminates the ionic current, the early conformational changes in the VSD do not require PIP2. This result indicates that the later conformational changes that couple VSD activation to the opening of the PGD are eliminated by PIP2 depletion. We continue this work by dissecting the molecular details of why PIP2 is required for opening of the PGD in response to VSD activation. These results may provide insights on the common principle of how Kv channels are modulated by PIP2.

Membrane docking of the C2 domain from protein kinase Cα as seen by polarized ATR-IR. The role of PIP2

We have used attenuated total internal reflection infrared spectroscopy (ATR-IR) spectroscopy to study the association of the C2 domain from protein kinase Cα (PKCα) with different phospholipid membranes, so as to characterise the mode of membrane docking and its modulation by the second-messenger lipid PIP2. In parallel, we have also examined the membrane interaction of the C2 domain from cytosolic phospholipase A2. PIP2 did not induce significant changes in secondary structure of the membrane-bound PKCα-C2 domain, nor did binding of the PKCα-C2 domain change the dichroic ratios of the lipid chains, whereas the C2 domain from phospholipase A2 did perturb the lipid chain orientation. Measurements of the dichroic ratios for the amide I and amide II protein bands were combined so as to distinguish the tilt of the β-sheets from that of the β-strands within the sheet. When associated with POPC/POPS membranes, the β-sandwich of the PKCα-C2 domain is inclined at an angle α=35° to the membrane normal, i.e., is oriented more nearly perpendicular than parallel to the membrane. In the process of membrane docking, the tilt angle increases to α=44° in the presence of PIP2, indicating that the β-sandwich comes closer to the membrane surface, so confirming the importance of this lipid in determining docking of the C2 domain and consequent activation of PKCα.

Phosphatidylinositol 4,5-Bisphosphate-Induced Conformational Change of Ezrin and Formation of Ezrin Oligomers

The plasma membrane-cytoskeleton interface is a dynamic structure involved in a variety of cellular events. Ezrin, a protein from the ERM family, provides a direct linkage between the cytoskeleton and the membrane via its interaction with phosphatidylinositol 4,5-bisphosphate (PIP₂). In this paper, we investigate the interaction between PIP₂ and ezrin in vitro using PIP₂ dispersed in a unimolecular way in buffer. We compared the results obtained with full-length ezrin to those obtained with an ezrin mutant, which was previously found not to be localized at the cell membrane, and with the N-terminal membrane binding domain (FERM domain) of ezrin. We show that PIP₂ induced a conformational change in full-length ezrin. PIP₂ was also found to induce, in vitro, the formation of oligomers of wild-type ezrin, but not of mutant ezrin. These oligomers had previously been observed in vivo, but their role is yet to be clarified. Our finding hints at a possible role for PIP₂ in the formation of ezrin oligomers.

Role Of Pip2 On Ca2+-Dependent Desensitization of Trpv2

TRPV2 is a member of the transient receptor potential superfamily of ion channels involved in chemical and thermal pain transduction. Unlike the related TRPV1 channel, TRPV2 does not appear to bind either calmodulin or ATP in its N-terminal ankyrin repeat domain. In addition, it does not contain a calmodulin-binding site in the distal C-terminal region, as has been proposed for TRPV1. Importantly, though, we have found that TRPV2 undergoes Ca2+-dependent desensitization similar to TRPV1, suggesting that the mechanism of desensitization may be conserved in the two channels. To elucidate the molecular mechanism underlying Ca2+-dependent desensitization in TRPV2 we used whole-cell recordings of F-11 cells transiently transfected with TRPV2. We found that prolonged applications of the TRPV2 agonist 2-APB led to nearly complete desensitization of the channel in the presence of extracellular Ca2+. In contrast, no desensitization was observed in the absence of Ca2+. TRPV2 desensitization was not altered in whole-cell recordings in the presence of calmodulin inhibitors or upon co-expression of mutant calmodulin, suggesting that CaM does not play a major role in Ca2+-dependent desensitization of TRPV2. Interestingly, simultaneous confocal imaging and electrophysiological recording of whole cells expressing TRPV2 and a fluorescent PI(4,5)P2 binding probe showed a high degree of temporal correlation between the Ca2+ induced desensitization and depletion of PI(4,5)P2. Thus, Ca2+ influx through TRPV2 is sufficient to trigger a dramatic decrease in PI(4,5)P2 levels, presumably by activating PLC. We propose that the decrease in PI(4,5)P2 levels upon channel activation underlies at least a major component of Ca2+-dependent desensitization of TRPV2.

Inhibition of transient receptor potential A1 channel by phosphatidylinositol-4,5-bisphosphate

Membrane phosphatidylinositol-4,5-bisphosphate (PIP2) is critical for the function of many transient receptor potential (TRP) ion channels. The role of PIP2 in TRPA1 function is not well known. The effect of PIP2 on TRPA1 was investigated by direct application of PIP2 and by using polylysine and PIP2 antibody that sequester PIP2. In inside-out patches from HeLa cells expressing mouse TRPA1, polytriphosphate (PPPi) was added to the bath solution to keep TRPA1 sensitive to allyl isothiocyanate (AITC; mustard oil). Direct application of PIP2 (10 microM) to inside-out patches did not activate TRPA1, but AITC and Delta(9)-tetrahydrocannabinol (THC) produced strong activation. In inside-out patches in which TRPA1 was first activated with AITC (in the presence of PPPi), further addition of PIP2 produced a concentration-dependent inhibition of TRPA1 [agonist concentration producing half-maximal activity (K(1/2)), 2.8 microM]. Consistent with the inhibition of TRPA1 by PIP2, AITC activated a large whole cell current when polylysine or PIP2 antibody was added to the pipette but a markedly diminished current when PIP2 was added to the pipette. In inside-out patches with PPPi in the bath solution, application of PIP2 antibody or polylysine caused activation of TRPA1, and this was blocked by PIP2. However, TRPA1 was not activated by polylysine and PIP2 antibody under whole cell conditions, suggesting a more complex regulation of TRPA1 by PIP2 in intact cells. These results show that PIP2 inhibits TRPA1 and reduces the sensitivity of TRPA1 to AITC.

Dissection of the components for PIP2 activation and thermosensation in TRP channels.

Phosphatidylinositol 4,5-bisphosphate (PIP2) plays a central role in the activation of several transient receptor potential (TRP) channels. The role of PIP2 on temperature gating of thermoTRP channels has not been explored in detail, and the process of temperature activation is largely unexplained. In this work, we have exchanged different segments of the C-terminal region between cold-sensitive (TRPM8) and heat-sensitive (TRPV1) channels, trying to understand the role of the segment in PIP2 and temperature activation. A chimera in which the proximal part of the C-terminal of TRPV1 replaces an equivalent section of TRPM8 C-terminal is activated by PIP2 and confers the phenotype of heat activation. PIP2, but not temperature sensitivity, disappears when positively charged residues contained in the exchanged region are neutralized. Shortening the exchanged segment to a length of 11 aa produces voltage-dependent and temperature-insensitive channels. Our findings suggest the existence of different activation domains for temperature, PIP2, and voltage. We provide an interpretation for channel-PIP2 interaction using a full-atom molecular model of TRPV1 and PIP2 docking analysis.

Role of Na/Ca exchange and the plasma membrane Ca 2+ ‐ATPase in cell function

The Fifth International Conference on Na/Ca Exchange took place between 23 and 27 August 2006 in Brussels, Belgium, and was organized by A. Herchuelz. Image taken from the meeting poster showing hypothetical models for the Na/Ca exchanger (left) and the plasma membrane Ca2+‐ATPase (right), courtesy of M. Hilge. ![][1] Cytosolic Ca2+ regulates several cellular processes and its concentration is, in turn, finely regulated by various channels, pumps and exchangers. The Na/Ca exchanger (NCX) and the plasma membrane Ca2+‐ATPase (PMCA) pump are two concurrent mechanisms for Ca2+ extrusion from the cell (Carafoli, 1994; Blaustein & Lederer, 1999; Philipson & Nicoll, 2000; Strehler & Treiman, 2004; Prasad et al , 2004). PMCA and NCX were cloned 17 and 19 years ago, respectively; four mammalian isoforms have been identified for the former (PMCA1–4) and three have been identified for the latter (NCX1–3; Carafoli, 1994; Philipson & Nicoll, 2000). Na/Ca exchange (NCX) is a unique mechanism that allows Ca2+ extrusion from the cell against its gradient without energy consumption. Indeed, it is the entry of Na+ along its electrochemical gradient that provides the energy for Ca2+ extrusion. In addition, because NCX is electrogenic and voltage sensitive, it can reverse during cellular activation and contribute to Ca2+ entry into the cell (Blaustein & Lederer, 1999). Recently, the crucial role of NCX in cellular Ca2+ homeostasis has been definitively shown by the generation of mice with functional destruction of one Ncx gene. Homozygous Ncx1 ‐deficient mice are not viable, and die between embryonic days 9 and 10, owing to the lack of development of the cardiovascular system (Wakimoto et al , 2000); therefore, NCX is essential for life. PMCA belongs to the P‐type family of transport ATPases, which form a phosphorylated intermediate … [1]: /embed/graphic-1.gif

From genotype to phenotype and back

From genotype to phenotype and back

PIP5Kγ-Null Mutation Induces Cytoskeletal Changes within Megakaryocytes.

All eukaryotic cells contain the phospholipid phosphatidylinositol-4, 5-bisphosphate (PIP2) that serves multiple roles in signaling cascades critical for actin dynamics. Phosphatidylinositol-4-phosphate 5 kinase (PIP5K) catalyzes the synthesis of PIP2 by phosphorylating PI4P. Although the classical PIP5K isoforms (α, β, and γ) generate the same phospholipid product, the isoforms have different primary structures, are expressed at different levels in various tissues, and localize in different intracellular compartments. Therefore, it is likely that the functions of these isoforms are different as well. PIP5Kγ is unique because it associates directly with talin and consequently localizes in focal adhesion. Given the role of PIP2 in actin dynamics, we generated a murine line containing a null mutation in the PIP5Kγ gene. We found that the targeted disruption of PIP5Kγ results in early prenatal mortality that is associated with pleotropic effects involving the cardiovascular, neurologic, and hematopoietic systems. This early lethality prevented studies of hematopoietic cells derived from the bone marrow or the liver. However, we were able to analyze yolk sac progenitor cells that were treated with thrombopoietin ex vivo, differentiating them into megakaryocytes. After 5 days in culture, many of the non-adherent yolk sac progenitor cells were multinucleated, and approximately 70% expressed CD41 (αIIb), a marker of the megakaryocyte lineage. We then examined their cytoskeletal content and dynamics. Absence of PIP5Kγ had no effect on the quantity of F-actin in unstimulated or thrombin-stimulated cells that were adherent to fibrinogen or were maintained in suspension in the absence of stimulation. Next, we examined membrane dynamics during cell adhesion in real time using spinning disk video confocal microscopy. Wild type megakaryocytes actively formed and contracted lamellipodia and rapidly spread upon the fibrinogen matrix. In contrast, PIP5Kγ-null megakaryocytes continuously extended and retracted membrane blebs, rather than lamellipodia, but eventually spread as much as wild type cells. This observation is consistent with the previous suggestion that PIP2 contributes to the stable association of the membrane with the cytoskeleton. It is also noteworthy that a similar phenotype has been described in cells with defective anchoring of the membrane to the cytoskeleton because of a lack of filamin. Accordingly, our data are consistent with the hypothesis that PIP5Kγ generates compartmentalized pools of PIP2 that contribute to the association of the membrane with the cytoskeleton.