Membrane PIP2 Research Articles

A high-avidity biosensor reveals plasma membrane PI(3,4)P2 is predominantly a class I PI3K signaling product.

Class I phosphoinositide 3-OH kinase (PI3K) signaling is central to animal growth and metabolism, and pathological disruption of this pathway affects cancer and diabetes. However, the specific spatial/temporal dynamics and signaling roles of its minor lipid messenger, phosphatidylinositol (3,4)-bisphosphate (PI(3,4)P2), are not well understood. This owes principally to a lack of tools to study this scarce lipid. Here we developed a high-sensitivity genetically encoded biosensor for PI(3,4)P2, demonstrating high selectivity and specificity of the sensor for the lipid. We show that despite clear evidence for class II PI3K in PI(3,4)P2-driven function, the overwhelming majority of the lipid accumulates through degradation of class I PI3K-produced PIP3 However, we show that PI(3,4)P2 is also subject to hydrolysis by the tumor suppressor lipid phosphatase PTEN. Collectively, our results show that PI(3,4)P2 is potentially an important driver of class I PI3K-driven signaling and provides powerful new tools to begin to resolve the biological functions of this lipid downstream of class I and II PI3K.

Pore- and voltage sensor-targeted KCNQ openers have distinct state-dependent actions.

Ion channels encoded by KCNQ2-5 generate a prominent K+ conductance in the central nervous system, referred to as the M current, which is controlled by membrane voltage and PIP2. The KCNQ2-5 voltage-gated potassium channels are targeted by a variety of activating compounds that cause negative shifts in the voltage dependence of activation. The underlying pharmacology of these effects is of growing interest because of possible clinical applications. Recent studies have revealed multiple binding sites and mechanisms of action of KCNQ activators. For example, retigabine targets the pore domain, but several compounds have been shown to influence the voltage-sensing domain. An important unexplored feature of these compounds is the influence of channel gating on drug binding or effects. In the present study, we compare the state-dependent actions of retigabine and ICA-069673 (ICA73, a voltage sensor-targeted activator). We assess drug binding to preopen states by applying drugs to homomeric KCNQ2 channels at different holding voltages, demonstrating little or no association of ICA73 with resting states. Using rapid solution switching, we also demonstrate that the rate of onset of ICA73 correlates with the voltage dependence of channel activation. Retigabine actions differ significantly, with prominent drug effects seen at very negative holding voltages and distinct voltage dependences of drug binding versus channel activation. Using similar approaches, we investigate the mechanistic basis for attenuation of ICA73 actions by the voltage-sensing domain mutation KCNQ2[A181P]. Our findings demonstrate different state-dependent actions of pore- versus voltage sensor-targeted KCNQ channel activators, which highlight that subtypes of this drug class operate with distinct mechanisms.

Metabotropic Acetylcholine and Glutamate Receptors Mediate PI(4,5)P2 Depletion and Oscillations in Hippocampal CA1 Pyramidal Neurons in situ

The sensitivity of many ion channels to phosphatidylinositol-4,5-bisphosphate (PIP2) levels in the cell membrane suggests that PIP2 fluctuations are important and general signals modulating neuronal excitability. Yet the PIP2 dynamics of central neurons in their native environment remained largely unexplored. Here, we examined the behavior of PIP2 concentrations in response to activation of Gq-coupled neurotransmitter receptors in rat CA1 hippocampal neurons in situ in acute brain slices. Confocal microscopy of the PIP2-selective molecular sensors tubbyCT-GFP and PLCδ1-PH-GFP showed that pharmacological activation of muscarinic acetylcholine (mAChR) or group I metabotropic glutamate (mGluRI) receptors induces transient depletion of PIP2 in the soma as well as in the dendritic tree. The observed PIP2 dynamics were receptor-specific, with mAChR activation inducing stronger PIP2 depletion than mGluRI, whereas agonists of other Gαq-coupled receptors expressed in CA1 neurons did not induce measureable PIP2 depletion. Furthermore, the data show for the first time neuronal receptor-induced oscillations of membrane PIP2 concentrations. Oscillatory behavior indicated that neurons can rapidly restore PIP2 levels during persistent activation of Gq and PLC. Electrophysiological responses to receptor activation resembled PIP2 dynamics in terms of time course and receptor specificity. Our findings support a physiological function of PIP2 in regulating electrical activity.

Regulation of PI4P levels by PI4KIIIα during G-protein-coupled PLC signaling in Drosophila photoreceptors.

The activation of phospholipase C (PLC) is a conserved mechanism of receptor-activated cell signaling at the plasma membrane. PLC hydrolyzes the minor membrane lipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], and continued signaling requires the resynthesis and availability of PI(4,5)P2 at the plasma membrane. PI(4,5)P2 is synthesized by the phosphorylation of phosphatidylinositol 4-phosphate (PI4P). Thus, a continuous supply of PI4P is essential to support ongoing PLC signaling. While the enzyme PI4KA has been identified as performing this function in cultured mammalian cells, its function in the context of an in vivo physiological model has not been established. In this study, we show that, in Drosophila photoreceptors, PI4KIIIα activity is required to support signaling during G-protein-coupled PLC activation. Depletion of PI4KIIIα results in impaired electrical responses to light, and reduced plasma membrane levels of PI4P and PI(4,5)P2 Depletion of the conserved proteins Efr3 and TTC7 [also known as StmA and L(2)k14710, respectively, in flies], which assemble PI4KIIIα at the plasma membrane, also results in an impaired light response and reduced plasma membrane PI4P and PI(4,5)P2 levels. Thus, PI4KIIIα activity at the plasma membrane generates PI4P and supports PI(4,5)P2 levels during receptor activated PLC signaling.

Multivalent Cation-Bridged PI(4,5)P2 Clusters Form at Very Low Concentrations

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2 or PIP2), is a key component of the inner leaflet of the plasma membrane in eukaryotic cells. In model membranes, PIP2 has been reported to form clusters, but whether these locally different conditions could give rise to distinct pools of unclustered and clustered PIP2 is unclear. By use of both fluorescence self-quenching and Förster resonance energy transfer assays, we have discovered that PIP2 self-associates at remarkably low concentrations starting below 0.05 mol% of total lipids. Formation of these clusters was dependent on physiological divalent metal ions, such as Ca2+, Mg2+, Zn2+, or trivalent ions Fe3+ and Al3+. Formation of PIP2 clusters was also headgroup-specific, being largely independent of the type of acyl chain. The similarly labeled phospholipids phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol exhibited no such clustering. However, six phosphoinositide species coclustered with PIP2. The degree of PIP2 cation clustering was significantly influenced by the composition of the surrounding lipids, with cholesterol and phosphatidylinositol enhancing this behavior. We propose that PIP2 cation-bridged cluster formation, which might be similar to micelle formation, can be used as a physical model for what could be distinct pools of PIP2 in biological membranes. To our knowledge, this study provides the first evidence of PIP2 forming clusters at such low concentrations. The property of PIP2 to form such clusters at such extremely low concentrations in model membranes reveals, to our knowledge, a new behavior of PIP2 proposed to occur in cells, in which local multivalent metal ions, lipid compositions, and various binding proteins could greatly influence PIP2 properties. In turn, these different pools of PIP2 could further regulate cellular events.

NEDD4-induced degradative ubiquitination of phosphatidylinositol 4-phosphate 5-kinase α and its implication in breast cancer cell proliferation.

Phosphatidylinositol 4‐phosphate 5‐kinase (PIP5K) family members generate phosphatidylinositol 4,5‐bisphosphate (PIP2), a critical lipid regulator of diverse physiological processes. The PIP5K‐dependent PIP2 generation can also act upstream of the oncogenic phosphatidylinositol 3‐kinase (PI3K)/Akt pathway. Many studies have demonstrated various mechanisms of spatiotemporal regulation of PIP5K catalytic activity. However, there are few studies on regulation of PIP5K protein stability. Here, we examined potential regulation of PIP5Kα, a PIP5K isoform, via ubiquitin‐proteasome system, and its implication for breast cancer. Our results showed that the ubiquitin ligase NEDD4 (neural precursor cell expressed, developmentally down‐regulated gene 4) mediated ubiquitination and proteasomal degradation of PIP5Kα, consequently reducing plasma membrane PIP2 level. NEDD4 interacted with the C‐terminal region and ubiquitinated the N‐terminal lysine 88 in PIP5Kα. In addition, PIP5Kα gene disruption inhibited epidermal growth factor (EGF)‐induced Akt activation and caused significant proliferation defect in breast cancer cells. Notably, PIP5Kα K88R mutant that was resistant to NEDD4‐mediated ubiquitination and degradation showed more potentiating effects on Akt activation by EGF and cell proliferation than wild‐type PIP5Kα. Collectively, these results suggest that PIP5Kα is a novel degradative substrate of NEDD4 and that the PIP5Kα‐dependent PIP2 pool contributing to breast cancer cell proliferation through PI3K/Akt activation is negatively controlled by NEDD4.

A PKC-MARCKS-PI3K regulatory module links Ca2+ and PIP3 signals at the leading edge of polarized macrophages

The leukocyte chemosensory pathway detects attractant gradients and directs cell migration to sites of inflammation, infection, tissue damage, and carcinogenesis. Previous studies have revealed that local Ca2+ and PIP3 signals at the leading edge of polarized leukocytes play central roles in positive feedback loop essential to cell polarization and chemotaxis. These prior studies showed that stimulation of the leading edge Ca2+ signal can strongly activate PI3K, thereby triggering a larger PIP3 signal, but did not elucidate the mechanistic link between Ca2+ and PIP3 signaling. A hypothesis explaining this link emerged, postulating that Ca2+-activated PKC displaces the MARCKS protein from plasma membrane PIP2, thereby releasing sequestered PIP2 to serve as the target and substrate lipid of PI3K in PIP3 production. In vitro single molecule studies of the reconstituted pathway on lipid bilayers demonstrated the feasibility of this PKC-MARCKS-PI3K regulatory module linking Ca2+ and PIP3 signals in the reconstituted system. The present study tests the model predictions in live macrophages by quantifying the effects of: (a) two pathway activators—PDGF and ATP that stimulate chemoreceptors and Ca2+ influx, respectively; and (b) three pathway inhibitors—wortmannin, EGTA, and Go6976 that inhibit PI3K, Ca2+ influx, and PKC, respectively; on (c) four leading edge activity sensors—AKT-PH-mRFP, CKAR, MARCKSp-mRFP, and leading edge area that report on PIP3 density, PKC activity, MARCKS membrane binding, and leading edge expansion/contraction, respectively. The results provide additional evidence that PKC and PI3K are both essential elements of the leading edge positive feedback loop, and strongly support the existence of a PKC-MARCKS-PI3K regulatory module linking the leading edge Ca2+ and PIP3 signals. As predicted, activators stimulate leading edge PKC activity, displacement of MARCKS from the leading edge membrane and increased leading edge PIP3 levels, while inhibitors trigger the opposite effects. Comparison of the findings for the ameboid chemotaxis of leukocytes with recently published findings for the mesenchymal chemotaxis of fibroblasts suggests that some features of the emerging leukocyte leading edge core pathway (PLC-DAG-Ca2+-PKC-MARCKS-PIP2-PI3K-PIP3) may well be shared by all chemotaxing eukaryotic cells, while other elements of the leukocyte pathway may be specialized features of these highly optimized, professional gradient-seeking cells. More broadly, the findings suggest a molecular mechanism for the strong links between phospho-MARCKS and many human cancers.

Molecular Mechanism of Voltage-Gated Ca2+ Channel Regulation by Membrane PIP2

Voltage-gated calcium (CaV) channel, CaV β subunit, phosphatidylinositol 4,5-bisphosphate (PIP2), molecular gating mechanism

Optically sensing phospholipid induced coil-helix transitions in the phosphoinositide-binding motif of gelsolin.

We present a systematic experimental and computational study of phospholipid induced peptide coil-helix transitions which are relevant in the context of proteins mediating cytoskeletal rearrangement via membrane binding. We developed a sensitive Förster resonance energy transfer (FRET) based assay to address whether coil-helix transitions in phospholipid binding motifs of actin-binding proteins can be induced by physiologically-relevant concentrations (1-20 μM) of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) phospholipids. Based on inter-residue distance constraints obtained from Molecular Dynamics (MD) simulations of a 20 residue peptide (Gel 150-169) from the actin-severing protein gelsolin, we synthetized and labeled the peptide with a tryptophan donor and IAEDANS acceptor pair. Upon addition of PI(4,5)P2 micelles and mixed vesicles containing PI(4,5)P2 and phosphatidylcholine to the peptide, we observed a decrease in the tryptophan emission intensity with increasing concentrations of PI(4,5)P2. The IAEDANS emission spectra showed a more complex profile exhibiting a blue shift of the emission peak and non-monotonic changes in the intensity profile with increasing concentrations of PI(4,5)P2. We showed that the IAEDANS acceptor emission response is a result of both intrinsic polarity sensitivity of the acceptor in the vicinity of the membrane surface and fluorescence energy transfer from the donor. Importantly, the fluorescence lifetime of the donor (tryptophan) showed a monotonous decrease with increasing mol% of PI(4,5)P2 from 1.13 ± 0.10 ns in the absence of phospholipids to 0.25 ± 0.03 ns in the presence of 100% PI(4,5)P2 micelles. We also showed a concomitant increase in FRET efficiency with increasing PI(4,5)P2 levels indicating a PI(4,5)P2 concentration dependent coil-helix transition. Our studies demonstrate that membrane PI(4,5)P2 concentrations as low as 2.5-5 μM can trigger helix-coil conformational changes in gelsolin relevant for triggering regulatory processes in the cell.

Phosphatidylinositol 4,5-bisphosphate, cholesterol, and fatty acids modulate the calcium-activated chloride channel TMEM16A (ANO1)

The TMEM16A-mediated Ca2+-activated Cl− current drives several important physiological functions. Membrane lipids regulate ion channels and transporters but their influence on members of the TMEM16 family is poorly understood. Here we have studied the regulation of TMEM16A by phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), cholesterol, and fatty acids using patch clamp, biochemistry and fluorescence microscopy. We found that depletion of membrane PI(4,5)P2 causes a decline in TMEM16A current that is independent of cytoskeleton, but is partially prevented by removing intracellular Ca2+. On the other hand, supplying PI(4,5)P2 to inside-out patches attenuated channel rundown and/or partially rescued activity after channel rundown. Also, depletion (with methyl-β-cyclodextrin M-βCD) or restoration (with M-βCD+cholesterol) of membrane cholesterol slows down the current decay observed after reduction of PI(4,5)P2. Neither depletion nor restoration of cholesterol change PI(4,5)P2 content. However, M-βCD alone transiently increases TMEM16A activity and dampens rundown whereas M-βCD+cholesterol increases channel rundown. Thus, PI(4,5)P2 is required for TMEM16A function while cholesterol directly and indirectly via a PI(4,5)P2-independent mechanism regulate channel function. Stearic, arachidonic, oleic, docosahexaenoic, and eicosapentaenoic fatty acids as well as methyl stearate inhibit TMEM16A in a dose- and voltage-dependent manner. Phosphatidylserine, a phospholipid whose hydrocarbon tails contain stearic and oleic acids also inhibits TMEM16A. Finally, we show that TMEM16A remains in the plasma membrane after treatment with M-βCD, M-βCD+cholesterol, oleic, or docosahexaenoic acids. Thus, we propose that lipids and fatty acids regulate TMEM16A channels through a membrane-delimited protein-lipid interaction.

Compartmentalized Gαq-Signaling in Adult Cardiac Myocytes

Gαq-coupled G-protein receptors (Gq-receptors), including α1-adrenergic and angiotensin receptors (α1-AR, AT-R), activate cardiac signaling pathways controlling hypertrophy, cell survival/death, and inotropy. Early studies in cell and animal models established the convention that Gq-signaling exacerbates heart failure (HF). However, clinically, AT-R antagonists improve HF outcomes, whereas α1-AR antagonists worsen HF, suggesting Gq-receptors are functionally unique. Our work indicates that α1-ARs are unique cardiac Gq-receptors by inducing cardioprotection and localizing to the nucleus in adult cardiac myocytes, suggesting that subcellular localization might form the mechanistic basis for unique Gq-receptor function. Here, we test the hypothesis that α1-ARs and AT-Rs localize to and initiate proximal signaling in distinct subcellular compartments. Using a novel fluorescent α1-ligand that improves the dynamics of defining receptor localization, we detected α1-ARs only at the nucleus in adult cardiac myocytes and isolated nuclei. Conversely, using a fluorescent AT-R ligand, we detected AT-Rs at the sarcolemma and the t-tubules. To measure proximal Gq-receptor signaling, we employed a fluorescent probe, GFP-PHD, that binds phosphatidylinositol-4,5-bisphosphate (PIP2) in the membrane and is released when phospholipase Cβ (PLCβ) cleaves PIP2. In adult cardiac myocytes, GFP-PHD localized to the sarcolemma and t-tubules, but not the nucleus, consistent with previous reports. Angiotensin, but not phenylephrine, induced movement of GFP-PHD off of the sarcolemma and t-tubules. The failure to detect GFP-PHD at the nucleus indicates that either PIP2 is excluded from nuclear membranes or that the probe fails to target the nucleus. Addition of a nuclear localization sequence to GFP-PHD (NLS-GFP-PHD) produced nuclear localization of GFP-PHD suggesting the presence of PIP2 in nuclear membranes in adult cardiac myocytes. Phenylephrine, and to a lesser extent, angiotensin induced movement of NLS-GFP-PHD off the nuclear membrane. In summary, our data demonstrate that α1-ARs and AT-Rs localize to and initiate proximal signaling in unique subcellular compartments.

Control of actin polymerization via the coincidence of phosphoinositides and high membrane curvature.

The conditional use of actin during clathrin-mediated endocytosis in mammalian cells suggests that the cell controls whether and how actin is used. Using a combination of biochemical reconstitution and mammalian cell culture, we elucidate a mechanism by which the coincidence of PI(4,5)P2 and PI(3)P in a curved vesicle triggers actin polymerization. At clathrin-coated pits, PI(3)P is produced by the INPP4A hydrolysis of PI(3,4)P2, and this is necessary for actin-driven endocytosis. Both Cdc42⋅guanosine triphosphate and SNX9 activate N-WASP-WIP- and Arp2/3-mediated actin nucleation. Membrane curvature, PI(4,5)P2, and PI(3)P signals are needed for SNX9 assembly via its PX-BAR domain, whereas signaling through Cdc42 is activated by PI(4,5)P2 alone. INPP4A activity is stimulated by high membrane curvature and synergizes with SNX9 BAR domain binding in a process we call curvature cascade amplification. We show that the SNX9-driven actin comets that arise on human disease-associated oculocerebrorenal syndrome of Lowe (OCRL) deficiencies are reduced by inhibiting PI(3)P production, suggesting PI(3)P kinase inhibitors as a therapeutic strategy in Lowe syndrome.

Defining the minimum substrate and charge recognition model of gamma-secretase

γ-Secretase is an intramembrane aspartyl protease that cleaves the C99 fragment of amyloid precursor protein to generate extracellular Aβ peptides. These peptides can oligomerize and aggregate to form amyloid plaques, processes that are widely believed to be causal for Alzheimer's disease. In spite of this critical function, it remains unknown how γ-secretase recognizes C99 and its other substrates, including Notch. In this study we determined E22-K55 as the minimal C99 fragment that was sufficient and required for initial cleavage. Within this fragment, we identified four determinants: (i) a transferable extracellular determinant that differed between C99 and Notch, and which included negative charge in the case of C99, (ii) the amino acid sequence of the C-terminal half of the transmembrane helix, (iii) an invariant lysine or arginine at the intracellular membrane border, and (iv) a positive charge cluster that included the invariant lysine/arginine. We demonstrated that the charge clusters of C99 and Notch receptors could directly bind phosphatidylinositol 4,5-bisphosphate (PIP2). The PIP2-binding cluster was required for γ-secretase cleavage, and modulation of membrane PIP2 levels strongly affected γ-secretase cleavage levels and the Aβ40/Aβ42 ratio, providing support for the importance of the PIP2 interaction in cells. Together, these studies provide critically needed insight into substrate recognition by γ-secretase.

Phosphatidylinositol (4,5)-bisphosphate dynamically regulates the K2P background K+ channel TASK-2

Two-pore domain K2P K+ channels responsible for the background K+ conductance and the resting membrane potential, are also finely regulated by a variety of chemical, physical and physiological stimuli. Hormones and transmitters acting through Gq protein-coupled receptors (GqPCRs) modulate the activity of various K2P channels but the signalling involved has remained elusive, in particular whether dynamic regulation by membrane PI(4,5)P2, common among other classes of K+ channels, affects K2P channels is controversial. Here we show that K2P K+ channel TASK-2 requires PI(4,5)P2 for activity, a dependence that accounts for its run down in the absence of intracellular ATP and its full recovery by addition of exogenous PI(4,5)P2, its inhibition by low concentrations of polycation PI scavengers, and inhibition by PI(4,5)P2 depletion from the membrane. Comprehensive mutagenesis suggests that PI(4,5)P2 interaction with TASK-2 takes place at C-terminus where three basic aminoacids are identified as being part of a putative binding site.

KCNQ-SMIT complex formation facilitates ion channel-solute transporter cross talk.

Voltage-gated potassium channels formed by KCNQ2 and KCNQ3 are essential for normal neuronal excitability. KCNQ2/3 channel activity is augmented in vivo by phosphatidylinositol 4,5-bisphosphate (PIP2), which is generated from myo-inositol, an osmolyte transported into cells by sodium-dependent myo-inositol transporters (SMITs). Here, we discovered that KCNQ2/3 channels isoform-specifically colocalize with SMIT1 and SMIT2 at sciatic nerve nodes of Ranvier and in axon initial segments, and form channel-transporter complexes in vitro and in vivo KCNQ2/3 coexpression protected SMIT1 activity from the otherwise inhibitory effects of cellular depolarization imposed by elevating extracellular [K+], and KCNQ2 was required for potentiation of SMIT activity by myo-inositol preincubation. Cytoskeletal disruption, which speeds PIP2 dispersion, attenuated potentiation of KCNQ2/3 currents by SMIT1-mediated myo-inositol uptake, suggesting close channel-transporter juxtaposition ensures KCNQ2/3 exposure to locally high myo-inositol-derived PIP2 concentrations. Thus, KCNQ2/3-SMIT1/2 coassembly permits cross talk via physical interaction, and may also be required for optimal, reciprocal indirect regulation via membrane potential and PIP2, especially within the specialized architecture of axons.-Neverisky, D. L., Abbott, G. W. KCNQ-SMIT complex formation facilitates ion channel-solute transporter cross talk.

Protein Kinase A-Dependent Phosphorylation Modulates the Affinity of Kv7.5 Potassium Channels to Phosphatidylinositol 4,5-Bisphosphate to Enhance Potassium Conductance

PKA-dependent phosphorylation of Kv7.5 potassium channels was previously found to enhance Kv7.5 currents, but the mechanism of the enhancement by PKA as well as putative PKA phosphorylation sites remained unknown. Activity of all known Kv7 channels critically depends on the presence of a minor membrane phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP2). In the present study, we tested the hypothesis that PKA-dependent phosphorylation increases the affinity of Kv7.5 potassium channels to PIP2. Application of wortmannin (10 µM), an inhibitor of PI-4 kinase that decreases the level of membrane PIP2, caused time-dependent inhibition of the endogenous Kv7.5 current in A7r5 rat aortic smooth muscle cells. Activation of PKA by co-application of forskolin (activator of adenylyl cyclase, 1 µM) with IBMX (phosphodiesterase inhibitor, 500 µM) significantly attenuated the rate and total amount of wortmannin-induced inhibition of endogenous Kv7.5 current. Co-expression of ci-VSP, a voltage dependent phosphatase that dephosphorylates PIP2, with human Kv7.5 (hKv7.5) in A7r5 cells caused pronounced voltage and time-dependent inhibition of hKv7.5 current at membrane voltages positive to 0 mV. Enhancement of the hKv7.5 current observed in the presence of forskolin (10 µM) and IBMX (500 µM) occurred in parallel with the positive shift of the ci-VSP-induced voltage-dependent inhibition of the Kv7.5 current. In the presence of forskolin and IBMX, ci-VSP induced inhibition of the Kv7.5 current at voltages positive to +60 mV. To identify PKA phosphorylation sites we mutated predicted PKA phosphorylation sites S406 and S412 (located near the putative PIP2 binding domain on the C-terminus of the Kv7.5 channels) to alanine. Currents through both mutant channels Kv7.5 S406A and Kv7.5 S412A were enhanced by co-application of forskolin with IBMX to the same degree as wild type Kv7.5 current. Thus, phosphorylation of S406 and S412 cannot account for PKA-dependent Kv7.5 current enhancement. Overall, experimental results support our hypothesis that enhancement of Kv7.5 currents caused by PKA activation is associated with increased affinity of the Kv7.5 potassium channels to PIP2.

Nanosecond pulsed electric field induced dose dependent phosphatidylinositol-4,5-bisphosphate signaling and intracellular electro-sensitization

Previously, it was demonstrated that nanometer-sized pores (nanopores) are formed in outer cellular membranes after exposure to nanosecond electric pulses (nsEPs). We reported that plasma membrane nanoporation affects phospholipids of the cell membrane, culminating in cascading phosphoinositide phosphatidylinositol-4,5-bisphosphate (PIP2) intracellular signaling. In the current study, we show that nsEPs initiated electric field (EF) dose-dependent PIP2 hydrolysis and/or depletion from the plasma membrane. This process was confirmed using fluorescent optical probes of PIP2 hydrolysis: PLCδ-PH-EGFP and GFP-C1-PKCγ-C1a. The 50% maximum response occurs with a single 600ns pulse achieving an effective dose (ED50) of EF~8kV/cm within our model cell system. At 16.2kV/cm, the ED50 for the pulse width was 484ns. Reduction of the pulse width or EF amplitude gradually reduced the observed effect, but twenty 60ns 16.2kV/cm pulses produced an effect similar to a single 600ns pulse of the same amplitude. Propidium iodide (PI) uptake after the nsEP exposure confirmed a strong relationship between EF-induced plasma membrane impact and PIP2 depletion. These results have expanded our current knowledge of nsEPs dependent cell physiological effects, and serve as a basis for model development of new exposure standards, providing novel tools for drug independent stimulation and approaches to differential modulation of key cellular functions.

Receptor Species-dependent Desensitization Controls KCNQ1/KCNE1 K+ Channels as Downstream Effectors of Gq Protein-coupled Receptors

Activation of Gq protein-coupled receptors (GqPCRs) might induce divergent cellular responses, related to receptor-specific activation of different branches of the Gq signaling pathway. Receptor-specific desensitization provides a mechanism of effector modulation by restricting the spatiotemporal activation of signaling components downstream of Gq We quantified signaling events downstream of GqPCR activation with FRET-based biosensors in CHO and HEK 293 cells. KCNQ1/KCNE1 channels (IKs) were measured as a functional readout of receptor-specific activation. Activation of muscarinic M1 receptors (M1-Rs) caused robust and reversible inhibition of IKs. In contrast, activation of α1B-adrenergic receptors (α1B-ARs) induced transient inhibition of IKs, which turned into delayed facilitation after agonist withdrawal. As a novel finding, we demonstrate that GqPCR-specific kinetics of IKs modulation are determined by receptor-specific desensitization, evident at the level of Gαq activation, phosphatidylinositol 4,5-bisphosphate (PIP2) depletion, and diacylglycerol production. Sustained IKs inhibition during M1-R stimulation is attributed to robust membrane PIP2 depletion, whereas the rapid desensitization of α1B-AR delimits PIP2 reduction and augments current activation by protein kinase C (PKC). Overexpression of Ca2+-independent PKCδ did not affect the time course of α1B-AR-induced diacylglycerol formation, excluding a contribution of PKCδ to α1B-AR desensitization. Pharmacological inhibition of Ca2+-dependent PKC isoforms abolished fast α1B receptor desensitization and augmented IKs reduction, but did not affect IKs facilitation. These data indicate a contribution of Ca2+-dependent PKCs to α1B-AR desensitization, whereas IKs facilitation is induced by Ca2+-independent PKC isoforms. In contrast, neither inhibition of Ca2+-dependent/Ca2+-independent isoforms nor overexpression of PKCδ induced M1 receptor desensitization, excluding a contribution of PKC to M1-R-induced IKs modulation.

Activation of the Ca2+-sensing receptors increases currents through inward rectifier K+ channels via activation of phosphatidylinositol 4-kinase.

Inward rectifier K+ channels are important for maintaining normal electrical function in many cell types. The proper function of these channels requires the presence of membrane phosphoinositide 4,5-bisphosphate (PIP2). Stimulation of the Ca2+-sensing receptor CaR, a pleiotropic G protein-coupled receptor, activates both Gq/11, which decreases PIP2, and phosphatidylinositol 4-kinase (PI-4-K), which, conversely, increases PIP2. How membrane PIP2 levels are regulated by CaR activation and whether these changes modulate inward rectifier K+ are unknown. In this study, we found that activation of CaR by the allosteric agonist, NPSR568, increased inward rectifier K+ current (I K1) in guinea pig ventricular myocytes and currents mediated by Kir2.1 channels exogenously expressed in HEK293T cells with a similar sensitivity. Moreover, using the fluorescent PIP2 reporter tubby-R332H-cYFP to monitor PIP2 levels, we found that CaR activation in HEK293T cells increased membrane PIP2 concentrations. Pharmacological studies showed that both phospholipase C (PLC) and PI-4-K are activated by CaR stimulation with the latter played a dominant role in regulating membrane PIP2 and, thus, Kir currents. These results provide the first direct evidence that CaR activation upregulates currents through inward rectifier K+ channels by accelerating PIP2 synthesis. The regulation of I K1 plays a critical role in the stability of the electrical properties of many excitable cells, including cardiac myocytes and neurons. Further, synthetic allosteric modulators that increase CaR activity have been used to treat hyperparathyroidism, and negative CaR modulators are of potential importance in the treatment of osteoporosis. Thus, our results provide further insight into the roles played by CaR in the cardiovascular system and are potentially valuable for heart disease treatment and drug safety.

Binding of Vinculin to Lipid Membranes in Its Inhibited and Activated States

Phosphoinositols are an important class of phospholipids that are involved in a myriad of cellular processes, from cell signaling to motility and adhesion. Vinculin (Vn) is a major adaptor protein that regulates focal adhesions in conjunction with PIP2 in lipid membranes and other cytoskeletal components. The binding and unbinding transitions of Vn at the membrane interface are an important link to understanding the coordination of cell signaling and motility. Using different biophysical tools, including atomic force microscopy combined with confocal fluorescence microscopy and Fourier transform infrared spectroscopy, we studied the nanoscopic interactions of activated and autoinhibited states of Vn with lipid membranes. We hypothesize that a weak interaction occurs between Vn and lipid membranes, which leads to binding of autoinhibited Vn to supported lipid bilayers, and to unbinding in freestanding lipid vesicles. Likely driving forces may include tethering of the C-terminus to the lipid membrane, as well as hydrophobic helix-membrane interactions. Conversely, activated Vn binds strongly to membranes through specific interactions with clusters of PIP2 embedded in lipid membranes. Activated Vn harbored on PIP2 clusters may form small oligomeric interaction platforms for further interaction partners, which is necessary for the proper function of focal adhesion points.