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.     

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.     

Regulation of TRPV1 Ion Channel by Phosphoinositide (4,5)-Bisphosphate: THE ROLE OF MEMBRANE ASYMMETRY*

Membrane asymmetry is essential for generating second messengers that act in the cytosol and for trafficking of membrane proteins and membrane lipids, but the role of asymmetry in regulating membrane protein function remains unclear. Here we show that the signaling lipid phosphoinositide 4,5-bisphosphate (PI(4,5)P₂) has opposite effects on the function of TRPV1 ion channels depending on which leaflet of the cell membrane it resides in. We observed potentiation of capsaicin-activated TRPV1 currents by PI(4,5)P₂ in the intracellular leaflet of the plasma membrane but inhibition of capsaicin-activated currents when PI(4,5)P₂ was in both leaflets of the membrane, although much higher concentrations of PI(4,5)P₂ in the extracellular leaflet were required for inhibition compared with the concentrations of PI(4,5)P₂ in the intracellular leaflet that produced activation. Patch clamp fluorometry using a synthetic PI(4,5)P₂ whose fluorescence reports its concentration in the membrane indicates that PI(4,5)P₂ must incorporate into the extracellular leaflet for its inhibitory effects to be observed. The asymmetry-dependent effect of PI(4,5)P₂ may resolve the long standing controversy about whether PI(4,5)P₂ is an activator or inhibitor of TRPV1. Our results also underscore the importance of membrane asymmetry and the need to consider its influence when studying membrane proteins reconstituted into synthetic bilayers.     

Short-Chain Phosphoinositide Partitioning into Plasma Membrane Models

Phosphoinositides are vital for many cellular signaling processes, and therefore a number of approaches to manipulating phosphoinositide levels in cells or excised patches of cell membranes have been developed. Among the most common is the use of “short-chain” phosphoinositides, usually dioctanoyl phosphoinositol phosphates. We use isothermal titration calorimetry to determine partitioning of the most abundant phosphoinositol phosphates, PI(4)P and PI(4,5)P₂ into models of the intracellular and extracellular facing leaflets of neuronal plasma membranes. We show that phosphoinositide mole fractions in the lipid membrane reach physiological levels at equilibrium with reasonable solution concentrations. Finally we explore the consequences of our results for cellular electrophysiology. In particular, we find that TRPV1 is more selective for PI(4,5)P₂ than PI(4)P and activated by extremely low membrane mole fractions of PIPs. We conclude by discussing how the logic of our work extends to other experiments with short-chain phosphoinositides. For delayed rectifier K⁺ channels, consideration of the membrane mole fraction of PI(4,5)P₂ lipids with different acyl chain lengths suggests a different mechanism for PI(4,5)P₂ regulation than previously proposed. Inward rectifier K⁺ channels apparent lack of selectivity for certain short-chain PIPs may require reinterpretation in view of the PIPs different membrane partitioning.     

Differential Regulation of Proton-Sensitive Ion Channels by Phospholipids: A Comparative Study between ASICs and TRPV1

Protons are released in pain-generating pathological conditions such as inflammation, ischemic stroke, infection, and cancer. During normal synaptic activities, protons are thought to play a role in neurotransmission processes. Acid-sensing ion channels (ASICs) are typical proton sensors in the central nervous system (CNS) and the peripheral nervous system (PNS). In addition to ASICs, capsaicin- and heat-activated transient receptor potential vanilloid 1 (TRPV1) channels can also mediate proton-mediated pain signaling. In spite of their importance in perception of pH fluctuations, the regulatory mechanisms of these proton-sensitive ion channels still need to be further investigated. Here, we compared regulation of ASICs and TRPV1 by membrane phosphoinositides, which are general cofactors of many receptors and ion channels. We observed that ASICs do not require membrane phosphatidylinositol 4-phosphate (PI(4)P) or phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) for their function. However, TRPV1 currents were inhibited by simultaneous breakdown of PI(4)P and PI(4,5)P₂. By using a novel chimeric protein, CF-PTEN, that can specifically dephosphorylate at the D3 position of phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P₃), we also observed that neither ASICs nor TRPV1 activities were altered by depletion of PI(3,4,5)P₃ in intact cells. Finally, we compared the effects of arachidonic acid (AA) on two proton-sensitive ion channels. We observed that AA potentiates the currents of both ASICs and TRPV1, but that they have different recovery aspects. In conclusion, ASICs and TRPV1 have different sensitivities toward membrane phospholipids, such as PI(4)P, PI(4,5)P₂, and AA, although they have common roles as proton sensors. Further investigation about the complementary roles and respective contributions of ASICs and TRPV1 in proton-mediated signaling is necessary.     

Determinants of molecular specificity in phosphoinositide regulation. Phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2) is the endogenous lipid regulating TRPV1

Once thought of as simply an oily barrier that maintains cellular integrity, lipids are now known to play an active role in a large variety of cellular processes. Phosphoinositides are of particular interest because of their remarkable ability to affect many signaling pathways. Ion channels and transporters are an important target of phosphoinositide signaling, but identification of the specific phosphoinositides involved has proven elusive. TRPV1 is a good example; although phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P(2)) can potently regulate its activation, we show that phosphatidylinositol (4)-phosphate (PI(4)P) and phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P(3)) can as well. To determine the identity of the endogenous phosphoinositide regulating TRPV1, we applied recombinant pleckstrin homology domains to inside-out excised patches. Although a PI(4,5)P(2)-specific pleckstrin homology domain inhibited TRPV1, a PI(3,4,5)P(3)-specific pleckstrin homology domain had no effect. Simultaneous confocal imaging and electrophysiological recording of whole cells expressing a rapamycin-inducible lipid phosphatase also demonstrates that depletion of PI(4,5)P(2) inhibits capsaicin-activated TRPV1 current; the PI(4)P generated by the phosphatases was not sufficient to support TRPV1 function. We conclude that PI(4,5)P(2), and not other phosphoinositides or other lipids, is the endogenous phosphoinositide regulating TRPV1 channels.     

Interplay between Calmodulin and Phosphatidylinositol 4,5-Bisphosphate in Ca2+-induced Inactivation of Transient Receptor Potential Vanilloid 6 Channels

The epithelial Ca²⁺ channel transient receptor potential vanilloid 6 (TRPV6) undergoes Ca²⁺-induced inactivation that protects the cell from toxic Ca²⁺ overload and may also limit intestinal Ca²⁺ transport. To dissect the roles of individual signaling pathways in this phenomenon, we studied the effects of Ca²⁺, calmodulin (CaM), and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) in excised inside-out patches. The activity of TRPV6 strictly depended on the presence of PI(4,5)P₂, and Ca²⁺-CaM inhibited the channel at physiologically relevant concentrations. Ca²⁺ alone also inhibited TRPV6 at high concentrations (IC₅₀ = ∼20 μm). A double mutation in the distal C-terminal CaM-binding site of TRPV6 (W695A/R699E) essentially eliminated inhibition by CaM in excised patches. In whole cell patch clamp experiments, this mutation reduced but did not eliminate Ca²⁺-induced inactivation. Providing excess PI(4,5)P₂ reduced the inhibition by CaM in excised patches and in planar lipid bilayers, but PI(4,5)P₂ did not inhibit binding of CaM to the C terminus of the channel. Overall, our data show a complex interplay between CaM and PI(4,5)P₂ and show that Ca²⁺, CaM, and the depletion of PI(4,5)P₂ all contribute to inactivation of TRPV6.     

Voltage- and temperature-dependent activation of TRPV3 channels is potentiated by receptor-mediated PI(4,5)P2 hydrolysis

TRPV3 is a thermosensitive channel that is robustly expressed in skin keratinocytes and activated by innocuous thermal heating, membrane depolarization, and chemical agonists such as 2-aminoethyoxy diphenylborinate, carvacrol, and camphor. TRPV3 modulates sensory thermotransduction, hair growth, and susceptibility to dermatitis in rodents, but the molecular mechanisms responsible for controlling TRPV3 channel activity in keratinocytes remain elusive. We show here that receptor-mediated breakdown of the membrane lipid phosphatidylinositol (4,5) bisphosphate (PI(4,5)P(2)) regulates the activity of both native TRPV3 channels in primary human skin keratinocytes and expressed TRPV3 in a HEK-293-derived cell line stably expressing muscarinic M(1)-type acetylcholine receptors. Stimulation of PI(4,5)P(2) hydrolysis or pharmacological inhibition of PI 4 kinase to block PI(4,5)P(2) synthesis potentiates TRPV3 currents by causing a negative shift in the voltage dependence of channel opening, increasing the proportion of voltage-independent current and causing thermal activation to occur at cooler temperatures. The activity of single TRPV3 channels in excised patches is potentiated by PI(4,5)P(2) depletion and selectively decreased by PI(4,5)P(2) compared with related phosphatidylinositol phosphates. Neutralizing mutations of basic residues in the TRP domain abrogate the effect of PI(4,5)P(2) on channel function, suggesting that PI(4,5)P(2) directly interacts with a specific protein motif to reduce TRPV3 channel open probability. PI(4,5)P(2)-dependent modulation of TRPV3 activity represents an attractive mechanism for acute regulation of keratinocyte signaling cascades that control cell proliferation and the release of autocrine and paracrine factors.     

Maintenance of hormone-sensitive phosphoinositide pools in the plasma membrane requires phosphatidylinositol 4-kinase IIIalpha

Type III phosphatidylinositol (PtdIns) 4-kinases (PI4Ks) have been previously shown to support plasma membrane phosphoinositide synthesis during phospholipase C activation and Ca²⁺ signaling. Here, we use biochemical and imaging tools to monitor phosphoinositide changes in the plasma membrane in combination with pharmacological and genetic approaches to determine which of the type III PI4Ks (α or β) is responsible for supplying phosphoinositides during agonist-induced Ca²⁺ signaling. Using inhibitors that discriminate between the α- and β-isoforms of type III PI4Ks, PI4KIIIα was found indispensable for the production of phosphatidylinositol 4-phosphate (PtdIns4P), phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P₂], and Ca²⁺ signaling in angiotensin II (AngII)-stimulated cells. Down-regulation of either the type II or type III PI4K enzymes by small interfering RNA (siRNA) had small but significant effects on basal PtdIns4P and PtdIns(4,5)P₂ levels in ³²P-labeled cells, but only PI4KIIIα down-regulation caused a slight impairment of PtdIns4P and PtdIns(4,5)P₂ resynthesis in AngII-stimulated cells. None of the PI4K siRNA treatments had a measurable effect on AngII-induced Ca²⁺ signaling. These results indicate that a small fraction of the cellular PI4K activity is sufficient to maintain plasma membrane phosphoinositide pools, and they demonstrate the value of the pharmacological approach in revealing the pivotal role of PI4KIIIα enzyme in maintaining plasma membrane phosphoinositides.     

Differential effects of the phosphatidylinositol 4-kinases, PI4KII�� and PI4KIII��, on Akt activation and apoptosis

In this study, we investigated the role of PI4P synthesis by the phosphatidylinositol 4-kinases, PI4KIIα and PI4KIIIβ, in epidermal growth factor (EGF)-stimulated phosphoinositide signaling and cell survival. In COS-7 cells, knockdown of either isozyme by RNA interference reduced basal levels of PI4P and PI(4,5)P₂, without affecting receptor activation. Only knockdown of PI4KIIα inhibited EGF-stimulated Akt phosphorylation, indicating that decreased PI(4,5)P₂ synthesis observed by loss of either isoform could not account for this PI4KIIα-specific effect. Phospholipase Cγ activation was also differentially affected by knockdown of either PI4K isozyme. Overexpression of kinase-inactive PI4KIIα, which induces defective endosomal trafficking without reducing PI(4,5)P₂ levels, also reduced Akt activation. Furthermore, PI4KIIα knockdown profoundly inhibited cell proliferation and induced apoptosis as evidenced by the cleavage of caspase-3 and its substrate poly(ADP-ribose) polymerase. However, in MDA-MB-231 breast cancer cells, apoptosis was observed subsequent to knockdown of either PI4KIIα or PI4KIIIβ and this correlated with enhanced proapoptotic Akt phosphorylation. The differential effects of phosphatidylinositol 4-kinase knockdown in the two cell lines lead to the conclusion that phosphoinositide turnover is inhibited through PI4P substrate depletion, whereas impaired antiapoptotic Akt signaling is an indirect consequence of dysfunctional endosomal trafficking.     

Phospholipid-binding Sites of Phosphatase and Tensin Homolog (PTEN): EXPLORING THE MECHANISM OF PHOSPHATIDYLINOSITOL 4,5-BISPHOSPHATE ACTIVATION*

The lipid phosphatase activity of the tumor suppressor phosphatase and tensin homolog (PTEN) is enhanced by the presence of its biological product, phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂). This enhancement is suggested to occur via the product binding to the N-terminal region of the protein. PTEN effects on short-chain phosphoinositide ³¹P linewidths and on the full field dependence of the spin-lattice relaxation rate (measured by high resolution field cycling ³¹P NMR using spin-labeled protein) are combined with enzyme kinetics with the same short-chain phospholipids to characterize where PI(4,5)P₂ binds on the protein. The results are used to model a discrete site for a PI(4,5)P₂ molecule close to, but distinct from, the active site of PTEN. This PI(4,5)P₂ site uses Arg-47 and Lys-13 as phosphate ligands, explaining why PTEN R47G and K13E can no longer be activated by that phosphoinositide. Placing a PI(4,5)P₂ near the substrate site allows for proper orientation of the enzyme on interfaces and should facilitate processive catalysis.     

Sac2/INPP5F is an inositol 4-phosphatase that functions in the endocytic pathway

The recruitment of inositol phosphatases to endocytic membranes mediates dephosphorylation of PI(4,5)P₂, a phosphoinositide concentrated in the plasma membrane, and prevents its accumulation on endosomes. The importance of the conversion of PI(4,5)P₂ to PtdIns during endocytosis is demonstrated by the presence of both a 5-phosphatase and a 4-phosphatase (Sac domain) module in the synaptojanins, endocytic PI(4,5)P₂ phosphatases conserved from yeast to humans and the only PI(4,5)P₂ phosphatases in yeast. OCRL, another 5-phosphatase that couples endocytosis to PI(4,5)P₂ dephosphorylation, lacks a Sac domain. Here we show that Sac2/INPP5F is a PI4P phosphatase that colocalizes with OCRL on endocytic membranes, including vesicles formed by clathrin-mediated endocytosis, macropinosomes, and Rab5 endosomes. An OCRL–Sac2/INPP5F interaction could be demonstrated by coimmunoprecipitation and was potentiated by Rab5, whose activity is required to recruit Sac2/INPP5F to endosomes. Sac2/INPP5F and OCRL may cooperate in the sequential dephosphorylation of PI(4,5)P₂ at the 5 and 4 position of inositol in a partnership that mimics that of the two phosphatase modules of synaptojanin.     

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

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

NHE3 Activity Is Dependent on Direct Phosphoinositide Binding at the N Terminus of Its Intracellular Cytosolic Region

The small intestinal BB Na⁺/H⁺ antiporter NHE3 accounts for the majority of intestinal sodium and water absorption. It is highly regulated with both postprandial inhibition and stimulation sequentially occurring. Phosphatidylinositide 4,5-bisphosphate (PI(4,5)P₂) and phosphatidylinositide 3,4,5-trisphosphate (PI(3,4,5)P₃) binding is involved with regulation of multiple transporters. We tested the hypothesis that phosphoinositides bind NHE3 under basal conditions and are necessary for its acute regulation. His₆ proteins were made from the NHE3 C-terminal region divided into four parts as follows: F1 (amino acids 475–589), F2 (amino acids 590–667), F3 (amino acids 668–747), and F4 (amino acids 748–832) and purified by a nickel column. Mutations were made in the F1 region of NHE3 and cloned in pet30a and pcDNA3.1 vectors. PI(4,5)P₂ and PI(3,4,5)P₃ bound only to the NHE3 F1 fusion protein (amino acids 475–589) on liposomal pulldown assays. Mutations were made in the putative lipid binding region of the F1 domain and studied for alterations in lipid binding and Na⁺/H⁺ exchange as follows: Y501A/R503A/K505A; F509A/R511A/R512A; R511L/R512L; R520/FR527F; and R551L/R552L. Our results indicate the following. 1) The F1 domain of the NHE3 C terminus has phosphoinositide binding regions. 2) Mutations of these regions alter PI(4,5)P₂ and PI(3,4,5)P₃ binding and basal NHE3 activity. 3) The magnitude of serum stimulation of NHE3 correlates with PI(4,5)P₂ and PI(3,4,5)P₃ binding of NHE3. 4) Wortmannin inhibition of PI3K did not correlate with PI(4,5)P₂ or PI(3,4,5)P₃ binding of NHE3. Two functionally distinct phosphoinositide binding regions (Tyr⁵⁰¹–Arg⁵¹² and Arg⁵²⁰–Arg⁵⁵²) are present in the NHE3 F1 domain; both regions are important for serum stimulation, but they display differences in phosphoinositide binding, and the latter but not the former alters NHE3 surface expression.     

The N-terminal homology (ENTH) domain of Epsin 1 is a sensitive reporter of physiological PI(4,5)P2 dynamics

Phospholipase Cβ (PLCβ)-induced depletion of phosphatidylinositol-(4,5)-bisphosphate (PI(4,5)P₂) transduces a plethora of signals into cellular responses. Importance and diversity of PI(4,5)P₂-dependent processes led to strong need for biosensors of physiological PI(4,5)P₂ dynamics applicable in live-cell experiments. Membrane PI(4,5)P₂ can be monitored with fluorescently-labelled phosphoinositide (PI) binding domains that associate to the membrane depending on PI(4,5)P₂ levels. The pleckstrin homology domain of PLCδ1 (PLCδ1-PH) and the C-terminus of tubby protein (tubby_CT) are two such sensors widely used to study PI(4,5)P₂ signaling. However, certain limitations apply to both: PLCδ1-PH binds cytoplasmic inositol-1,4,5-trisphosphate (IP₃) produced from PI(4,5)P₂ through PLCβ, and tubby_CT responses do not faithfully report on PLCβ-dependent PI(4,5)P₂ dynamics. In searching for an improved biosensor, we fused N-terminal homology domain of Epsin1 (ENTH) to GFP and examined use of this construct as genetically-encoded biosensor for PI(4,5)P₂ dynamics in living cells. We utilized recombinant tools to manipulate PI or G_q protein-coupled receptors (G_qPCR) to stimulate PLCβ signaling and characterized PI binding properties of ENTH-GFP with total internal reflection (TIRF) and confocal microscopy. ENTH-GFP specifically recognized membrane PI(4,5)P₂ without interacting with IP₃, as demonstrated by dialysis of cells with the messenger through a patch pipette. Utilizing Ci-VSP to titrate PI(4,5)P₂ levels, we found that ENTH-GFP had low PI(4,5)P₂ affinity. Accordingly, ENTH-GFP was highly sensitive to PLCβ-dependent PI(4,5)P₂ depletion, and in contrast to PLCδ1-PH, overexpression of ENTH-GFP did not attenuate G_qPCR signaling. Taken together, ENTH-GFP detects minute changes of PI(4,5)P₂ levels and provides an important complementation of experimentally useful reporters of PI(4,5)P₂ dynamics in physiological pathways.     

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.     

ADF/Cofilin Binds Phosphoinositides in a Multivalent Manner to Act as a PIP2-Density Sensor

Actin-depolymerizing-factor (ADF)/cofilins have emerged as key regulators of cytoskeletal dynamics in cell motility, morphogenesis, endocytosis, and cytokinesis. The activities of ADF/cofilins are regulated by membrane phospholipid PI(4,5)P₂ in vitro and in cells, but the mechanism of the ADF/cofilin-PI(4,5)P₂ interaction has remained controversial. Recent studies suggested that ADF/cofilins interact with PI(4,5)P₂ through a specific binding pocket, and that this interaction is dependent on pH. Here, we combined systematic mutagenesis with biochemical and spectroscopic methods to elucidate the phosphoinositide-binding mechanism of ADF/cofilins. Our analysis revealed that cofilin does not harbor a specific PI(4,5)P₂-binding pocket, but instead interacts with PI(4,5)P₂ through a large, positively charged surface of the molecule. Cofilin interacts simultaneously with multiple PI(4,5)P₂ headgroups in a cooperative manner. Consequently, interactions of cofilin with membranes and actin exhibit sharp sensitivity to PI(4,5)P₂ density. Finally, we show that cofilin binding to PI(4,5)P₂ is not sensitive to changes in the pH at physiological salt concentration, although the PI(4,5)P₂-clustering activity of cofilin is moderately inhibited at elevated pH. Collectively, our data demonstrate that ADF/cofilins bind PI(4,5)P₂ headgroups through a multivalent, cooperative mechanism, and suggest that the actin filament disassembly activity of ADF/cofilin can be accurately regulated by small changes in the PI(4,5)P₂ density at cellular membranes.     

Wasted TMEM16A channels are rescued by phosphatidylinositol 4,5-bisphosphate

Recently there has been a flurry of interest in the regulation of the homo-dimeric calcium-activated chloride channel ANO1 (also known as TMEM16A) by phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P₂). These recent studies show that upon Ca²⁺ binding, PI(4,5)P₂ cooperates to maintain the conductive state of ANO1. PI(4,5)P₂ does so by binding to sites or modules on the protein’s cytosolic side. These findings add a new function to the PI(4,5)P₂ repertoire and a new dimension to ANO1 gating.     

Interaction between the human immunodeficiency virus type 1 Gag matrix domain and phosphatidylinositol-(4,5)-bisphosphate is essential for efficient gag membrane binding

Human immunodeficiency virus type 1 (HIV-1) particle assembly mediated by the viral structural protein Gag occurs predominantly on the plasma membrane (PM). Although it is known that the matrix (MA) domain of Gag plays a major role in PM localization, molecular mechanisms that determine the location of assembly remain to be elucidated. We observed previously that overexpression of polyphosphoinositide 5-phosphatase IV (5ptaseIV) that depletes PM phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P₂] impairs virus particle production and redirects processed Gag to intracellular compartments. In this study, we examined the impact of PI(4,5)P₂ depletion on the subcellular localization of the entire Gag population using Gag-fluorescent protein chimeras. Upon 5ptaseIV overexpression, in addition to perinuclear localization, Gag also showed a hazy cytosolic signal, suggesting that PI(4,5)P₂ depletion impairs Gag membrane binding. Indeed, Gag was less membrane bound in PI(4,5)P₂-depleted cells, as assessed by biochemical analysis. These observations are consistent with the hypothesis that Gag interacts with PI(4,5)P₂. To examine a putative Gag interaction with PI(4,5)P₂, we developed an in vitro binding assay using full-length myristoylated Gag and liposome-associated PI(4,5)P₂. Using this assay, we observed that PI(4,5)P₂ significantly enhances liposome binding of wild-type Gag. In contrast, a Gag derivative lacking MA did not require PI(4,5)P₂ for efficient liposome binding. To analyze the involvement of MA in PI(4,5)P₂ binding further, we examined MA basic amino acid substitution mutants. These mutants, previously shown to localize in perinuclear compartments, bound PI(4,5)P₂-containing liposomes weakly. Altogether, these results indicate that HIV-1 Gag binds PI(4,5)P₂ on the membrane and that the MA basic domain mediates this interaction.     

GABARAP-mediated targeting of PI4K2A/PI4KIIα to autophagosomes regulates PtdIns4P-dependent autophagosome-lysosome fusion

For decades, phosphatidylinositol 4-phosphate (PtdIns4P) was considered primarily as a precursor in the synthesis of phosphatidylinositol(4,5)bisphosphate (PtdIns(4,5)P₂). More recently, specific functions for PtdIns4P itself have been identified, particularly in the regulation of intracellular membrane trafficking. PI4K2A/PI4KIIα (phosphatidylinositol 4-kinase type 2 α), one of the 4 enzymes that catalyze PtdIns4P production in mammalian cells, promotes vesicle formation from the trans-Golgi network (TGN) and endosomes. We recently identified a novel function for PI4K2A-derived PtdIns4P, as a facilitator of autophagosome-lysosome (A-L) fusion. We further showed that that this function requires the presence of the autophagic adaptor protein GABARAP (GABA[A] receptor-associated protein), which binds to PI4K2A and recruits it to autophagosomes. The mechanism whereby GABARAP-PI4K2A-PtdIns4P promotes A-L fusion remains to be defined. Based on other examples of phosphoinositide involvement in membrane trafficking, we speculate that it acts by recruiting elements of the membrane docking and fusion machinery.