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.     

Mechanism for phosphoinositide selectivity and activation of TRPV1 ion channels

Although PI(4,5)P₂ is believed to play an essential role in regulating the activity of numerous ion channels and transporters, the mechanisms by which it does so are unknown. Here, we used the ability of the TRPV1 ion channel to discriminate between PI(4,5)P₂ and PI(4)P to localize the region of TRPV1 sequence that interacts directly with the phosphoinositide. We identified a point mutation in the proximal C-terminal region after the TRP box, R721A, that inverted the selectivity of TRPV1. Although the R721A mutation produced only a 30% increase in the EC₅₀ for activation by PI(4,5)P₂, it decreased the EC₅₀ for activation by PI(4)P by more than two orders of magnitude. We used chemically induced and voltage-activated phosphatases to determine that PI(4)P continued to support TRPV1 activity even after depletion of PI(4,5)P₂ from the plasma membrane. Our data cannot be explained by a purely electrostatic mechanism for interaction between the phosphoinositide and the protein, similar to that of the MARCKS (myristoylated alanine-rich C kinase substrate) effector domain or the EGF receptor. Rather, conversion of a PI(4,5)P₂-selective channel to a PI(4)P-selective channel indicates that a structured phosphoinositide-binding site mediates the regulation of TRPV1 activity and that the amino acid at position 721 likely interacts directly with the moiety at the 5′ position of the phosphoinositide.     

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.     

Enteropathogenic Escherichia coli subverts phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate upon epithelial cell infection

Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] and phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P₃] are phosphoinositides (PIs) present in small amounts in the inner leaflet of the plasma membrane (PM) lipid bilayer of host target cells. They are thought to modulate the activity of proteins involved in enteropathogenic Escherichia coli (EPEC) infection. However, the role of PI(4,5)P₂ and PI(3,4,5)P₃ in EPEC pathogenesis remains obscure. Here we show that EPEC induces a transient PI(4,5)P₂ accumulation at bacterial infection sites. Simultaneous actin accumulation, likely involved in the construction of the actin-rich pedestal, is also observed at these sites. Acute PI(4,5)P₂ depletion partially diminishes EPEC adherence to the cell surface and actin pedestal formation. These findings are consistent with a bimodal role, whereby PI(4,5)P₂ contributes to EPEC association with the cell surface and to the maximal induction of actin pedestals. Finally, we show that EPEC induces PI(3,4,5)P₃ clustering at bacterial infection sites, in a translocated intimin receptor (Tir)-dependent manner. Tir phosphorylated on tyrosine 454, but not on tyrosine 474, forms complexes with an active phosphatidylinositol 3-kinase (PI3K), suggesting that PI3K recruited by Tir prompts the production of PI(3,4,5)P₃ beneath EPEC attachment sites. The functional significance of this event may be related to the ability of EPEC to modulate cell death and innate immunity.     

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.     

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

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

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.     

Effect of H-Bond Donor Lipids on Phosphatidylinositol-3,4,5-Trisphosphate Ionization and Clustering

The phosphoinositide, phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P₃), is a key signaling lipid in the inner leaflet of the cell plasma membrane, regulating diverse signaling pathways including cell growth and migration. In this study we investigate the impact of the hydrogen-bond donor lipids phosphatidylethanolamine (PE) and phosphatidylinositol (PI) on the charge and phase behavior of PI(3,4,5)P₃. PE and PI can interact with PI(3,4,5)P₃ through hydrogen-bond formation, leading to altered ionization behavior and charge distribution within the PI(3,4,5)P₃ headgroup. We quantify the altered PI(3,4,5)P₃ ionization behavior using a multistate ionization model to obtain micro-pK_a values for the ionization of each phosphate group. The presence of PE leads to a decrease in the pK_a values for the initial deprotonation of PI(3,4,5)P₃, which describes the removal of the first proton of the three protons remaining at the phosphomonoester groups at pH 4.0. The decrease in these micro-pK_a values thus leads to a higher charge at low pH. Additionally, the charge distribution changes lead to increased charge on the 3- and 5-phosphates. In the presence of PI, the final deprotonation of PI(3,4,5)P₃ is delayed, leading to a lower charge at high pH. This is due to a combination of hydrogen-bond formation between PI and PI(3,4,5)P₃, and increased surface charge due to the addition of the negatively charged PI. The interaction between PI and PI(3,4,5)P₃ leads to the formation of PI and PI(3,4,5)P₃-enriched domains within the membrane. These domains may have a critical impact on PI(3,4,5)P₃-signaling. We also reevaluate results for all phosphatidylinositol bisphosphates as well as for PI(4,5)P₂ in complex lipid mixtures with the multistate ionization model.     

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

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

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.     

The phosphoinositide sensitivity of the KV channel family

Recently, we screened several K_V channels for possible dependence on plasma membrane phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂). The channels were expressed in tsA-201 cells and the PI(4,5)P₂ was depleted by several manipulations in whole-cell experiments with parallel measurements of channel activity. In contrast to reports on excised-patches using Xenopus laevis oocytes, we found only K_V7, but none of the other tested K_V channels, to be strongly dependent on PI(4,5)P₂. We now have extended our study to K_V1.2 channels, a K_V channel we had not previously tested, because a new published study on excised patches showed regulation of the voltage-dependence of activation by PI(4,5)P₂. In full agreement with those published results, we found a reduction of current amplitude by ~20% after depletion of PI(4,5)P₂ and a small left shift in the activation curve of K_V1.2 channels. We also found a small reduction of K_V11.1 (hERG) currents that was not accompanied by a gating shift. In conclusion, our whole-cell methods yield a PI(4,5)P₂-dependence of K_V1.2 currents in tsA-201 cells that is comparable to findings from excised patches of Xenopus laevis oocytes. We discuss possible physiological rationales for PI(4,5)P₂ sensitivity of some ion channels and insensitivity of others.     

Rab27a controls HIV-1 assembly by regulating plasma membrane levels of phosphatidylinositol 4,5-bisphosphate

During the late stages of the HIV-1 replication cycle, the viral polyprotein Pr55^Gag is recruited to the plasma membrane (PM), where it binds phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) and directs HIV-1 assembly. We show that Rab27a controls the trafficking of late endosomes carrying phosphatidylinositol 4-kinase type 2 α (PI4KIIα) toward the PM of CD4⁺ T cells. Hence, Rab27a promotes high levels of PM phosphatidylinositol 4-phosphate and the localized production of PI(4,5)P₂, therefore controlling Pr55^Gag membrane association. Rab27a also controls PI(4,5)P₂ levels at the virus-containing compartments of macrophages. By screening Rab27a effectors, we identified that Slp2a, Slp3, and Slac2b are required for the association of Pr55^Gag with the PM and that Slp2a cooperates with Rab27a in the recruitment of PI4KIIα to the PM. We conclude that by directing the trafficking of PI4KIIα-positive endosomes toward the PM, Rab27a controls PI(4,5)P₂ production and, consequently, HIV-1 replication.     

Hydrolysis of phosphatidylinositol 4,5-bisphosphate mediates calcium-induced inactivation of TRPV6 channels

TRPV6 is a member of the transient receptor potential superfamily of ion channels that facilitates Ca(2+) absorption in the intestines. These channels display high selectivity for Ca(2+), but in the absence of divalent cations they also conduct monovalent ions. TRPV6 channels have been shown to be inactivated by increased cytoplasmic Ca(2+) concentrations. Here we studied the mechanism of this Ca(2+)-induced inactivation. Monovalent currents through TRPV6 substantially decreased after a 40-s application of Ca(2+), but not Ba(2+). We also show that Ca(2+), but not Ba(2+), influx via TRPV6 induces depletion of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2) or PIP(2)) and the formation of inositol 1,4,5-trisphosphate. Dialysis of DiC(8) PI(4,5)P(2) through the patch pipette inhibited Ca(2+)-dependent inactivation of TRPV6 currents in whole-cell patch clamp experiments. PI(4,5)P(2) also activated TRPV6 currents in excised patches. PI(4)P, the precursor of PI(4,5)P(2), neither activated TRPV6 in excised patches nor had any effect on Ca(2+)-induced inactivation in whole-cell experiments. Conversion of PI(4,5)P(2) to PI(4)P by a rapamycin-inducible PI(4,5)P(2) 5-phosphatase inhibited TRPV6 currents in whole-cell experiments. Inhibiting phosphatidylinositol 4 kinases with wortmannin decreased TRPV6 currents and Ca(2+) entry into TRPV6-expressing cells. We propose that Ca(2+) influx through TRPV6 activates phospholipase C and the resulting depletion of PI(4,5)P(2) contributes to the inactivation of TRPV6.     

Alterations in the MA and NC Domains Modulate Phosphoinositide-Dependent Plasma Membrane Localization of the Rous Sarcoma Virus Gag Protein

Retroviral Gag proteins direct virus particle assembly from the plasma membrane (PM). Phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P₂] plays a role in PM targeting of several retroviral Gag proteins. Here we report that depletion of intracellular PI(4,5)P₂ and phosphatidylinositol-(3,4,5)-triphosphate [PI(3,4,5)P₃] levels impaired Rous sarcoma virus (RSV) Gag PM localization. Gag mutants deficient in nuclear trafficking were less sensitive to reduction of intracellular PI(4,5)P₂ and PI(3,4,5)P₃, suggesting a possible connection between Gag nuclear trafficking and phosphoinositide-dependent PM targeting.     

The inositol polyphosphate 5-phosphatase, PIPP, Is a novel regulator of phosphoinositide 3-kinase-dependent neurite elongation

The spatial activation of phosphoinositide 3-kinase (PI3-kinase) signaling at the axon growth cone generates phosphatidylinositol 3,4,5 trisphosphate (PtdIns(3,4,5)P₃), which localizes and facilitates Akt activation and stimulates GSK-3β inactivation, promoting microtubule polymerization and axon elongation. However, the molecular mechanisms that govern the spatial down-regulation of PtdIns(3,4,5)P₃ signaling at the growth cone remain undetermined. The inositol polyphosphate 5-phosphatases (5-phosphatase) hydrolyze the 5-position phosphate from phosphatidylinositol 4,5 bisphosphate (PtdIns(4,5)P₂) and/or PtdIns(3,4,5)P₃. We demonstrate here that PIPP, an uncharacterized 5-phosphatase, hydrolyzes PtdIns(3,4,5)P₃ forming PtdIns(3,4)P₂, decreasing Ser473-Akt phosphorylation. PIPP is expressed in PC12 cells, localizing to the plasma membrane of undifferentiated cells and the neurite shaft and growth cone of NGF-differentiated neurites. Overexpression of wild-type, but not catalytically inactive PIPP, in PC12 cells inhibited neurite elongation. Targeted depletion of PIPP using RNA interference (RNAi) resulted in enhanced neurite differentiation, associated with neurite hyperelongation. Inhibition of PI3-kinase activity prevented neurite hyperelongation in PIPP-deficient cells. PIPP targeted-depletion resulted in increased phospho-Ser473-Akt and phospho-Ser9-GSK-3β, specifically at the neurite growth cone, and accumulation of PtdIns(3,4,5)P₃ at this site, associated with enhanced microtubule polymerization in the neurite shaft. PIPP therefore inhibits PI3-kinase-dependent neurite elongation in PC12 cells, via regulation of the spatial distribution of phospho-Ser473-Akt and phospho-Ser9-GSK-3β signaling.     

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.     

Regulation of the transient receptor potential channel TRPM3 by phosphoinositides

The transient receptor potential (TRP) channel TRPM3 is a calcium-permeable cation channel activated by heat and by the neurosteroid pregnenolone sulfate (PregS). TRPM3 is highly expressed in sensory neurons, where it plays a key role in heat sensing and inflammatory hyperalgesia, and in pancreatic β cells, where its activation enhances glucose-induced insulin release. However, despite its functional importance, little is known about the cellular mechanisms that regulate TRPM3 activity. Here, we provide evidence for a dynamic regulation of TRPM3 by membrane phosphatidylinositol phosphates (PIPs). Phosphatidylinositol 4,5-bisphosphate (PI[4,5]P₂) and ATP applied to the intracellular side of excised membrane patches promote recovery of TRPM3 from desensitization. The stimulatory effect of cytosolic ATP on TRPM3 reflects activation of phosphatidylinositol kinases (PI-Ks), leading to resynthesis of PIPs in the plasma membrane. Various PIPs directly enhance TRPM3 activity in cell-free inside-out patches, with a potency order PI(3,4,5)P₃ > PI(3,5)P₂ > PI(4,5)P₂ ≈ PI(3,4)P₂ >> PI(4)P_. Conversely, TRPM3 activity is rapidly and reversibly inhibited by activation of phosphatases that remove the 5-phosphate from PIPs. Finally, we show that recombinant TRPM3, as well as the endogenous TRPM3 in insuloma cells, is rapidly and reversibly inhibited by activation of phospholipase C–coupled muscarinic acetylcholine receptors. Our results reveal basic cellular mechanisms whereby membrane receptors can regulate TRPM3 activity.     

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

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

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.     

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

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