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.     

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.     

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.     

A conserved gating element in TRPV6 channels

The Ca²⁺-selective tetrameric Transient Receptor Potential Vanilloid 6 (TRPV6) channel is an inwardly rectifying ion channel. The constitutive current endures Ca²⁺-induced inactivation as a result of the activation of phospholipase C followed depletion of phosphatidylinositol 4,5-bisphosphate, and calmodulin binding. Replacing a glycine residue within the cytosolic S4-S5 linker of the human TRPV6 protein, glycine 516, which is conserved in all TRP channel proteins, by a serine residue forces the channels into an open conformation thereby enhancing constitutive Ca²⁺ entry and preventing inactivation. Introduction of a second mutation (T621A) into TRPV6_G516S reduces constitutive activity and partially rescues the TRPV6 function. According to the recently revealed crystal structure of the rat TRPV6 the T621 is adjacent to the distal end of the transmembrane segment 6 (S6) within a short linker between S6 and the helix formed by the TRP domain. These results indicate that the S4-S5 linker and the S6-TRP-domain linker are critical constituents of TRPV6 channel gating and that disturbance of their sequences foster constitutive Ca²⁺ entry.     

Conserved residues within the putative S4-S5 region serve distinct functions among thermosensitive vanilloid transient receptor potential (TRPV) channels

The vanilloid transient receptor potential channel TRPV1 is a tetrameric six-transmembrane segment (S1-S6) channel that can be synergistically activated by various proalgesic agents such as capsaicin, protons, heat, or highly depolarizing voltages, and also by 2-aminoethoxydiphenyl borate (2-APB), a common activator of the related thermally gated vanilloid TRP channels TRPV1, TRPV2, and TRPV3. In these channels, the conserved charged residues in the intracellular S4-S5 region have been proposed to constitute part of a voltage sensor that acts in concert with other stimuli to regulate channel activation. The molecular basis of this gating event is poorly understood. We mutated charged residues all along the S4 and the S4-S5 linker of TRPV1 and identified four potential voltage-sensing residues (Arg(557), Glu(570), Asp(576), and Arg(579)) that, when specifically mutated, altered the functionality of the channel with respect to voltage, capsaicin, heat, 2-APB, and/or their interactions in different ways. The nonfunctional charge-reversing mutations R557E and R579E were partially rescued by the charge-swapping mutations R557E/E570R and D576R/R579E, indicating that electrostatic interactions contribute to allosteric coupling between the voltage-, temperature- and capsaicin-dependent activation mechanisms. The mutant K571E was normal in all aspects of TRPV1 activation except for 2-APB, revealing the specific role of Lys(571) in chemical sensitivity. Surprisingly, substitutions at homologous residues in TRPV2 or TRPV3 had no effect on temperature- and 2-APB-induced activity. Thus, the charged residues in S4 and the S4-S5 linker contribute to voltage sensing in TRPV1 and, despite their highly conserved nature, regulate the temperature and chemical gating in the various TRPV channels in different ways.     

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.     

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.     

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.     

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

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

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.     

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.     

Energetics and Location of Phosphoinositide Binding in Human Kir2.1 Channels

Kir2.1 channels are uniquely activated by phosphoinositide 4,5-bisphosphate (PI(4,5)P₂) and can be inhibited by other phosphoinositides (PIPs). Using biochemical and computational approaches, we assess PIP-channel interactions and distinguish residues that are energetically critical for binding from those that alter PIP sensitivity by shifting the open-closed equilibrium. Intriguingly, binding of each PIP is disrupted by a different subset of mutations. In silico ligand docking indicates that PIPs bind to two sites. The second minor site may correspond to the secondary anionic phospholipid site required for channel activation. However, 96–99% of PIP binding localizes to the first cluster, which corresponds to the general PI(4,5)P₂ binding location in recent Kir crystal structures. PIPs can encompass multiple orientations; each di- and triphosphorylated species binds with comparable energies and is favored over monophosphorylated PIPs. The data suggest that selective activation by PI(4,5)P₂ involves orientational specificity and that other PIPs inhibit this activation through direct competition.     

Eisosomes Regulate Phosphatidylinositol 4,5-Bisphosphate (PI(4,5)P2) Cortical Clusters and Mitogen-activated Protein (MAP) Kinase Signaling upon Osmotic Stress

Eisosomes are multiprotein structures that generate linear invaginations at the plasma membrane of yeast cells. The core component of eisosomes, the BAR domain protein Pil1, generates these invaginations through direct binding to lipids including phosphoinositides. Eisosomes promote hydrolysis of phosphatidylinositol 4,5 bisphosphate (PI(4,5)P₂) by functioning with synaptojanin, but the cellular processes regulated by this pathway have been unknown. Here, we found that PI(4,5)P₂ regulation by eisosomes inhibits the cell integrity pathway, a conserved MAPK signal transduction cascade. This pathway is activated by multiple environmental conditions including osmotic stress in the fission yeast Schizosaccharomyces pombe. Activation of the MAPK Pmk1 was impaired by mutations in the phosphatidylinositol (PI) 5-kinase Its3, but this defect was suppressed by removal of eisosomes. Using fluorescent biosensors, we found that osmotic stress induced the formation of PI(4,5)P₂ clusters that were spatially organized by eisosomes in both fission yeast and budding yeast cells. These cortical clusters contained the PI 5-kinase Its3 and did not assemble in the its3-1 mutant. The GTPase Rho2, an upstream activator of Pmk1, also co-localized with PI(4,5)P₂ clusters under osmotic stress, providing a molecular link between these novel clusters and MAPK activation. Our findings have revealed that eisosomes regulate activation of MAPK signal transduction through the organization of cortical lipid-based microdomains.     

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

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

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

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

Regulation of voltage-gated potassium channels by PI(4,5)P2

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) regulates activities of numerous ion channels including inwardly rectifying potassium (K_ir) channels, KCNQ, TRP, and voltage-gated calcium channels. Several studies suggest that voltage-gated potassium (K_V) channels might be regulated by PI(4,5)P₂. Wide expression of K_V channels in different cells suggests that such regulation could have broad physiological consequences. To study regulation of K_V channels by PI(4,5)P₂, we have coexpressed several of them in tsA-201 cells with a G protein–coupled receptor (M₁R), a voltage-sensitive lipid 5-phosphatase (Dr-VSP), or an engineered fusion protein carrying both lipid 4-phosphatase and 5-phosphatase activity (pseudojanin). These tools deplete PI(4,5)P₂ with application of muscarinic agonists, depolarization, or rapamycin, respectively. PI(4,5)P₂ at the plasma membrane was monitored by Förster resonance energy transfer (FRET) from PH probes of PLCδ1 simultaneously with whole-cell recordings. Activation of Dr-VSP or recruitment of pseudojanin inhibited K_V7.1, K_V7.2/7.3, and K_ir2.1 channel current by 90–95%. Activation of M₁R inhibited K_V7.2/7.3 current similarly. With these tools, we tested for potential PI(4,5)P₂ regulation of activity of K_V1.1/K_Vβ1.1, K_V1.3, K_V1.4, and K_V1.5/K_Vβ1.3, K_V2.1, K_V3.4, K_V4.2, K_V4.3 (with different KChIPs and DPP6-s), and hERG/KCNE2. Interestingly, we found a substantial removal of inactivation for K_V1.1/K_Vβ1.1 and K_V3.4, resulting in up-regulation of current density upon activation of M₁R but no changes in activity upon activating only VSP or pseudojanin. The other channels tested except possibly hERG showed no alteration in activity in any of the assays we used. In conclusion, a depletion of PI(4,5)P₂ at the plasma membrane by enzymes does not seem to influence activity of most tested K_V channels, whereas it does strongly inhibit members of the K_V7 and K_ir families.     

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.     

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.     

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.     

Profilin Interaction with Phosphatidylinositol (4,5)-Bisphosphate Destabilizes the Membrane of Giant Unilamellar Vesicles

Profilin, a small cytoskeletal protein, and phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P₂] have been implicated in cellular events that alter the cell morphology, such as endocytosis, cell motility, and formation of the cleavage furrow during cytokinesis. Profilin has been shown to interact with PI(4,5)P₂, but the role of this interaction is still poorly understood. Using giant unilamellar vesicles (GUVs) as a simple model of the cell membrane, we investigated the interaction between profilin and PI(4,5)P₂. A number and brightness analysis demonstrated that in the absence of profilin, molar ratios of PI(4,5)P₂ above 4% result in lipid demixing and cluster formations. Furthermore, adding profilin to GUVs made with 1% PI(4,5)P₂ leads to the formation of clusters of both profilin and PI(4,5)P₂. However, due to the self-quenching of the dipyrrometheneboron difluoride-labeled PI(4,5)P₂, we were unable to determine the size of these clusters. Finally, we show that the formation of these clusters results in the destabilization and deformation of the GUV membrane.