Regulation of transient receptor potential (TRP) channels by phosphoinositides

This review summarizes the modulation of transient receptor potential (TRP) channels, by phosphoinositides. TRP channels are characterized by polymodal activation and a surprising complexity of regulation mechanisms. Possibly, most if not all TRP channels are modulated by phosphoinositides. Modulation by phosphatidylinositol 4,5-biphosphate (PIP₂) has been shown in detail for TRP vanilloid (TRPV) 1, TRPV5, TRP melastatin (TRPM) 4, TRPM5, TRPM7, TRPM8, TRP polycystin 2, and the Drosophila TPR-like (TRPL) channels. This review describes mechanisms of modulation of TRP channels mainly by PIP₂ and discusses some future challenges of this fascinating topic.     

Phosphoinositide-mediated gating of inwardly rectifying K+ channels

Phosphoinositides, such as phosphatidylinositol-bisphosphate (PIP₂), control the activity of many ion channels in yet undefined ways. Inwardly, rectifying potassium (Kir) channels were the first shown to be dependent on direct interactions with phosphoinositides. Alterations in channel-PIP₂ interactions affect Kir single-channel gating behavior. Aberrations in channel-PIP₂ interactions can lead to human disease. As the activity of all Kir channels depends on their interactions with phosphoinositides, future research will aim to understand the molecular events that occur from phosphoinositide binding to channel gating. The determination of atomic resolution structures for several mammalian and bacterial Kir channels provides great promise towards this goal. We have mapped onto the three-dimensional channel structure the position of basic residues identified through mutagenesis studies that contribute to the sensitivity of a Kir channel to PIP₂. The localization of these putative PIP₂-interacting residues relative to the channel’s permeation pathway has given rise to a testable model, which could account for channel activation by PIP₂.     

Regulation of the actin cytoskeleton by phosphatidylinositol 4-phosphate 5 kinases

Phosphatidylinositol (4,5)-bisphosphate (PIP₂) is an important lipid mediator that has multiple regulatory functions. There is now increasing evidence that the phosphatidylinositol 4-phosphate 5 kinases (PIP5Ks), which synthesize PIP₂, are regulated spatially and temporally and that they have isoform-specific functions and regulations. This review will summarize the highlights of recent developments in understanding how the three major PIP5K isoforms regulate the actin cytoskeleton and other important cellular processes.     

Lipid components in the detergent-resistant membrane microdomain (DRM) obtained from the synaptic plasma membrane of rat brain

Lateral association of sphingolipids and cholesterol is considered to form membrane microdomains such as “lipid rafts” obtainable as a detergent-resistant membrane microdomain (DRM) fraction after solubilization with a non-ionic detergent and density gradient centrifugation. Since not only sphinogolipids and cholesterol, but also functional lipids such as phosphatidylinositol 4,5-bisphosphate (PIP₂) are reported to be localized in DRM prepared from several cultured cells, this domain is considered to be a platform mediating lipid-signaling. Although PIP₂ is considered to have pivotal roles in the nervous system, little information is available on the localization of PIP₂ in the DRM within the synaptic plasma membrane (SPM) obtained from matured rat brains. In this study, in order to know the localization of PIP₂ in SPM-derived DRM, we measured the amount of PIP₂ in SPM and SPM-derived DRM, by the thin-layer chromatography blotting method, using a GST-fusion protein of the pleckstrin-homology domain of phospholipase Cδ1 as a PIP₂ binding probe. About 10% of the PIP₂ in SPM was recovered in DRM. In contrast, over 40% recovery was observed for the membrane cholesterol and sphingomyelin, and about 30% recovery was observed for phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine in the DRM were detected using the thin-layer chromatography method. Since the recovery of proteins in DRM was about 10%, the result indicates that there occurs no enrichment of PIP₂ in DRM prepared from SPM.     

Complex functions of phosphatidylinositol 4,5-bisphosphate in regulation of TRPC5 cation channels

The canonical transient receptor potential (TRPC) proteins have been recognized as key players in calcium entry pathways activated through phospholipase-C-coupled receptors. While it is clearly demonstrated that members of the TRPC3/6/7 subfamily are activated by diacylglycerol, the mechanism by which phospholipase C activates members of the TRPC1/4/5 subfamily remains a mystery. In this paper, we provide evidence for both negative and positive modulatory roles for membrane polyphosphoinositides in the regulation of TRPC5 channels. Depletion of polyphosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate (PIP₂) through inhibition of phosphatidylinositol 4-kinase activates calcium entry and membrane currents in TRPC5-expressing but not in TRPC3- or TRPC7-expressing cells. Inclusion of polyphosphatidylinositol 4-phosphate or PIP₂, but not phosphatidylinositol 3,4,5-trisphosphate, in the patch pipette inhibited TRPC5 currents. Paradoxically, depletion of PIP₂ with a directed 5-phosphatase strategy inhibited TRPC5. Furthermore, when the activity of single TRPC5 channels was examined in excised patches, the channels were robustly activated by PIP₂. These findings indicate complex functions for regulation of TRPC5 by PIP₂, and we propose that membrane polyphosphoinositides may have at least two distinct functions in regulating TRPC5 channel activity. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Mohamed Trebak and Loic Lemonnier contributed equally to this work.     

Molecular characteristics of phosphoinositide binding

Phosphoinositides in general and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂ or PIP₂) in particular have been recently found to function as important regulators of ion channels. Yet, while specific residues have been identified that affect channel–PIP₂ interactions, the precise binding site of PIP₂ has not been determined in any case. In addition to binding ion channels, however, phosphoinositides interact with a plethora of other proteins, and in a number of cases, the crystallographic structures of the complexes have been determined. Based on a database of 25 complexed crystallographic structures, we have addressed the molecular characteristics of phosphoinositide binding to proteins. Implications to phosphoinositide binding to ion channels are also discussed.     

Modulation of cyclic nucleotide-regulated HCN channels by PIP2 and receptors coupled to phospholipase C

Recent results indicate that phosphoinositides, including phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂), directly enhance the opening of hyperpolarization-activated, cyclic nucleotide-regulated (HCN) channels by shifting their activation gating to more positive voltages. This contrasts with the action of phosphoinositides to inhibit the opening of the related cyclic nucleotide-gated (CNG) channels involved in sensory signaling. We both review previous studies and present new experiments that investigate whether HCN channels may be regulated by dynamic changes in PI(4,5)P₂ levels caused by the receptor-mediated activation of phospholipase C (PLC). We coexpressed HCN1 or HCN2 channels in Xenopus oocytes with the PLC-coupled bradykinin BK₂ receptor, the muscarinic M1 receptor, or the TrkA receptor. Activation of all three receptors produced a positive shift in HCN channel voltage gating, the opposite of the effect expected for PI(4,5)P₂ depletion. This action was not caused by alterations in cAMP as the effect was preserved in HCN mutant channels that fail to bind cAMP. The receptor effects were mediated by PLC activity, but did not depend on signaling through the downstream products of PI(4,5)P₂ hydrolysis: IP₃ or diacylglycerol (DAG). Importantly, the modulatory effects on gating were blocked by inhibitors of phosphatidylinositol (PI) kinases, suggesting a role for increased PI(4,5)P₂ synthesis. Finally, we found that bradykinin exerted a similar PI kinase-dependent effect on the gating of native HCN channels in cardiac sinoatrial node cells, suggesting that this pathway may represent a novel, physiologically relevant mechanism for enhancing HCN channel function.     

Regulation of the epithelial sodium channel by phosphatidylinositides: experiments, implications, and speculations

Recent studies suggest that the activity of epithelial sodium channels (ENaC) is increased by phosphatidylinositides, especially phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) and phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P₃). Stimulation of phospholipase C by either adenosine triphosphate (ATP)-activation of purinergic P2Y receptors or epidermal growth factor (EGF)-activation of EGF receptors reduces membrane PI(4,5)P₂, and consequently decreases ENaC activity. Since ATP and EGF may be trapped in cysts formed by the distal tubule, it is possible that ENaC inhibition induced by ATP and EGF facilitates cyst formation in polycystic kidney diseases (PKD). However, some results suggest that ENaC activity is increased in PKD. In contrast to P2Y and EGF receptors, stimulation of insulin-like growth factor-1 (IGF-1) receptor by aldosterone or insulin produces PI(3,4,5)P₃, and consequently increases ENaC activity. The acute effect of aldosterone on ENaC activity through PI(3,4,5)P₃ possibly accounts for the initial feedback for blood volume recovery after hypovolemic hypotension. PI(4,5)P₂ and PI(3,4,5)P₃, respectively, interacts with the N terminus of β-ENaC and the C terminus of γ-ENaC. However, whether ENaC selectively binds to PI(4,5)P₂ and PI(3,4,5)P₃ over other anionic phospholipids remains unclear.     

From cardiac cation channels to the molecular dissection of the transient receptor potential channel TRPM4

In 2006, we celebrate not only the milestone paper on the patch-clamp technique [14] but also the publication of the first single-channel measurements in cardiac cells revealing a Ca²⁺-activated, nonselective cation channel [6]. Considerable effort has been undertaken since this time to identify molecular candidates for this class of cation channels that can be found in a variety of tissues. Recent work has shown that this channel is very likely TRPM4, a member of the TRPM ion channel family. The current review links the epochal Colquhoun et al. paper to the detailed molecular knowledge and structure function aspects of this TRP channel. It will be shown that TRPM4 is a Ca²⁺- and voltage-activated channel, which is dramatically modulated by the phospholipid phosphatidyl inositol bisphosphate (PIP₂) and belongs to the heat-activated thermoTRPs. A functional hallmark of TRPM4, as for several TRP channels, is a dramatic shift of its voltage dependence towards negative, physiologically meaningful potentials.     

Does diacylglycerol regulate KCNQ channels?

Some ion channels are regulated by inositol phospholipids and by the products of cleavage by phospholipase C (PLC). KCNQ channels (Kv7) require membrane phosphatidylinositol 4,5-bisphosphate (PIP₂) and are turned off when muscarinic receptors stimulate cleavage of PIP₂ by PLC. We test whether diacylglycerols are also important in the regulation of KCNQ2/KCNQ3 channels using electrophysiology and fluorescent translocation probes as indicators for PIP₂ and diacylglycerol in tsA cells. The cells are transfected with M₁ muscarinic receptors, channel subunits, and translocation probes. Although they cause translocation of a fluorescent probe with a diacylglycerol-binding C1 domain, exogenously applied diacylglycerol (oleoyl-acetyl-glycerol and dioctanoyl glycerol) and phorbol ester do not mimic or occlude the suppression of KCNQ current by muscarinic agonist. Blocking the metabolism of endogenous diacylglycerol by inhibiting diacylglycerol kinase with R59022 or R59949 slows the decay of diacylglycerol twofold but does not mimic or occlude muscarinic regulation and recovery of current. Blocking diacylglycerol lipase with RHC-80267 also does not occlude muscarinic modulation of current. We conclude that the diacylglycerol produced during activation of PLC, any activation of protein kinase C that it may stimulate, and downstream products of its metabolism are not essential players in the acute muscarinic modulation of KCNQ channels.     

The lipid connection–regulation of voltage-gated Ca2+ channels by phosphoinositides

Recent findings have revealed a pivotal role for phospholipids phosphatidylinositol -4,5-biphosphate (PIP₂) and phosphatidylinositol -3,4,5-trisphosphate (PIP₃) in the regulation of high voltage-activated (HVA) Ca²⁺ channels. PIP₂ exerts two opposing actions on HVA Ca²⁺ channels: It stabilizes their activity but also produces a voltage-dependent inhibition that can be antagonized by protein kinase A (PKA) phosphorylation. PIP₂ depletion and arachidonic acid together mediate the slow, voltage-independent inhibition of HVA Ca²⁺ channels by G _q/11 -coupled receptors in neurons. A sufficient level of plasma membrane PIP₂ also appears to be necessary for G _βγ -mediated inhibition. On the other hand, increased production of PIP₃ by PI-3 kinases promotes trafficking of HVA Ca²⁺ channels to the plasma membrane. This review discusses these findings and their implications.     

Up- and down-regulation of the mechano-gated K2P channel TREK-1 by PIP2 and other membrane phospholipids

TREK-1 is an unconventional K⁺ channel that is activated by both physical and chemical stimuli. In this study, we show that the inner leaflet membrane phospholipids, including PIP₂, exert a mixed stimulatory and inhibitory effect on TREK-1. Intra-cellular phospholipids inhibit basal channel activity and activation by membrane stretch, intra-cellular acidosis and arachidonic acid. However, binding of endogenous negative inner leaflet phospholipids with poly-lysine reduces inhibition and reveals channel stimulation by exogenous intra-cellular phospholipids. A similar effect is observed with PI, PE, PS and PA, unlike DG, demonstrating that the phosphate at position 3 is required although the global charge of the molecule is not critical. Inhibition depends on the distal C-terminal domain that conditions channel mechano-sensitivity, but is independent of the positively charged PIP₂ stimulatory site in the proximal C-terminal domain. This is, to our knowledge, the first report of an ion channel dually regulated by membrane phospholipids.     

Channelopathies linked to plasma membrane phosphoinositides

The plasma membrane phosphoinositide phosphatidylinositol 4,5-bisphosphate (PIP₂) controls the activity of most ion channels tested thus far through direct electrostatic interactions. Mutations in channel proteins that change their apparent affinity to PIP₂ can lead to channelopathies. Given the fundamental role that membrane phosphoinositides play in regulating channel activity, it is surprising that only a small number of channelopathies have been linked to phosphoinositides. This review proposes that for channels whose activity is PIP₂-dependent and for which mutations can lead to channelopathies, the possibility that the mutations alter channel-PIP₂ interactions ought to be tested. Similarly, diseases that are linked to disorders of the phosphoinositide pathway result in altered PIP₂ levels. In such cases, it is proposed that the possibility for a concomitant dysregulation of channel activity also ought to be tested. The ever-growing list of ion channels whose activity depends on interactions with PIP₂ promises to provide a mechanism by which defects on either the channel protein or the phosphoinositide levels can lead to disease.     

Phototransduction and retinal degeneration in Drosophila

Drosophila visual transduction is the fastest known G-protein-coupled signaling cascade and has therefore served as a genetically tractable animal model for characterizing rapid responses to sensory stimulation. Mutations in over 30 genes have been identified, which affect activation, adaptation, or termination of the photoresponse. Based on analyses of these genes, a model for phototransduction has emerged, which involves phosphoinoside signaling and culminates with opening of the TRP and TRPL cation channels. Many of the proteins that function in phototransduction are coupled to the PDZ containing scaffold protein INAD and form a supramolecular signaling complex, the signalplex. Arrestin, TRPL, and Gα_q undergo dynamic light-dependent trafficking, and these movements function in long-term adaptation. Other proteins play important roles either in the formation or maturation of rhodopsin, or in regeneration of phosphatidylinositol 4,5-bisphosphate (PIP₂), which is required for the photoresponse. Mutation of nearly any gene that functions in the photoresponse results in retinal degeneration. The underlying bases of photoreceptor cell death are diverse and involve mechanisms such as excessive endocytosis of rhodopsin due to stable rhodopsin/arrestin complexes and abnormally low or high levels of Ca²⁺. Drosophila visual transduction appears to have particular relevance to the cascade in the intrinsically photosensitive retinal ganglion cells in mammals, as the photoresponse in these latter cells appears to operate through a remarkably similar mechanism.     

Short- and long-term roles of phosphatidylinositol 4,5-bisphosphate PIP2 on Cav3.1- and Cav3.2-T-type calcium channel current

T-type calcium (Ca²⁺) channels play important physiological functions in excitable cells including cardiomyocyte. Phosphatidylinositol-4,5-bisphosphate (PIP₂) has recently been reported to modulate various ion channels’ function. However the actions of PIP₂ on the T-type Ca²⁺ channel remain unclear. To elucidate possible effects of PIP₂ on the T-type Ca²⁺ channel, we applied patch clamp method to investigate recombinant Ca_V3.1- and Ca_V3.2-T-type Ca²⁺ channels expressed in mammalian cell lines with PIP₂ in acute- and long-term potentiation. Short- and long-term potentiation of PIP₂ shifted the activation and the steady-state inactivation curve toward the hyperpolarization direction of Ca_V3.1-I_Ca.T without affecting the maximum inward current density. Short- and long-term potentiation of PIP₂ also shifted the activation curve toward the hyperpolarization direction of Ca_V3.2-I_Ca.T without affecting the maximum inward current density. Conversely, long-term but not short-term potentiation of PIP₂ shifted the steady-state inactivation curve toward the hyperpolarization direction of Ca_V3.2-I_Ca.T. Long-term but not short-term potentiation of PIP₂ blunted the voltage-dependency of current decay Ca_V3.1-I_Ca.T. PIP₂ modulates Ca_V3.1- and Ca_V3.2-I_Ca.T not by their current density but by their channel gating properties possibly through its membrane-delimited actions.     

Store-operated Ca2+ entry in platelets occurs independently of transient receptor potential (TRP) C1

Changes in [Ca²⁺]_i are a central step in platelet activation. In nonexcitable cells, receptor-mediated depletion of intracellular Ca²⁺ stores triggers Ca²⁺ entry through store-operated calcium (SOC) channels. Stromal interaction molecule 1 (STIM1) has been identified as an endoplasmic reticulum (ER)-resident Ca²⁺ sensor that regulates store-operated calcium entry (SOCE), but the identity of the SOC channel in platelets has been controversially debated. Some investigators proposed transient receptor potential (TRP) C1 to fulfil this function based on the observation that antibodies against the channel impaired SOCE in platelets. However, others could not detect TRPC1 in the plasma membrane of platelets and raised doubts about the specificity of the inhibiting anti-TRPC1 antibodies. To address the role of TRPC1 in SOCE in platelets, we analyzed mice lacking TRPC1. Platelets from these mice display fully intact SOCE and also otherwise unaltered calcium homeostasis compared to wild-type. Furthermore, platelet function in vitro and in vivo is not altered in the absence of TRPC1. Finally, studies on human platelets revealed that the presumably inhibitory anti-TRPC1 antibodies have no specific effect on SOCE and fail to bind to the protein. Together, these results provide evidence that SOCE in platelets is mediated by channels other than TRPC1. David Varga-Szabo and Kalwant S. Authi contributed equally to this article.     

CaV2.1 channels are modulated by muscarinic M1 receptors through phosphoinositide hydrolysis in neostriatal neurons

In adult neostriatal projection neurons, the intracellular Ca²⁺ supplied by Ca_V2.1 (P/Q) Ca²⁺ channels is in charge of both the generation of the afterhyperpolarizing potential (AHP) and the release of GABA from their synaptic terminals, thus being a major target for firing pattern and transmitter release modulations. We have shown that activation of muscarinic M₁-class receptors modulates Ca_V2.1 channels in these neurons in rats. This modulation is reversible, is not membrane delimited, is blocked by the specific M₁-class muscarinic antagonist muscarine toxin 7 (MT-7), and is neither mediated by protein kinase C (PKC) nor by protein phosphatase 2B (PP-2B). Hence, the signaling mechanism of muscarinic Ca_V2.1 channel modulation has remained elusive. The present paper shows that inactivation of phospholipase C (PLC) abolishes this modulation while inhibition of phosphoinositide kinases, PI-3K and PI-4K, prevents its reversibility, suggesting that the reconstitution of muscarinic modulation depends on phosphoinositide rephosphorylation. In support of this hypothesis, the supply of intracellular phosphatidylinositol (4,5) bisphosphate [PI(4,5)P₂] blocked all muscarinic modulation of this channel. The results indicate that muscarinic M₁ modulation of Ca_V2.1 Ca²⁺ channels in these neurons involves phosphoinositide hydrolysis.     

Mechanisms for Kir channel inhibition by quinacrine: acute pore block of Kir2.x channels and interference in PIP<Subscript>2</Subscript> interaction with Kir2.x and Kir6.2 channels

Cardiac inward rectifier potassium currents determine the resting membrane potential and contribute repolarization capacity during phase 3 repolarization. Quinacrine is a cationic amphiphilic drug. In this work, the effects of quinacrine were studied on cardiac Kir channels expressed in HEK 293 cells and on the inward rectifier potassium currents, I_K1 and I_KATP, in cardiac myocytes. We found that quinacrine differentially inhibited Kir channels, Kir6.2 ∼ Kir2.3 > Kir2.1. In addition, we found in cardiac myocytes that quinacrine inhibited I_KATP > I_K1. We presented evidence that quinacrine displays a double action towards strong inward rectifier Kir2.x channels, i.e., direct pore block and interference in phosphatidylinositol 4,5-bisphosphate, PIP₂–Kir channel interaction. Pore block is evident in Kir2.1 and 2.3 channels as rapid block; channel block involves residues E224 and E299 facing the cytoplasmic pore of Kir2.1. The interference of the drug with the interaction of Kir2.x and Kir6.2/SUR2A channels and PIP₂ is suggested from four sources of evidence: (1) Slow onset of current block when quinacrine is applied from either the inside or the outside of the channel. (2) Mutation of Kir2.3(I213L) and mutation of Kir6.2(C166S) increase their affinity for PIP₂ and lowers its sensitivity for quinacrine. (3) Mutations of Kir2.1(L222I and K182Q) which decreased its affinity for PIP₂ increased its sensitivity for quinacrine. (4) Co-application of quinacrine with PIP₂ lowers quinacrine-mediated current inhibition. In conclusion, our data demonstrate how an old drug provides insight into a dual a blocking mechanism of Kir carried inward rectifier channels.     

Modulation of the transient receptor potential channel TRPA1 by phosphatidylinositol 4,5-biphosphate manipulators

The transient receptor potential channel of the ankyrin-binding repeat subfamily, TRPA1, is a Ca(2+)-permeable non-selective cation channel that depolarizes the plasma membrane and causes Ca(2+) influx. A typical feature of TRPA1 is its rapid desensitization following activation by agonists such as mustard oil (MO), cinnamaldehyde, and a high intracellular Ca(2+) concentration. In whole-cell recordings on Chinese hamster ovary (CHO) cells expressing TRPA1, desensitization was delayed when phosphatidylinositol 4,5-biphosphate (PIP(2)) was supplemented via the patch pipette, whereas the PIP(2) scavenger neomycin accelerated desensitization. Preincubation with the PI-4 kinase inhibitor wortmannin reduced both constitutive TRPA1 channels activity and the response to MO. Run down was also accelerated by high intracellular Mg(2+) concentrations, whereas chelating intracellular Mg(2+) with 10 mM ethylenedinitrilotetraacetic acid (EDTA) increased the basal channel activity. In inside-out patches, we observed a rapid run down of TRPA1 activity, which could be prevented by application of diC8-PIP(2) or 2 mM Mg-ATP but not Na(2)-ATP to the cytosolic side of the excised patches. In isolated trigeminal ganglion neurons, preincubation with wortmannin resulted in inhibition of endogenous TRPA1 activation by MO. Taken together, our data indicate that PIP(2) modulates TRPA1, albeit to a lesser extent than other known PIP(2)-dependent TRP channels, and that tools modifying the plasma membrane PIP(2) content often have direct effects on this channel.     

New developments in the signaling mechanisms of the store-operated calcium entry pathway

The most ubiquitous pathway for regulated calcium (Ca²⁺) entry into the cells is the store-operated Ca²⁺ (SOC) entry pathway (also called capacitative Ca²⁺ entry) that is conserved from lower organisms such as yeast, worms, and flies to man. The SOC concept was proposed over two decades ago, and SOC channels are defined by their activation in response to depletion of the internal Ca²⁺ stores. Influx through SOC channels is necessary for the replenishment of the Ca²⁺ stores and is also involved in cell signaling to the nucleus. Despite intensive investigations, most of which are focusing on transient receptor potential (TRP) channels as molecular candidates for SOC channels, the mechanisms of activation and the identity of the key molecular players participating in this signaling pathway have long remained elusive. In the last 2–3 years, however, the improvements of RNA silencing protocols combined with high throughput platforms have yielded significant breakthroughs, with the identification of Stim1 as the Ca²⁺ store sensor and Orai1 (CRACM1) as the pore-forming subunit of the archetypical SOC channel, CRAC. This review summarizes the recent advances in the mechanisms of activation of SOC channels and their molecular composition, with emphasis on the roles of Stim, Orai, and TRP proteins.