Derived (mutated)-types of TRPV6 channels elicit greater Ca²+ influx into the cells than ancestral-types of TRPV6: evidence from Xenopus oocytes and mammalian cell expression system

The frequency of the allele containing three derived nonsynonymous SNPs (157C, 378M, 681M) of the gene encoding calcium permeable TRPV6 channels expressed in the intestine has been increased by positive selection in non-African populations. To understand the nature of these SNPs, we compared the properties of Ca2+ influx of ancestral (in African populations) and derived-TRPV6 (in non-African populations) channels with electrophysiological, Ca2+-imaging, and morphological methods using both the Xenopus oocyte and mammalian cell expression systems. Functional electrophysiological and Ca2+-imaging analyses indicated that the derived-TRPV6 elicited more Ca2+ influx than the ancestral one in TRPV6-expressing cells where both channels were equally expressed in the cells. Ca2+-inactivation properties in the ancestral- and derived-TRPV6 were almost the same. Furthermore, fluorescence resonance energy transfer (FRET) analysis showed that both channels have similar multimeric formation properties, suggesting that derived-TRPV6 itself could cause higher Ca2+ influx. These findings suggest that populations having derived-TRPV6 in non-African areas may absorb higher Ca2+ from the intestine than ancestral-TRPV6 in the African area.     

TRP channels in lymphocytes

TRP proteins form ion channels that are activated following receptor stimulation. Several members of the TRP family are likely to be expressed in lymphocytes. However, in many studies, messenger RNA (mRNA) but not protein expression was analyzed and cell lines but not primary human or murine lymphocytes were used. Among the expressed TRP mRNAs are TRPC1, TRPC3, TRPM2, TRPM4, TRPM7, TRPV1, and TRPV2. Regulation of Ca2+ entry is a key process for lymphocyte activation, and TRP channels may both increase Ca2+ influx (such as TRPC3) or decrease Ca2+ influx through membrane depolarization (such as TRPM4). In the future, linking endogenous Ca2+/cation channels in lymphocytes with TRP proteins should lead to a better molecular understanding of lymphocyte activation.     

TRP channels in platelet function

Ca2+ entry forms an essential component of platelet activation; however, the mechanisms associated with this process are not understood. Ca2+ entry upon receptor activation occurs as a consequence of intracellular store depletion (referred to as store-operated Ca2+ entry or SOCE), a direct action of second messengers on cation entry channels or the direct occupancy of a ligand-gated P2(Xi) receptor. The molecular identity of the SOCE channel has yet to be established. Transient receptor potential (TRP) proteins are candidate cation entry channels and are classified into a number of closely related subfamilies including TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPP (polycystin) and TRPML (mucolipins). From the TRPC family, platelets have been shown to express TRPC6 and TRPC1, and are likely to express other TRPC and other TRP members. TRPC6 is suggested to be involved with receptor-activated, diacyl-glycerol-mediated cation entry. TRPC1 has been suggested to be involved with SOCE, though many of the suggested mechanisms remain controversial. As no single TRP channel has the properties described for SOCE in platelets, it is likely that it is composed of a heteromeric association of TRP and related subunits, some of which may be present in intracellular compartments in the resting cell.     

Capsaicin stimulates the non-store-operated Ca2+ entry but inhibits the store-operated Ca2+ entry in neutrophils

Rat neutrophils express the mRNA encoding for transient receptor potential (TRP) V1. However, capsaicin-stimulated [Ca2+]i elevation occurred only at high concentrations (> or = 100 microM). This response was substantially decreased in a Ca2+-free medium. Vanilloids displayed similar patterns of Ca2+ response with the rank order of potency as follows: scutigeral>resiniferatoxin>capsazepine>capsaicin=olvanil>isovelleral. Arachidonyl dopamine (AAD), an endogenous ligand for TRPV1, failed to desensitize the subsequent capsaicin challenge. Capsaicin-induced Ca2+ response was not affected by 8-bromo-cyclic ADP-ribose (8-Br-cADPR), the ryanodine receptor blocker, but was slightly attenuated by 1-[6-[17beta-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U-73122), the inhibitor of phospholipase C-coupled processes, 1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole (SKF-96365), the blocker of receptor-gated and store-operated Ca2+ (SOC) channels, 2-aminoethyldiphenyl borate (2-APB), the blocker of D-myo-inositol 1,4,5-trisphospahte (IP3) receptor and Ca2+ influx, and by ruthenium red, a blocker of TRPV channels, and enhanced by the Ca2+ channels blocker, cis-N-(2-phenylcyclopentyl)azacyclotridec-1-en-2-amine (MDL-12330A) and Na+-deprivation. In addition, capsaicin had no effect on the plasma membrane Ca2+-ATPase activity or the production of nitric oxide (NO) and reactive oxygen intermediates (ROI) or on the total thiols content. Capsaicin (> or = 100 microM) inhibited the cyclopiazonic acid (CPA)-induced store-operated Ca2+ entry (SOCE). In the absence of external Ca2+, the robust Ca2+ entry after subsequent addition of Ca2+ was decreased by capsaicin in CPA-activated cells. Capsaicin alone increased the actin cytoskeleton, and also increased the actin filament content in cell activation with CPA. These results indicate that capsaicin activates a TRPV1-independent non-SOCE pathway in neutrophils. The reorganization of the actin cytoskeleton is probably involved in the capsaicin inhibition of SOCE.     

TRPV4

TRPV4 is a non-selective cation channel subunit expressed in a wide variety of tissues. TRP channels are formed by a tetrameric complex of channel subunits. The available evidence suggests that TRPV4 cannot form heteromultimers with other TRPV isoforms, and that TRPV4-containing channels are homotetramers. These channels have a characteristic outwardly rectifying current-voltage relation, and are 5-10 times more permeable for Ca2+ than for Na+. TRPV4 can be activated by a wide range of stimuli including physical (cell swelling, heat, mechanical stimulation) and chemical stimuli (endocannabinoids, arachidonic acid, and, surprisingly, 4alpha-phorbol esters). Activation by swelling and endocannabinoids involves cytochrome P450 epoxygenase-dependent arachidonic acid metabolism to the epoxyeicosatrienoic acids (EETs). Heat and 4alpha-phorbol esters also seem to share a common mechanism of activation, but the endogenous messenger involved in the response to heat has not yet been identified. Ca2+ acting from the intracellular side can have both potentiating and inhibitory effects on channel activity and is involved in channel activation and inactivation. Given its wide expression and the variety of activatory stimuli, TRPV4 is likely to play a number of physiological roles. Studies with TRPV4(-/-) mice suggest a role for the channel in the regulation of body osmolarity, mechanosensation, temperature sensing, vascular regulation and, possibly, hearing.     

TRPV5, the gateway to Ca2+ homeostasis

Ca2+ homeostasis in the body is tightly controlled, and is a balance between absorption in the intestine, excretion via the urine, and exchange from bone. Recently, the epithelial Ca2+ channel (TRPV5) has been identified as the gene responsible for the Ca2+ influx in epithelial cells of the renal distal convoluted tubule. TRPV5 is unique within the family of transient receptor potential (TRP) channels due to its high Ca2+ selectivity. Ca2+ flux through TRPV5 is controlled in three ways. First, TRPV5 gene expression is regulated by calciotropic hormones such as vitamin D3 and parathyroid hormone. Second, Ca2+ transport through TRPV5 is controlled by modulating channel activity. Intracellular Ca2+, for example, regulates channel activity by feedback inhibition. Third, TRPV5 is controlled by mobilization of the channel through trafficking toward the plasma membrane. The newly identified anti-aging hormone Klotho regulates TRPV5 by cleaving off sugar residues from the extracellular domain of the protein, resulting in a prolonged expression of TRPV5 at the plasma membrane. Inactivation of TRPV5 in mice leads to severe hypercalciuria, which is compensated by increased intestinal Ca2+ absorption due to augmented vitamin D3 levels. Furthermore, TRPV5 deficiency in mice is associated with polyuria, urine acidification, and reduced bone thickness. Some pharmaceutical compounds, such as the immunosuppressant FK506, affect the Ca2+ balance by modulating TRPV5 gene expression. This underlines the importance of elucidating the role of TRPV5 in Ca(2+)-related disorders, thereby enhancing the possibilities for pharmacological intervention. This chapter describes a unique TRP channel and highlights its regulation and function in renal Ca2+ reabsorption and overall Ca2+ homeostasis.     

TRPV channels' role in osmotransduction and mechanotransduction

In signal transduction of metazoan cells, transient receptor potential (TRP) ion channels have been identified that respond to diverse external and internal stimuli, among them osmotic and mechanical stimuli. This chapter will summarize findings on the TRPV subfamily, both its vertebrate and invertebrate members. Of the six mammalian TRPV channels, TRPV1, -V2, and -V4 were demonstrated to function in transduction of osmotic and/or mechanical stimuli. TRPV channels have been found to function in cellular as well as systemic osmotic homeostasis in vertebrates. Invertebrate TRPV channels, five in Caenorhabditis elegans and two in Drosophila, have been shown to play a role in mechanosensation, such as hearing and proprioception in Drosophila and nose touch in C. elegans, and in the response to osmotic stimuli in C. elegans. In a striking example of evolutionary conservation of function, mammalian TRPV4 has been found to rescue mechanosensory and osmosensory deficits of the TRPV mutant line osm-9 in C. elegans, despite no more than 26% orthology of the respective amino acid sequences.     

TRP channels in normal and dystrophic skeletal muscle

TRP proteins constitute non-selective cation-permeable ion channels, most of which are permeable to Ca2?. In skeletal muscle, several isoforms of the TRPC (Canonical), TRPV (Vanilloid) and TRPM (Melastatin) subfamilies are expressed. In particular, TRPC1, C3 and C6, TRPV2 and V4, TRPM4 and TRPM7 have been consistently found in cultured myoblasts or in adult muscles. These channels seem to directly or indirectly respond to membrane stretch or to Ca2? stores depletion; some isoforms might also constitute unregulated Ca2? leak channels. Their function is largely unknown. TRPC1 and C3 have been involved in muscle development, in particular in myoblasts migration and differentiation. TRPC1 and V4 might allow a basal influx of Ca2? at rest. Their lack has consequences on muscle fatigue. TRPV2 seems to be stretch-sensitive. It localizes mainly in intracellular pools at rest, and translocates to the plasma membrane upon IGF-1 stimulation. TRP channels seem to be involved in the pathophysiology of muscle disorders. In particular in Duchenne muscular dystrophy, the lack of the cytoskeletal protein dystrophin induces a disregulation of several ion channels leading to an abnormal influx of Ca2?. We discuss here, the possible involvement of TRP channels in this abnormal influx of Ca2?.     

TRP channels as a newly emerging non-voltage-gated CA2+ entry channel superfamily

It has long been known that many chemical and physical stimuli imposed on the cell from its exterior environments elicit a long-lasting Ca2+ influx through yet poorly elucidated transmembrane pathways distinct from voltage-gated and fast ligand-gated Ca2+ entry channels, thereby activating and modulating a variety of cellular functions. Recent progress in molecularly identifying these pathways, initiated from the discovery of Drosophila's visual transduction mutants transient receptor potential (TRP) proteins, has begun to reveal the presence of an enormous superfamily of non-voltage-gated Ca2+ channels. The mammalian members of TRP superfamily are (except for two members) Ca2+-permeable non-selective cation channels which are constitutively active or gated by a multitude of physicochemical stimuli such as receptor stimulation, phospholipids, oxidants, pheromones, cell volume change/shear stress, exogenous compounds affecting sensations, and changes in ambient temperature, acidity and osmolarity and cellular metabolic status. Owing to these diversities in activation and their broad distribution from brain to peripheral organs and tissues, TRP channels are now thought to be involved in divergent physiological functions including; pain and taste transductions; thermo- and mechano-sensations; regulation of mineral absorption/reabsorption; blood pressure, gut motility and airway responsiveness; cell proliferation/death, some of which seem tightly associated with specific genetic disorders. These features will render TRP channels the attractive novel molecular targets for future drug therapy. This paper briefly overviews the current knowledge available for these channels with a main interest in their possible linkage with in vivo function.     

化疗引起的神经痛治疗靶点温度选择性TRP通道的研究进展

作为钙离子渗透性的瞬时受体电位(TRP),5种通道(TRPV1~4和TRPM2)被不同的高温激活,两种通道(TRPV1和TRPV8)被低温激活。越来越多的证据表明,TRPA1和TRPM8拮抗剂可预防顺铂、奥沙利铂和紫杉醇诱导的线粒体氧化应激、炎症、冷痛和痛觉过敏。TRPV1在顺铂引起的感觉神经元热痛觉和机械异常中有应答。TRPA1、TRPM8和TRPV2蛋白表达水平主要通过这些治疗方法在背根(DRG)和三叉神经节中增加。主要总结了5种温度调节TRP通道(TRPA1、TRPM8、TRPV1、TRPV2和TRPV4)。     

Activation of the melastatin-related cation channel TRPM3 by D-erythro-sphingosine [corrected

TRPM3, a member of the melastatin-like transient receptor potential channel subfamily (TRPM), is predominantly expressed in human kidney and brain. TRPM3 mediates spontaneous Ca2+ entry and nonselective cation currents in transiently transfected human embryonic kidney 293 cells. Using measurements with the Ca2+-sensitive fluorescent dye fura-2 and the whole-cell patch-clamp technique, we found that D-erythro-sphingosine, a metabolite arising during the de novo synthesis of cellular sphingolipids, activated TRPM3. Other transient receptor potential (TRP) channels tested [classic or canonical TRP (TRPC3, TRPC4, TRPC5), vanilloid-like TRP (TRPV4, TRPV5, TRPV6), and melastatin-like TRP (TRPM2)] did not significantly respond to application of sphingosine. Sphingosine-induced TRPM3 activation was not mediated by inhibition of protein kinase C, depletion of intracellular Ca2+ stores, and intracellular conversion of sphingosine to sphingosine-1-phosphate. Although sphingosine-1-phosphate and ceramides had no effect, two structural analogs of sphingosine, dihydro-D-erythro-sphingosine and N,N-dimethyl-D-erythro-sphingosine, also activated TRPM3. Sphingolipids, including sphingosine, are known to have inhibitory effects on a variety of ion channels. Thus, TRPM3 is the first ion channel activated by sphingolipids.     

TRPC channels: interacting proteins

TRP channels, in particular the TRPC and TRPV subfamilies, have emerged as important constituents of the receptor-activated Ca2+ influx mechanism triggered by hormones, growth factors, and neurotransmitters through activation ofphospholipase C (PLC). Several TRPC channels are also activated by passive depletion of endoplasmic reticulum (ER) Ca2+. Although in several studies the native TRP channels faithfully reproduce the respective recombinant channels, more often the properties of Ca2+ entry and/or the store-operated current are strikingly different from that of the TRP channels expressed in the same cells. The present review aims to discuss this disparity in the context of interaction of TRPC channels with auxiliary proteins that may alter the permeation and regulation of TRPC channels.     

Receptor-activated Ca2+ influx: capacitative Ca2+ entry and TRP proteins]

Receptor-activated Ca2+ channels (RACC) are triggered in response to activation of G protein-coupled receptors or tyrosine kinase-coupled receptors. RACCs, together with voltage-dependent Ca2+ channels, form physiologically the most important Ca2+ influx pathways, being highly diverged in activation mechanisms and Ca2+ permeability. Characterization of mammalian homologues of Drosophila TRP proteins has been an important clue for understanding molecular mechanisms underlying receptor-activated Ca2+ influx in vertebrate cells. Recent issues have been whether any members of the TRP family form capacitative Ca2+ entry (CCE) channels activated by release of Ca2+ from internal stores and their depletion. We have isolated cDNAs that encode seven mouse TRP homologues, TRP1-7. TRP homologues are distributed differently among tissues, although they are all abundant in the brain. Functional characterization of TRP proteins recombinantly expressed in HEK cells indicate that TRP5 is highly permeable to Ca2+, while TRP3 and 7 are non-selective cation channels. The results demonstrate that TRP3,5,7 are capable of generating Ca2+ currents after desensitization of the stimulated G-protein-coupled receptors and replenishment of stores, suggesting that store depletion is not necessary to maintain activity of the TRP homologues. Ca2+ positively regulates TRP channels through Ca(2+)-calmodulin pathways, but via different Ca(2+)-calmodulin-dependent enzymes. Thus, activation of TRP channels is not tightly coupled with store depletion as CCE, suggesting that CCE (or CRAC) channels are molecular entities separate from TRP.     

The TRPM ion channel subfamily: molecular, biophysical and functional features

Significant progress in the molecular and functional characterization of a subfamily of genes that encode melastatin-related transient receptor potential (TRPM) cation channels has been made during the past few years. This subgroup of the TRP superfamily of ion channels contains eight mammalian members and has isoforms in most eukaryotic organisms. The individual members of the TRPM subfamily have specific expression patterns and ion selectivity, and their specific gating and regulatory mechanisms are tailored to integrate multiple signaling pathways. The diverse functional properties of these channels have a profound effect on the regulation of ion homoeostasis by mediating direct influx of Ca2+, controlling Mg2+ entry, and determining the potential of the cell membrane. TRPM channels are involved in several physiological and pathological conditions in electrically excitable and non-excitable cells, which make them exciting targets for drug discovery.     

Transient receptor potential (TRP) channels, vascular tone and autoregulation of cerebral blood flow

Members of the transient receptor potential (TRP) channel superfamily are present in vascular smooth muscle cells and play important roles in the regulation of vascular contractility. The TRPC3 and TRPC6 channels are activated by stimulation of several excitatory receptors in vascular smooth muscle cells. Activation of these channels leads to myocyte depolarization, which stimulates Ca2+ entry via voltage-dependent Ca2+ channels (VDCC), leading to vasoconstriction. The TRPV4 channels in arterial myocytes are activated by epoxyeicosatrienoic acids, and activation of the channels enhances Ca2+ spark and transient Ca2+-sensitive K+ channel activity, thereby hyperpolarizing and relaxing vascular smooth muscle cells. The TRPC6 and TRPM4 channels are activated by mechanical stimulation of cerebral artery myocytes. Subsequent depolarization and activation of VDCC Ca2+ entry is directly linked to the development of myogenic tone in vitro and to autoregulation of cerebral blood flow in vivo. These findings imply a fundamental importance of TRP channels in the regulation of vascular smooth muscle tone and suggest that TRP channels could be important targets for drug therapy under conditions in which vascular contractility is disturbed (e.g. hypertension, stroke, vasospasm).     

Involvement of transient receptor potential vanilloid subtype 1 in analgesic action of methylsalicylate

Methylsalicylate (MS) is a naturally occurring compound that is used as a major active ingredient of balms and liniments supplied as topical analgesics. Despite the common use of MS as a pain reliever, the underlying molecular mechanism is not fully understood. Here we characterize the action of MS on transient receptor potential V1 (TRPV1). In human embryonic kidney 293 cells expressing human TRPV1 (hTRPV1), MS evoked increases of [Ca(2+)](i), which declined regardless of its continuous presence, indicative of marked desensitization. TRPV1 antagonists dose-dependently suppressed the MS-induced [Ca(2+)](i) increase. MS simultaneously elicited an inward current and increase of [Ca(2+)](i) in the voltage-clamped cells, suggesting that MS promoted Ca(2+) influx through the activation of TRPV1 channels. MS reversibly inhibited hTRPV1 activation by polymodal stimuli such as capsaicin, protons, heat, anandamide, and 2-aminoethoxydiphenyl borate. Because both the stimulatory and inhibitory actions of MS were exhibited in capsaicin- and allicin-insensitive mutant channels, MS-induced hTRPV1 activation was mediated by distinct channel regions from capsaicin and allicin. In cultured rat sensory neurons, MS elicited a [Ca(2+)](i) increase in cells responding to capsaicin. MS significantly suppressed nocifensive behavior induced by intraplantar capsaicin in rats. The present data indicate that MS has both stimulatory and inhibitory actions on TRPV1 channels and suggest that the latter action may partly underlie the analgesic effects of MS independent of inhibition of cyclooxygenases in vivo.     

Surfactant-induced TRPV1 activity--a novel mechanism for eye irritation?

The pain receptor transient receptor potential vanilloid type 1 (TRPV1) has been reported as one of the key components in the pain pathway. Activation of the receptor causes a Ca2+ influx with secondary effects leading to neurogenic inflammation. Here we report specific activation of TRPV1 by detergent-containing hygiene products measured as intracellular Ca2+ influxes in stably TRPV1-expressing neuroblastoma SH-SY5Y cells. Children products marketed as "painless" (containing lower concentration of detergents), and conditioners (without detergents) did not induce specific TRPV1 activation. Furthermore, low concentrations of the detergent sodium lauryl sulfate dose-dependently induced Ca2+ influxes that could be addressed to TRPV1. These results reveal a novel mechanistic pathway for surfactant-induced nociception, which may be an important endpoint in in vitro test batteries as alternatives to Draize's rabbit eye test for classification of eye irritating products.     

The M-channel blocker linopirdine is an agonist of the capsaicin receptor TRPV1

Linopirdine is a well known blocker of voltage-gated potassium channels from the Kv7 (or KCNQ) family that generate the so called M current in mammalian neurons. Kv7 subunits are also expressed in pain-sensing neurons in dorsal root ganglia, in which they modulate neuronal excitability. In this study we demonstrate that linopirdine acts as an agonist of TRPV1 (transient receptor potential vanilloid type 1), another ion channel expressed in nociceptors and involved in pain signaling. Linopirdine induces increases in intracellular calcium concentration in human embryonic kidney 293 (HEK293) cells expressing TRPV1, but not TRPA1 and TRPM8 or in wild-type HEK293 cells. Linopirdine also activates an inward current in TRPV1-expressing HEK293 cells that is almost completely blocked by the selective TRPV1 antagonist capsazepine. At low concentrations linopirdine sensitizes both recombinant and native TRPV1 channels to heat, in a manner that is not prevented by the Kv7-channel opener flupirtine. Taken together, these results indicate that linopirdine exerts an excitatory action on mammalian nociceptors not only through inhibition of the M current but also through activation of the capsaicin receptor TRPV1.     

TRPC3: a multifunctional, pore-forming signalling molecule

TRPC3 represents one of the first identified mammalian relatives of the Drosophila trp gene product. Despite intensive biochemical and biophysical characterization as well as numerous attempts to uncover its physiological role in native cell systems, this channel protein still represents one of the most enigmatic members of the transient receptor potential (TRP) superfamily. TRPC3 is significantly expressed in brain and heart and likely to play a role in both non-excitable as well as excitable cells, being potentially involved in a wide spectrum of Ca2+ signalling mechanisms. Its ability to associate with a variety of partner proteins apparently enables TRPC3 to form different cation channels in native cells. TRPC3 cation channels display unique gating and regulatory properties that allow for recognition and integration of multiple input stimuli including lipid mediators and cellular Ca2+ gradients as well as redox signals. The physiological/pathophysiological functions of this highly versatile cation channel protein are as yet barely delineated. Here we summarize current knowledge on properties and possible signalling functions of TRPC3 and discuss the potential biological relevance of this signalling molecule.     

TRP channels in neurogastroenterology: opportunities for therapeutic intervention

The members of the superfamily of transient receptor potential (TRP) cation channels are involved in a plethora of cellular functions. During the last decade, a vast amount of evidence is accumulating that attributes an important role to these cation channels in different regulatory aspects of the alimentary tract. In this review we discuss the expression patterns and roles of TRP channels in the regulation of gastrointestinal motility, enteric nervous system signalling and visceral sensation, and provide our perspectives on pharmacological targeting of TRPs as a strategy to treat various gastrointestinal disorders. We found that the current knowledge about the role of some members of the TRP superfamily in neurogastroenterology is rather limited, whereas the function of other TRP channels, especially of those implicated in smooth muscle cell contractility (TRPC4, TRPC6), visceral sensitivity and hypersensitivity (TRPV1, TRPV4, TRPA1), tends to be well established. Compared with expression data, mechanistic information about TRP channels in intestinal pacemaking (TRPC4, TRPC6, TRPM7), enteric nervous system signalling (TRPCs) and enteroendocrine cells (TRPM5) is lacking. It is clear that several different TRP channels play important roles in the cellular apparatus that controls gastrointestinal function. They are involved in the regulation of gastrointestinal motility and absorption, visceral sensation and visceral hypersensitivity. TRP channels can be considered as interesting targets to tackle digestive diseases, motility disorders and visceral pain. At present, TRPV1 antagonists are under development for the treatment of heartburn and visceral hypersensitivity, but interference with other TRP channels is also tempting. However, their role in gastrointestinal pathophysiology first needs to be further elucidated.