TRPP2: Ca2+-permeable cation channel and more.

TRPP2 (polycystin-2) is a member of the TRP family of non-selective cation channels that is mutated in human autosomal polycystic kidney disease. It is thought to function together with polycystin-1 (PKD1), a large plasma membrane integral protein, as part of a multiprotein complex involved in transducing Ca2+-dependent mechanosensitive information in renal epithelial cells. TRPP2 has been implicated in Ca2+-dependent pathways in a variety of biological functions and species, including cell proliferation, sperm fertilization, mating behavior and asymmetric gene expression. Although its function as a Ca2+-permeable cation channel is well established, its precise role, regulation and subcellular localization in plasma membrane, endoplasmic reticulum and cilium have remained controversial. The present review summarizes the most pertinent recent evidence regarding the structural and functional properties of TRPP2 channels, focusing on the regulation and physiology of mammalian TRPP2.

The lipid-activated two-pore domain K+ channel TREK-1 is resistant to hypoxia: implication for ischaemic neuroprotection.

TREK-1 is a member of the two-pore domain potassium (K(2P)) channel family that is mechano-, heat, pH, voltage and lipid sensitive. It is highly expressed in the central nervous system and probably encodes one of the previously described arachidonic acid-activated K(+) channels. Polyunsaturated fatty acids and lysophospholipids protect the brain against global ischaemia. Since both lipids are openers of TREK-1, it has been suggested that this K(2P) channel is directly involved in neuroprotection. Recently, however, this view has been challenged by a report claiming that TREK-1 and its activation by arachidonic acid is inhibited by hypoxia. In the present study, we demonstrate that the bubbling of saline with gases results in the loss of arachidonic acid from solution. Using experimental conditions which obviate this experimental artefact we demonstrate that TREK-1 is resistant to hypoxia and is strongly activated by arachidonic acid even at low P(O(2)) (< 4 Torr). Furthermore, hypoxia fails to affect basal as well as 2,4,6-trinitrophenol- and acid-stimulated TREK-1 currents. These data are supportive for a possible role of TREK-1 in ischaemic neuroprotection and in cell signalling via arachidonic acid.

Molecular physiology of oxygen-sensitive potassium channels.

Physiological adaptation to acute hypoxia involves oxygen-sensing by a variety of specialized cells including carotid body type I cells, pulmonary neuroepithelial body cells, pulmonary artery myocytes and foetal adrenomedullary chromaffin cells. Hypoxia induces depolarization by closing a specific set of potassium channels and triggers cellular responses. Molecular biology strategies have recently allowed the identification of the K+ channel subunits expressed in these specialized cells. Several voltage-gated K+ channel subunits comprising six transmembrane segments and a single pore domain (Kv1.2, Kv1.5, Kv2.1, Kv3.1, Kv3.3, Kv4.2 and Kv9.3) are reversibly blocked by hypoxia when expressed in heterologous expression systems. Additionally, the background K+ channel subunit TASK-1, which comprises four transmembrane segments and two pore domains, is also involved in both oxygen- and acid-sensing in peripheral chemoreceptors. Progress is currently being made to identify the oxygen sensors. Regulatory beta subunits may play an important role in the modulation of Kv channel subunits by oxygen.

Lipid and mechano-gated 2P domain K(+) channels.

The two pore domain K(+) channels TREK and TRAAK are opened by membrane stretch. The activating mechanical force comes from the bilayer membrane and is independent of the cytoskeleton. Emerging work shows that mechano-gated TREK and TRAAK are opened by various lipids, including long chain polyunsaturated anionic fatty acids and neutral cone-shaped lysophospholipids. TREK-1 shares the properties of the Aplysia neuronal S channel, a presynaptic background K(+) channel involved in behavioral sensitization, a simple form of learning.

Properties and modulation of mammalian 2P domain K+ channels.

Mammalian 2P domain K(+) channels are responsible for background or 'leak' K(+) currents. These channels are regulated by various physical and chemical stimuli, including membrane stretch, temperature, acidosis, lipids and inhalational anaesthetics. Furthermore, channel activity is tightly controlled by membrane receptor stimulation and second messenger phosphorylation pathways. Several members of this novel family of K(+) channels are highly expressed in the central and peripheral nervous systems in which they are proposed to play an important physiological role. The pharmacological modulation of this novel class of ion channels could be of interest for both general anaesthesia and ischaemic neuroprotection.

The endocannabinoid anandamide is a direct and selective blocker of the background K(+) channel TASK-1.

TASK-1 encodes an acid- and anaesthetic-sensitive background K(+) current, which sets the resting membrane potential of both cerebellar granule neurons and somatic motoneurons. We demonstrate that TASK-1, unlike the other two pore (2P) domain K(+) channels, is directly blocked by submicromolar concentrations of the endocannabinoid anandamide, independently of the CB1 and CB2 receptors. In cerebellar granule neurons, anandamide also blocks the TASK-1 standing-outward K(+) current, IKso, and induces depolarization. Anandamide-induced neurobehavioural effects are only partly reversed by antagonists of the cannabinoid receptors, suggesting the involvement of alternative pathways. TASK-1 constitutes a novel sensitive molecular target for this endocannabinoid.

TWIK-2, an inactivating 2P domain K+ channel.

We cloned human and rat TWIK-2 and expressed this novel 2P domain K(+) channel in transiently transfected COS cells. TWIK-2 is highly expressed in the gastrointestinal tract, the vasculature, and the immune system. Rat TWIK-2 currents are about 15 times larger than human TWIK-2 currents, but both exhibit outward rectification in a physiological K(+) gradient and mild inward rectification in symmetrical K(+) conditions. TWIK-2 currents are inactivating at depolarized potentials, and the kinetic of inactivation is highly temperature-sensitive. TWIK-2 shows an extremely low conductance, which prevents the visualization of discrete single channel events. The inactivation and rectification are intrinsic properties of TWIK-2 channels. In a physiological K(+) gradient, TWIK-2 is half inhibited by 0.1 mm Ba(2+), quinine, and quinidine. Finally, cysteine 53 in the M1P1 external loop is required for functional expression of TWIK-2 but is not critical for subunit self-assembly. TWIK-2 is the first reported 2P domain K(+) channel that inactivates. The base-line, transient, and delayed activities of TWIK-2 suggest that this novel 2P domain K(+) channel may play an important functional role in cell electrogenesis.

TREK-1 is a heat-activated background K(+) channel.

Peripheral and central thermoreceptors are involved in sensing ambient and body temperature, respectively. Specialized cold and warm receptors are present in dorsal root ganglion sensory fibres as well as in the anterior/preoptic hypothalamus. The two-pore domain mechano-gated K(+) channel TREK-1 is highly expressed within these areas. Moreover, TREK-1 is opened gradually and reversibly by heat. A 10 degrees C rise enhances TREK-1 current amplitude by approximately 7-fold. Prostaglandin E2 and cAMP, which are strong sensitizers of peripheral and central thermoreceptors, reverse the thermal opening of TREK-1 via protein kinase A-mediated phosphorylation of Ser333. Expression of TREK-1 in peripheral sensory neurons as well as in central hypothalamic neurons makes this K(+) channel an ideal candidate as a physiological thermoreceptor.

An oxygen-, acid- and anaesthetic-sensitive TASK-like background potassium channel in rat arterial chemoreceptor cells.

The biophysical and pharmacological properties of an oxygen-sensitive background K+ current in rat carotid body type-I cells were investigated and compared with those of recently cloned two pore domain K+ channels. Under symmetrical K+ conditions the oxygen-sensitive whole cell K+ current had a linear dependence on voltage indicating a lack of intrinsic voltage sensitivity. Single channel recordings identified a K+ channel, open at resting membrane potentials, that was inhibited by hypoxia. This channel had a single channel conductance of 14 pS, flickery kinetics and showed little voltage sensitivity except at extreme positive potentials. Oxygen-sensitive current was inhibited by 10 mM barium (57% inhibition), 200 microM zinc (53% inhibition), 200 microM bupivacaine (55% inhibition) and 1 mM quinidine (105 % inhibition). The general anaesthetic halothane (1.5%) increased the oxygen-sensitive K+ current (by 176%). Halothane (3 mM) also stimulated single channel activity in inside-out patches (by 240%). Chloroform had no effect on background K+ channel activity. Acidosis (pH 6.4) inhibited the oxygen-sensitive background K+ current (by 56%) and depolarised type-I cells. The pharmacological and biophysical properties of the background K+ channel are, therefore, analogous to those of the cloned channel TASK-1. Using in situ hybridisation TASK-1 mRNA was found to be expressed in type-I cells. We conclude that the oxygen- and acid-sensitive background K+ channel of carotid body type-I cells is likely to be an endogenous TASK-1-like channel.

Lysophospholipids open the two-pore domain mechano-gated K(+) channels TREK-1 and TRAAK.

The two-pore (2P) domain K(+) channels TREK-1 and TRAAK are opened by membrane stretch as well as arachidonic acid (AA) (Patel, A. J., Honoré, E., Maingret, F., Lesage, F., Fink, M., Duprat, F., and Lazdunski, M. (1998) EMBO J. 17, 4283-4290; Maingret, F., Patel, A. J., Lesage, F., Lazdunski, M., and Honoré, E. (1999) J. Biol. Chem. 274, 26691-26696; Maingret, F., Fosset, M., Lesage, F., Lazdunski, M. , and Honoré, E. (1999) J. Biol. Chem. 274, 1381-1387. We demonstrate that lysophospholipids (LPs) and platelet-activating factor also produce large specific and reversible activations of TREK-1 and TRAAK. LPs activation is a function of the size of the polar head and length of the acyl chain but is independent of the charge of the molecule. Bath application of lysophosphatidylcholine (LPC) immediately opens TREK-1 and TRAAK in the cell-attached patch configuration. In excised patches, LPC activation is lost, whereas AA still produces maximal opening. The carboxyl-terminal region of TREK-1, but not the amino terminus and the extracellular loop M1P1, is critically required for LPC activation. LPC activation is indirect and may possibly involve a cytosolic factor, whereas AA directly interacts with either the channel proteins or the bilayer and mimics stretch. Opening of TREK-1 and TRAAK by fatty acids and LPs may be an important switch in the regulation of synaptic function and may also play a protective role during ischemia and inflammation.

Pharmacokinetics of ibuprofen after intravenous and oral administration and assessment of safety of administration to healthy foals.

OBJECTIVE: To determine pharmacokinetics of ibuprofen in healthy foals and to determine clinical effects after oral administration for 6 days. ANIMALS: 7 healthy 5- to 10-week-old foals. PROCEDURE: Serum concentrations of ibuprofen were measured after IV and oral (nasogastric tube) administration at dosages of 10 and 25 mg/kg of body weight. Foals were given ibuprofen (25 mg/kg, PO, q 8 h) as a paste for 6 days. Serum and urine were obtained before and after the 6-day period. RESULTS: Half-life of elimination (Kel t1/2) of IV-administered ibuprofen (ie, 10 and 25 mg/kg), was 79 and 108 minutes, maximal serum concentration (C(MAX)) was 82 and 160 microg/ml, and clearance was 0.003 and 0.002 L/kg/min, respectively. At the higher dosage, clearance was significantly lower and C(MAX) was significantly higher. Ibuprofen given via nasogastric tube resulted in Kel t1/2 of 81 and 100 minutes and C(MAX) of 22 and 52 microg/ml for 10 and 25 mg/kg, respectively. The absorption half-life was 13 minutes, and bioavailability ranged from 71 to 100%. Foals remained healthy during oral administration of ibuprofen. Serum urea nitrogen, creatinine, and L-iditol dehydrogenase values increased significantly, and gamma-glutamyltransferase (GGT) activity and osmolality decreased, but all measurements remained within reference ranges. Urine GGT activity doubled. Necropsy did not reveal gross or histologic renal lesions attributable to ibuprofen. Acute gastric ulcers were evident in 1 foal, although clinical signs of ulcers were not observed. CONCLUSIONS AND CLINICAL RELEVANCE: Ibuprofen can be given safely to healthy foals at dosages < or = 25 mg/kg every 8 hours for up to 6 days.

Mechano- or acid stimulation, two interactive modes of activation of the TREK-1 potassium channel.

TREK-1 is a member of the novel structural class of K(+) channels with four transmembrane segments and two pore domains in tandem (1,2). TREK-1 is opened by membrane stretch and arachidonic acid. It is also an important target for volatile anesthetics (2,3). Here we show that internal acidification opens TREK-1. Indeed, lowering pH(i) shifts the pressure-activation relationship toward positive values and leads to channel opening at atmospheric pressure. The pH(i)-sensitive region in the carboxyl terminus of TREK-1 is the same that is critically involved in mechano-gating as well as arachidonic acid activation. A convergence, which is dependent on the carboxyl terminus, occurs between mechanical, fatty acids and acidic stimuli. Intracellular acidosis, which occurs during brain and heart ischemia, will induce TREK-1 opening with subsequent K(+) efflux and hyperpolarization.

Inhalational anesthetics activate two-pore-domain background K+ channels.

Volatile anesthetics produce safe, reversible unconsciousness, amnesia and analgesia via hyperpolarization of mammalian neurons. In molluscan pacemaker neurons, they activate an inhibitory synaptic K+ current (IKAn), proposed to be important in general anesthesia. Here we show that TASK and TREK-1, two recently cloned mammalian two-P-domain K+ channels similar to IKAn in biophysical properties, are activated by volatile general anesthetics. Chloroform, diethyl ether, halothane and isoflurane activated TREK-1, whereas only halothane and isoflurane activated TASK. Carboxy (C)-terminal regions were critical for anesthetic activation in both channels. Thus both TREK-1 and TASK are possibly important target sites for these agents.

TRAAK is a mammalian neuronal mechano-gated K+ channel.

The novel structural class of mammalian channels with four transmembrane segments and two pore regions comprise background K+ channels (TWIK-1, TREK-1, TRAAK, TASK, and TASK-2) with unique physiological functions (1-6). Unlike its counterparts, TRAAK is only expressed in neuronal tissues, including brain, spinal cord, and retina (1). This report shows that TRAAK, which was known to be activated by arachidonic acid (3), is also opened by membrane stretch. Mechanical activation of TRAAK is induced by a convex curvature of the plasma membrane and can be mimicked by the amphipathic membrane crenator trinitrophenol. Cytoskeletal elements are negative tonic regulators of TRAAK. Membrane depolarization and membrane crenation synergize with stretch-induced channel opening. Finally, TRAAK is reversibly blocked by micromolar concentrations of gadolinium, a well known blocker of stretch-activated channels. Mechanical activation of TRAAK in the central nervous system may play an important role during growth cone motility and neurite elongation.

Identification of the receptor subtype involved in the analgesic effect of neurotensin.

The neuropeptide neurotensin (NT) elicits hypothermic and naloxone-insensitive analgesic responses after brain injection. Recent pharmacological evidence obtained with NT agonists and antagonists suggests that these effects are mediated by a receptor distinct from the initially cloned high-affinity NT receptor (NTR1). The recent cloning of a second NT receptor (NTR2) prompted us to evaluate its role in NT-induced analgesia. Intracerebroventricular injections in mice of two different antisense oligodeoxynucleotides from the NTR2 markedly decreased NTR2 mRNA and protein and reduced NT-induced analgesia. This effect was specific, because NTR1 levels were unaffected, and sense or scramble oligodeoxynucleotides had no effect. Structure-activity studies revealed a close correlation between the analgesic potency of NT analogs and their affinity for the NTR2 and disclosed potent and selective agonists of this receptor. These data confirm that NTR1 is involved in the NT-elicited turning behavior and demonstrate that the NTR2 mediates NT-induced analgesia.

Coadministration of nomegestrol acetate does not diminish the beneficial effects of estradiol on coronary artery dilator responses in nonhuman primates (Macaca fascicularis).

OBJECTIVE: Our purpose was to examine the effect of coadministered nomegestrol acetate on estradiol-induced dilator responses of coronary arteries. STUDY DESIGN: In this prospective randomized trial, ovariectomized monkeys were fed a moderately atherogenic diet for 3 months while being treated with (1) no hormone replacement (control, n = 12), (2) estradiol (1.5 mg/d equivalent) added to the diet (n = 12), or (3) estradiol (1.5 mg/d equivalent) plus nomegestrol acetate (3.75 mg/d equivalent) (n = 12) added to the diet. Effects of treatment were measured with analysis of variance. Post hoc analyses were done by multiple comparison tests with Bonferroni corrections. RESULTS: Constrictor responses of epicardial coronary arteries (measured with quantitative angiography) and decreased coronary blood velocity (measured with Doppler ultrasonography) to acetylcholine (10(-6) mol/L) were less in the estradiol-treated monkeys (with or without cotreatment with nomegestrol acetate) than in the untreated monkeys (P <.05). Typical estrogenic responses were induced by estradiol in the endometrium (ie, increased proliferation [Ki-67 expression] [P <.04] and increased hormone receptor expression). These effects were antagonized by nomegestrol acetate. CONCLUSIONS: Although nomegestrol acetate has typical progestin-like effects on the uterus, it does not diminish the beneficial effects of estrogen on acetylcholine-induced dilator responses of coronary arteries.

A mammalian two pore domain mechano-gated S-like K+ channel.

Aplysia S-type K+ channels of sensory neurons play a dominant role in presynaptic facilitation and behavioural sensitization. They are closed by serotonin via cAMP-dependent phosphorylation, whereas they are opened by arachidonic acid, volatile general anaesthetics and mechanical stimulation. We have identified a cloned mammalian two P domain K+ channel sharing the properties of the S channel. In addition, the recombinant channel is opened by lipid bilayer amphipathic crenators, while it is closed by cup-formers. The cytoplasmic C-terminus contains a charged region critical for chemical and mechanical activation, as well as a phosphorylation site required for cAMP inhibition.

Disruption of mitochondrial respiration inhibits volume-regulated anion channels and provokes neuronal cell swelling.

Hypoxia and inhibitors of mitochondrial respiration impair the regulatory volume decrease (RVD) of cerebellar granule neurons after hypotonic swelling. RVD is linked to the opening of volume-regulated anion channels (VRACs). VRACs are outwardly rectifying, inactivate slowly during maintained depolarization, and are permeable to the cellular organic osmolyte taurine. Channel activation requires nonhydrolytic ATP binding and is not modulated by intracellular ADP. VRAC opening is reversibly depressed by hypoxia and by mitochondrial inhibitors such as oligomycin, rotenone, and antimycin A. These results demonstrate that neuronal VRAC activation and swelling are both tightly linked to cellular energy. Moreover, the findings reported in this work may have a particular significance for inherited mitochondrial human diseases, such as mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), which cause brain swelling and edema.