contributed equally to this work 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.
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 ®bres 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°C rise enhances TREK-1 current amplitude by~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.
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.The near completion of the sequencing of the nematode Caenorhabditis elegans genome recently identified more than 80 K ϩ channel genes divided into three major structural classes: (i) the inward rectifiers with two TMS 1 and a single P domain; (ii) the Shaker types with six TMS and a single P domain comprising the voltage-gated Kvs, the calcium-activated Slo, the calcium-regulated SK, the Eag/Erg, and the KQT channels; and (iii) the two P types with 4TMS being the largest structural class (about 50 genes) (4 -6). Despite an overall similar 4TMS/2P structure, the sequence identity between these channels is very low (less than 30%) (5, 6).The mammalian family of 4TMS/2P K ϩ channels comprises TWIK-1, TWIK-2, TASK-1, TASK-2, TREK-1, and TRAAK (1, 7-11). TWIK-1 and TWIK-2 are widely distributed and encode K ϩ -selective channels with a characteristic weak inward rectification (8, 11). TASK-1 is found principally in the pancreas, placenta, lung, brain, and heart (9, 12, 13). TASK-1 lacks intrinsic voltage sensitivity and is thus a pure background K ϩ -selective channel. Moreover, TASK-1 is extremely sensitive to variations of extracellular pH in a narrow physiological range, with 90% of the maximum current recorded at pH 7.7 and only 10% at pH 6.7 (9). TASK-2, another background K ϩ channel recently isolated from human kidney, shares the external pH sensitivity of TASK-1 (10). Unlike the other 4TMS/2P channels, TASK-2 is almost absent from the brain and is mainly expressed in the kidney. Murine TREK-1 is widely distributed with a strong expression in the brain and in the heart (1). It is activated by membrane stretch, by AA as well as inhalational anesthetics, while it is inhibited by a cAMP-dependent phosphorylation (2, 3). Interestingly, TREK-1 shares the properties of the Aplysia S-type K ϩ channel, which is involved in presynaptic facilitation underlying a simple form of learning (14, 15). TRAAK, another mouse mechano-gated AA-sensitive 4TMS/2P K ϩ channel, is only expressed in neuronal tissues including brain, spinal cord, and retina and lacks sensitivity to cAMP (7, 16).The mammalian mechano-gated K ϩ channels that have been previously described...
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.
. 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.
The 2P domain K + channel TREK-1 is widely expressed in the nervous system. It is opened by a variety of physical and chemical stimuli including membrane stretch, intracellular acidosis and polyunsaturated fatty acids. This activation can be reversed by PKAmediated phosphorylation. The C-terminal domain of TREK-1 is critical for its polymodal function. We demonstrate that the conversion of a speci®c glutamate residue (E306) to an alanine in this region locks TREK-1 in the open con®guration and abolishes the cAMP/PKA down-modulation. The E306A substitution mimics intracellular acidosis and rescues both lipid-and mechano-sensitivity of a loss-of-function truncated TREK-1 mutant. We conclude that protonation of E306 tunes the TREK-1 mechanical setpoint and thus sets lipid sensitivity.
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.
Altered function of Na+ channels is responsible for increased hyperexcitability of primary afferent neurons that may underlie pathological pain states. Recent evidence suggests that the Nav1.9 subunit is implicated in inflammatory but not acute pain. However, the contribution of Nav1.9 channels to the cellular events underlying nociceptor hyperexcitability is still unknown, and there remains much uncertainty as to the biophysical properties of Nav1.9 current and its modulation by inflammatory mediators. Here, we use gene targeting strategy and computer modeling to identify Nav1.9 channel current signature and its impact on nociceptors' firing patterns. Recordings using internal fluoride in small DRG neurons from wild-type and Nav1.9-null mutant mice demonstrated that Nav1.9 subunits carry the TTX-resistant “persistent” Na+ current called NaN. Nav1.9−/− nociceptors showed no significant change in the properties of the slowly inactivating TTX-resistant SNS/Nav1.8 current. The loss in Nav1.9-mediated Na+ currents was associated with the inability of small DRG neurons to generate a large variety of electrophysiological behaviors, including subthreshold regenerative depolarizations, plateau potentials, active hyperpolarizing responses, oscillatory bursting discharges, and bistable membrane behaviors. We further investigated, using CsCl- and KCl-based pipette solutions, whether G-protein signaling pathways and inflammatory mediators upregulate the NaN/Nav1.9 current. Bradykinin, ATP, histamine, prostaglandin-E2, and norepinephrine, applied separately at maximal concentrations, all failed to modulate the Nav1.9 current. However, when applied conjointly as a soup of inflammatory mediators they rapidly potentiated Nav1.9 channel activity, generating subthreshold amplification and increased excitability. We conclude that Nav1.9 channel, the molecular correlate of the NaN current, is potentiated by the concerted action of inflammatory mediators that may contribute to nociceptors' hyperexcitability during peripheral inflammation.
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