Acetylcholine-dependent upregulation of TASK-1 channels in thalamic interneurons by a smooth muscle-like signalling pathway.

KEY POINTS: The ascending brainstem transmitter acetylcholine depolarizes thalamocortical relay neurons while it induces hyperpolarization in local circuit inhibitory interneurons. Sustained K+ currents are modulated in thalamic neurons to control their activity modes; for the interneurons the molecular nature of the underlying ion channels is as yet unknown. Activation of TASK-1 K+ channels results in hyperpolarization of interneurons and suppression of their action potential firing. The modulation cascade involves a non-receptor tyrosine kinase, c-Src. The present study identifies a novel pathway for the activation of TASK-1 channels in CNS neurons that resembles cholinergic signalling and TASK-1 current modulation during hypoxia in smooth muscle cells. ABSTRACT: The dorsal part of the lateral geniculate nucleus (dLGN) is the main thalamic site for state-dependent transmission of visual information. Non-retinal inputs from the ascending arousal system and inhibition provided by γ-aminobutyric acid (GABA)ergic local circuit interneurons (INs) control neuronal activity within the dLGN. In particular, acetylcholine (ACh) depolarizes thalamocortical relay neurons by inhibiting two-pore domain potassium (K2P ) channels. Conversely, ACh also hyperpolarizes INs via an as-yet-unknown mechanism. By using whole cell patch-clamp recordings in brain slices and appropriate pharmacological tools we here report that stimulation of type 2 muscarinic ACh receptors induces IN hyperpolarization by recruiting the G-protein ßγ subunit (Gßγ), class-1A phosphatidylinositol-4,5-bisphosphate 3-kinase, and cellular and sarcoma (c-Src) tyrosine kinase, leading to activation of two-pore domain weakly inwardly rectifying K+ channel (TWIK)-related acid-sensitive K+ (TASK)-1 channels. The latter was confirmed by the use of TASK-1-deficient mice. Furthermore inhibition of phospholipase Cß as well as an increase in the intracellular level of phosphatidylinositol-3,4,5-trisphosphate facilitated the muscarinic effect. Our results have uncovered a previously unknown role of c-Src tyrosine kinase in regulating IN function in the brain and identified a novel mechanism by which TASK-1 channels are activated in neurons.

Differential phospholipase C-dependent modulation of TASK and TREK two-pore domain K+ channels in rat thalamocortical relay neurons.

KEY POINTS: During the behavioural states of sleep and wakefulness thalamocortical relay neurons fire action potentials in high frequency bursts or tonic sequences, respectively. The modulation of specific K(+) channel types, termed TASK and TREK, allows these neurons to switch between the two modes of activity. In this study we show that the signalling lipids phosphatidylinositol 4,5-bisphosphate (PIP2) and diacylglycerol (DAG), which are components of their membrane environment, switch on and shut off TREK and TASK channels, respectively. These channel modulations contribute to a better understanding of the molecular basis of the effects of neurotransmitters such as ACh which are released by the brainstem arousal system. The present report introduces PIP2 and DAG as new elements of signal transduction in the thalamus. The activity of two-pore domain potassium channels (K2P ) regulates the excitability and firing modes of thalamocortical (TC) neurons. In particular, the inhibition of two-pore domain weakly inwardly rectifying K(+) channel (TWIK)-related acid-sensitive K(+) (TASK) channels and TWIK-related K(+) (TREK) channels, as a consequence of the stimulation of muscarinic ACh receptors (MAChRs) which are coupled to phosphoinositide-specific phospholipase C (PLCß), induces a shift from burst to tonic firing. By using a whole cell patch-clamp approach, the contribution of the membrane-bound second messenger molecules phosphatidylinositol 4,5-bisphosphate (PIP2 ) and diacylglycerol (DAG) acting downstream of PLCß was probed. The standing outward current (ISO ) was used to monitor the current through TASK and TREK channels in TC neurons. By exploiting different manoeuvres to change the intracellular PIP2 level in TC neurons, we here show that the scavenging of PIP2 (by neomycin) results in an increased muscarinic effect on ISO whereas increased availability of PIP2 (inclusion to the patch pipette; histone-based carrier) decreased muscarinic signalling. The degree of muscarinic inhibition specifically depends on phosphatidylinositol phosphate (PIP) and PIP2 but no other phospholipids (phosphatidic acid, phosphatidylserine). The use of specific blockers revealed that PIP2 is targeting TREK but not TASK channels. Furthermore, we demonstrate that the inhibition of TASK channels is induced by the application of the DAG analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG). Under current clamp conditions the activation of MAChRs and PLCß as well as the application of OAG resulted in membrane depolarization, while PIP2 application via histone carrier induced a hyperpolarization. These results demonstrate a differential role of PIP2 and DAG in K2P channel modulation in native neurons which allows a fine-tuned inhibition of TREK (via PIP2 depletion) and TASK (via DAG) channels following MAChR stimulation.

TASK Channel Deletion Reduces Sensitivity to Local Anesthetic-induced Seizures.

BACKGROUND: Local anesthetics (LAs) are typically used for regional anesthesia but can be given systemically to mitigate postoperative pain, supplement general anesthesia, or prevent cardiac arrhythmias. However, systemic application or inadvertent intravenous injection can be associated with substantial toxicity, including seizure induction. The molecular basis for this toxic action remains unclear. METHODS: We characterized inhibition by different LAs of homomeric and heteromeric K channels containing TASK-1 (K2P3.1, KCNK3) and TASK-3 (K2P9.1, KCNK9) subunits in a mammalian expression system. In addition, we used TASK-1/TASK-3 knockout mice to test the possibility that TASK channels contribute to LA-evoked seizures. RESULTS: LAs inhibited homomeric and heteromeric TASK channels in a range relevant for seizure induction; channels containing TASK-1 subunits were most sensitive and IC50 values indicated a rank order potency of bupivacaine > ropivacaine >> lidocaine. LAs induced tonic-clonic seizures in mice with the same rank order potency, but higher LA doses were required to evoke seizures in TASK knockout mice. For bupivacaine, which produced the longest seizure times, seizure duration was significantly shorter in TASK knockout mice; bupivacaine-induced seizures were associated with an increase in electroencephalogram power at frequencies less than 5 Hz in both wild-type and TASK knockout mice. CONCLUSIONS: These data suggest that increased neuronal excitability associated with TASK channel inhibition by LAs contributes to seizure induction. Because all LAs were capable of evoking seizures in TASK channel deleted mice, albeit at higher doses, the results imply that other molecular targets must also be involved in this toxic action.

Long-chain polyunsaturated fatty acids as anti-HIV supplementation during breastfeeding.

OBJECTIVE: Breastfeeding by human immunodeficiency virus (HIV)-positive mothers is an unavoidable practice in some very poor countries. It has been suggested that long-chain polyunsaturated fatty acids (LC-PUFAs) in breast milk, such as arachidonic acid, act as natural, protective ingredients against HIV transmission. The objective of this study was to identify the protective mechanism of LC-PUFAs in cells susceptible to HIV infection (e.g. human CD4+ T cells, HeLa cells). RESULTS: LC-PUFAs are bioactive molecules capable of activating the cellular protective machinery via modulation of endogenous background K+ or KCNK channels. KCNK channel expression contributes significantly to the stability of the cell membrane potential. During HIV-1 infection, degradation of the KCNK channel is accelerated, and the cell membrane potential becomes pathologically depolarized. From studying functionally distinct KCNK mutants, we found that the degree of membrane potential depolarization was directly proportional to the release efficiency of HIV-1 virions. On the other hand, supplementation of KCNK channel modulators such as arachidonic acid (AA) and docosahexaenoic acid (DHA) at micromolar doses could restore hyperpolarization and stability of the cell membrane potential when endogenous KCNK channels are partially knocked down (mimicking the depolarized state of an HIV-1-infected cell). CONCLUSION: The protective mechanism of LC-PUFAs against HIV spread involves stimulation of the endogenous KCNK channels. Our work suggests that supplementation with AA and DHA may be beneficial in reducing the risk of HIV-1 transmission, particularly during the period of breastfeeding.

The mechano-gated K(2P) channel TREK-1.

The versatility of neuronal electrical activity is largely conditioned by the expression of different structural and functional classes of K+ channels. More than 80 genes encoding the main K+ channel alpha subunits have been identified in the human genome. Alternative splicing, heteromultimeric assembly, post-translational modification and interaction with auxiliary regulatory subunits further increase the molecular and functional diversity of K+ channels. Mammalian two-pore domain K+ channels (K(2P)) make up one class of K+ channels along with the inward rectifiers and the voltage- and/or calcium-dependent K+ channels. Each K(2P) channel subunit is made up of four transmembrane segments and two pore-forming (P) domains, which are arranged in tandem and function as either homo- or heterodimeric channels. This novel structural arrangement is associated with unusual gating properties including "background" or "leak" K+ channel activity, in which the channels show constitutive activity at rest. In this review article, we will focus on the lipid-sensitive mechano-gated K(2P) channel TREK-1 and will emphasize on the polymodal function of this "unconventional" K+ channel.

Pungent agents from Szechuan peppers excite sensory neurons by inhibiting two-pore potassium channels.

In traditional folk medicine, Xanthoxylum plants are referred to as 'toothache trees' because their anesthetic or counter-irritant properties render them useful in the treatment of pain. Psychophysical studies have identified hydroxy-alpha-sanshool as the compound most responsible for the unique tingling and buzzing sensations produced by Szechuan peppercorns or other Xanthoxylum preparations. Although it is generally agreed that sanshool elicits its effects by activating somatosensory neurons, the underlying cellular and molecular mechanisms remain a matter of debate. Here we show that hydroxy-alpha-sanshool excites two types of sensory neurons, including small-diameter unmyelinated cells that respond to capsaicin (but not mustard oil) as well as large-diameter myelinated neurons that express the neurotrophin receptor TrkC. We found that hydroxy-alpha-sanshool excites neurons through a unique mechanism involving inhibition of pH- and anesthetic-sensitive two-pore potassium channels (KCNK3, KCNK9 and KCNK18), providing a framework for understanding the unique and complex psychophysical sensations associated with the Szechuan pepper experience.

Sipatrigine could have therapeutic potential for major depression and bipolar depression through antagonism of the two-pore-domain K+ channel TREK-1.

Major depressive disorder (MDD) is a chronic, recurring and potentially life-threatening mental illness. Current treatments are inadequate - many depression medications, although safe and effective, generally have a slow onset of clinical benefit and around half of the MDD patients do not show full remission with optimized treatment. Therefore, there is still a need for the development of faster-acting and more effective medication for MDD. Recent studies have demonstrated that the TREK-1 protein, one of the 17 members of the two-pore domain K+ (K2P) potassium channel family, is inhibited by the antidepressant fluoxetine. Deletion of TREK-1 in mice caused a substantially reduced elevation of corticosterone levels under stress, and produced behaviour similar to that of naive animals treated with fluoxetine in various behavioural tests. These findings suggested that the blocker of the TREK-1 channel might potentially be a new type of antidepressant. Sipatrigine (BW619C89), a neuroprotective agent, has been found to be a potent antagonist of TREK-1. Its related compound, lamotrigine, has been approved for the treatment of bipolar depression and is used to supplement antidepressant medication in patients with treatment-resistant depression. Furthermore, in addition to its antagonistic effect on TREK-1, sipatrigine is also a glutamate release inhibitor. Excessive glutamatergic neurotransmission is associated with depressive-like behaviours and inhibiting glutamate neurotransmission may be implicated in antidepressant therapeutic mechanisms. From the above findings of the effects of sipatrigine on TREK-1 and glutamate neurotransmission, it is hypothesised that sipatrigine could have potential therapeutic effects for MDD or bipolar depression. Further evaluation of its antidepressant therapeutic and toxic effects in animal models is needed before clinical application.

Differential effects of volatile and intravenous anesthetics on the activity of human TASK-1.

Volatile anesthetics have been shown to activate various two-pore (2P) domain K(+) (K(2P)) channels such as TASK-1 and TREK-1 (TWIK-related acid-sensitive K(+) channel), and mice deficient in these channels are resistant to halothane-induced anesthesia. Here, we investigated whether K(2P) channels were also potentially important targets of intravenous anesthetics. Whole cell patch-clamp techniques were used to determine the effects of the commonly used intravenous anesthetics etomidate and propofol on the acid-sensitive K(+) current in rat ventricular myocytes (which strongly express TASK-1) and selected human K(2P) channels expressed in Xenopus laevis oocytes. In myocytes, etomidate decreased both inward rectifier K(+) (K(ir)) current (I(K1)) and acid-sensitive outward K(+) current at positive potentials, suggesting that this drug may inhibit TASK channels. Indeed, in addition to inhibiting guinea pig Kir2.1 expressed in oocytes, etomidate inhibited human TASK-1 (and TASK-3) in a concentration-dependent fashion. Propofol had no effect on human TASK-1 (or TASK-3) expressed in oocytes. Moreover, we showed that, similar to the known effect of halothane, sevoflurane and the purified R-(-)- and S-(+)-enantiomers of isoflurane, without stereoselectivity, activated human TASK-1. We conclude that intravenous and volatile anesthetics have dissimilar effects on K(2P) channels. Human TASK-1 (and TASK-3) are insensitive to propofol but are inhibited by supraclinical concentrations of etomidate. In contrast, stimulatory effects of sevoflurane and enantiomeric isoflurane on human TASK-1 can be observed at clinically relevant concentrations.

Up- and down-regulation of the mechano-gated K(2P) channel TREK-1 by PIP (2) 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 PIP(2), 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 PIP(2) 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.

Membrane resting potential of thalamocortical relay neurons is shaped by the interaction among TASK3 and HCN2 channels.

By combining molecular biological, electrophysiological, immunological, and computer modeling techniques, we here demonstrate a counterbalancing contribution of TASK channels, underlying hyperpolarizing K+ leak currents, and HCN channels, underlying depolarizing Ih, to the resting membrane potential of thalamocortical relay (TC) neurons. RT-PCR experiments revealed the expression of TASK1, TASK3, and HCN1-4. Quantitative determination of mRNA expression levels and immunocytochemical staining demonstrated that TASK3 and HCN2 channels represent the dominant thalamic isoforms and are coexpressed in TC neurons. Extracellular acidification, a standard procedure to inhibit TASK channels, blocked a TASK current masked by additional action on HCN channels. Only in the presence of the HCN blocker ZD7288 was the pH-sensitive component typical for a TASK current, i.e., outward rectification and current reversal at the K+ equilibrium potential. In a similar way extracellular acidification was able to shift the activity pattern of TC neurons from burst to tonic firing only during block of Ih or genetic knock out of HCN channels. A single compartmental computer model of TC neurons simulated the counterbalancing influence of TASK and HCN on the resting membrane potential. It is concluded that TASK3 and HCN2 channels stabilize the membrane potential by a mutual functional interaction, that the most efficient way to regulate the membrane potential of TC neurons is the converse modulation of TASK and HCN channels, and that TC neurons are potentially more resistant to insults accompanied by extracellular pH shifts in comparison to other CNS regions.

Deafness associated changes in expression of two-pore domain potassium channels in the rat cochlear nucleus.

Two-pore domain potassium channels (K(2PD)+) play an important role in setting resting membrane potential by regulating background leakage of potassium ions, which in turn controls neuronal excitability. To determine whether these channels contribute to activity-dependent plasticity following deafness, we used quantitative real-time PCR to examine the expression of 10 K(2PD)+ subunits in the rat cochlear nucleus at 3 days, 3 weeks and 3 months after bilateral cochlear ablation. There was a large sustained decrease in the expression of TASK-5, a subunit that is predominantly expressed in auditory brain stem neurons, and in the TASK-1 subunit which is highly expressed in several types of cochlear nucleus neurons. TWIK-1 and THIK-2 also showed significant decreases in expression that were maintained across all time points. TWIK-2, TREK-1 and TREK-2 showed no significant change in expression at 3 days but showed large decreases at 3 weeks and 3 months following deafness. TRAAK and TASK-3 subunits showed significant decreases at 3 days and 3 weeks following deafness, but these differences were no longer significant at 3 months. Dramatic changes in expression of K(2PD)+ subunits suggest these channels may play a role in deafness-associated changes in the excitability of cochlear nucleus neurons.

Dendritic excitability of mouse frontal cortex pyramidal neurons is shaped by the interaction among HCN, Kir2, and Kleak channels.

Dendritically placed, voltage-sensitive ion channels are key regulators of neuronal synaptic integration. In several cell types, hyperpolarization/cyclic nucleotide gated (HCN) cation channels figure prominently in dendritic mechanisms controlling the temporal summation of excitatory synaptic events. In prefrontal cortex, the sustained activity of pyramidal neurons in working memory tasks is thought to depend on the temporal summation of dendritic excitatory inputs. Yet we know little about how this is accomplished in these neurons and whether HCN channels play a role. To gain a better understanding of this process, layer V-VI pyramidal neurons in slices of mouse prelimbic and infralimbic cortex were studied. Somatic voltage-clamp experiments revealed the presence of rapidly activating and deactivating cationic currents attributable to HCN1/HCN2 channels. These channels were open at the resting membrane potential and had an apparent half-activation voltage near -90 mV. In the same voltage range, K+ currents attributable to Kir2.2/2.3 and K+-selective leak (Kleak) channels were prominent. Computer simulations grounded in the biophysical measurements suggested a dynamic interaction among Kir2, Kleak, and HCN channel currents in shaping membrane potential and the temporal integration of synaptic potentials. This inference was corroborated by experiment. Blockade of Kir2/Kleak channels caused neurons to depolarize, leading to the deactivation of HCN channels, the initiation of regular spiking (4-5 Hz), and enhanced temporal summation of EPSPs. These studies show that HCN channels are key regulators of synaptic integration in prefrontal pyramidal neurons but that their functional contribution is dependent on a partnership with Kir2 and Kleak channels.

The selectivity filter of the tandem pore potassium channel TASK-1 and its pH-sensitivity and ionic selectivity.

We have studied pH sensitivity and ionic selectivity of the tandem pore K(+) channel TASK-1 heterologously expressed in Xenopus oocytes. We fit pH sensitivity assuming that only one of the two residues H98 need be protonated for channels to be shut. The effect of protons was weakly voltage dependent with a p K(a) of 6.02 at +40 mV. Replacement of His (H98D, H98N) reduced pH sensitivity but did not abolish it. Use of a concatameric channel permitted replacement of one His residue only; this concatamer was fully pH-sensitive. Increasing the number of His residues to 4 (mutant D204H) abolished pH sensitivity over the physiological range. The implication that D204 plays a role in pH-sensitivity was confirmed by the finding that pH sensitivity over the physiological range was also abolished in the mutant D204N. Ionic selectivity was also altered in D204H, D204N and H98D mutants. P(Rb)/ P(K) was increased from 0.80+/-0.04 (n=19) in wild type to 1.06+/-0.04 (n=19) in D204H. H98D, D204H and D204N were permeable to Na(+) with P(Na)/ P(K)=0.39+/-0.03 (n=14) in H98D, 0.64+/-0.04 (n=18) in D204H and 0.33+/-0.07 (n=3) in D204N. Thus, the arrangement of ring of residues HDHD appears to optimise both pH sensitivity and ionic selectivity.