Emma L. Veale

The sfr6 mutation in Arabidopsis suppresses low-temperature induction of genes dependent on the CRT/DRE sequence motif

The sfr mutations, which result in sensitivity to freezing after cold acclimation, define genes that are required for freezing tolerance. We tested plants homozygous for mutations sfr2 to sfr7 for cold-induced gene expression and found that sfr6 plants were deficient in cold-inducible expression of the genes KIN1, COR15a, and LTI78, which all contain the C repeat/dehydration-responsive element (CRT/DRE) motif in their promoters. Similarly, sfr6 plants failed to induce KIN1 normally in response to either osmotic stress or the application of abscisic acid. In contrast, cold-inducible expression of genes CBF1, CBF2, CBF3, and ATP5CS1, which lack the CRT/DRE motif, was not affected. The freezing-sensitive phenotype that defines sfr6 also was found to be tightly linked to the gene expression phenotype. To determine whether the failure of cold induction of CRT/DRE–containing genes in sfr6 was due to altered low-temperature calcium signaling, cold-induced cytosolic-free calcium ([Ca2+]cyt) elevations were investigated in the sfr6 mutant, but these were found to be indistinguishable from those of the wild type. We discuss the possibilities that CRT/DRE binding proteins (such as CBF1) require activation to play a role in transcription and that the SFR6 protein is a vital component of their activation.

Modifying the Subunit Composition of TASK Channels Alters the Modulation of a Leak Conductance in Cerebellar Granule Neurons

Two-pore domain potassium (K2P) channel expression is believed to underlie the developmental emergence of a potassium leak conductance [IK(SO)] in cerebellar granule neurons (CGNs), suggesting that K2P function is an important determinant of the input conductance and resting membrane potential. To investigate the role that different K2P channels may play in the regulation of CGN excitability, we generated a mouse lacking TASK-1, a K2P channel known to have high expression levels in CGNs. In situ hybridization and real-time PCR studies in wild-type and TASK-1 knock-outs (KOs) demonstrated that the expression of other K2P channels was unaltered in CGNs. TASK-1 knock-out mice were healthy and bred normally but exhibited compromised motor performance consistent with altered cerebellar function. Whole-cell recordings from adult cerebellar slice preparations revealed that the resting excitability of mature CGNs was no different in TASK-1 KO and littermate controls. However, the modulation of IK(SO) by extracellular Zn2+, ruthenium red, and H+ was altered. The IK(SO) recorded from TASK-1 knock-out CGNs was no longer sensitive to alkalization and was blocked by Zn2+ and ruthenium red. These results suggest that a TASK-1-containing channel population has been replaced by a homodimeric TASK-3 population in the TASK-1 knock-out. These data directly demonstrate that TASK-1 channels contribute to the properties of IK(SO) in adult CGNs. However, TASK channel subunit composition does not alter the resting excitability of CGNs but does influence sensitivity to endogenous modulators such as Zn2+ and H+.

Inhibition of the human two-pore domain potassium channel, TREK-1, by fluoxetine and its metabolite norfluoxetine.

1 Block of the human two‐pore domain potassium (2‐PK) channel TREK‐1 by fluoxetine (ProzacR) and its active metabolite, norfluoxetine, was investigated using whole‐cell patch‐clamp recording of currents through recombinant channels in tsA 201 cells. 2 Fluoxetine produced a concentration‐dependent inhibition of TREK‐1 current that was reversible on wash. The IC50 for block was 19 μM. Block by fluoxetine was voltage‐independent. Fluoxetine (100 μM) produced an 84% inhibition of TREK‐1 currents, but only a 31% block of currents through a related 2‐PK channel, TASK‐3. 3 Norfluoxetine was a more potent inhibitor of TREK‐1 currents with an IC50 of 9 μM. Block by norfluoxetine was also voltage‐independent. 4 Truncation of the C‐terminus of TREK‐1 (Δ89) resulted in a loss of channel function, which could be restored by intracellular acidification or the mutation E306A. The mutation E306A alone increased basal TREK‐1 current and resulted in a loss of the slow phase of TREK‐1 activation. 5 Progressive deletion of the C‐terminus of TREK‐1 had no effect on the inhibition of the channel by fluoxetine. The E306A mutation, on the other hand, reduced the magnitude of fluoxetine inhibition, with 100 μM producing only a 40% inhibition. 6 It is concluded that fluoxetine and norfluoxetine are potent inhibitors of TREK‐1. Block of TREK‐1 by fluoxetine may have important consequences when the drug is used clinically in the treatment of depression.

TASK-3 Two-Pore Domain Potassium Channels Enable Sustained High-Frequency Firing in Cerebellar Granule Neurons

The ability of neurons, such as cerebellar granule neurons (CGNs), to fire action potentials (APs) at high frequencies during sustained depolarization is usually explained in relation to the functional properties of voltage-gated ion channels. Two-pore domain potassium (K2P) channels are considered to simply hyperpolarize the resting membrane potential (RMP) by increasing the potassium permeability of the membrane. However, we find that CGNs lacking the TASK-3 type K2P channel exhibit marked accommodation of action potential firing. The accommodation phenotype was not associated with any change in the functional properties of the underlying voltage-gated sodium channels, nor could it be explained by the more depolarized RMP that resulted from TASK-3 channel deletion. A functional rescue, involving the introduction of a nonlinear leak conductance with a dynamic current clamp, was able to restore wild-type firing properties to adult TASK-3 knock-out CGNs. Thus, in addition to the accepted role of TASK-3 channels in limiting neuronal excitability, by increasing the resting potassium conductance TASK-3 channels also increase excitability by supporting high-frequency firing once AP threshold is reached.

The in Vivo Contributions of TASK-1-Containing Channels to the Actions of Inhalation Anesthetics, the alpha~2 Adrenergic Sedative Dexmedetomidine, and Cannabinoid Agonists

Inhalation anesthetics activate and cannabinoid agonists inhibit TWIK-related acid-sensitive K+ channels (TASK)-1 two-pore domain leak K+ channels in vitro. Many neuromodulators, such as noradrenaline, might also manifest some of their actions by modifying TASK channel activity. Here, we have characterized the basal behavioral phenotype of TASK-1 knockout mice and tested their sensitivity to the inhalation anesthetics halothane and isoflurane, the α2 adrenoreceptor agonist dexmedetomidine, and the cannabinoid agonist WIN55212-2 mesylate [R-(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3,-de]-1,4-benzoxazinyl]-(1-naphtalenyl)methanone mesylate)]. TASK-1 knockout mice had a largely normal behavioral phenotype. Male, but not female, knockout mice displayed an enhanced acoustic startle response. The knockout mice showed increased sensitivity to thermal nociception in a hot-plate test but not in a tail-flick test. The analgesic, sedative, and hypothermic effects of WIN55212-2 (2–6 mg/kg s.c.) were reduced in TASK-1 knockout mice. These results implicate TASK-1-containing channels in supraspinal pain pathways, in particular those modulated by endogenous cannabinoids. TASK-1 knockout mice were less sensitive to the anesthetic effects of halothane and isoflurane than wild-type littermates, requiring higher anesthetic concentrations to induce immobility as reflected by loss of the tail-withdrawal reflex. Our results support the idea that the activation of multiple background K+ channels is crucial for the high potency of inhalation anesthetics. Furthermore, TASK-1 knockout mice were less sensitive to the sedative effects of dexmedetomidine (0.03 mg/kg s.c.), suggesting a role for the TASK-1 channels in the modulation of function of the adrenergic locus coeruleus nuclei and/or other neuronal systems.

Selective block of the human 2-P domain potassium channel, TASK-3, and the native leak potassium current, IKSO, by zinc

Background potassium channels control the resting membrane potential of neurones and regulate their excitability. Two‐pore‐domain potassium (2‐PK) channels have been shown to underlie a number of such neuronal background currents. Currents through human TASK‐1, TASK‐2 and TASK‐3 channels expressed in Xenopus oocytes were inhibited by extracellular acidification. For TASK‐3, mutation of histidine 98 to aspartate or alanine considerably reduced this effect of pH. Zinc was found to be a selective blocker of TASK‐3 with virtually no effect on TASK‐1 or TASK‐2. Zinc had an IC50 of 19.8 μm for TASK‐3, at +80 mV, with little voltage dependence associated with this inhibition. TASK‐3 H98A had a much reduced sensitivity to zinc suggesting this site is important for zinc block. Surprisingly, TASK‐1 also has histidine in position 98 but is insensitive to zinc block. TASK‐3 and TASK‐1 differ at position 70 with glutamate for TASK‐3 and lysine for TASK‐1. TASK‐3 E70K also had a much reduced sensitivity to zinc while the corresponding reverse mutation in TASK‐1, K70E, induced zinc sensitivity. A TASK‐3–TASK‐1 concatamer channel was comparatively zinc insensitive. For TASK‐3, it is concluded that positions E70 and H98 are both critical for zinc block. The native cerebellar granule neurone (CGN) leak current, IKSO, is sensitive to block by zinc, with current reduced to 0.58 of control values in the presence of 100 μm zinc. This suggests that TASK‐3 channels underlie a major component of IKSO. It has recently been suggested that zinc is released from inhibitory synapses onto CGNs. Therefore it is possible that inhibition of IKSO in cerebellar granule cells by synaptically released zinc may have important physiological consequences.

Gαq-Mediated Regulation of TASK3 Two-Pore Domain Potassium Channels: The Role of Protein Kinase C

The TASK subfamily of two pore domain potassium channels (K2P) gives rise to leak potassium currents, which contribute to the resting membrane potential of many neurons and regulate their excitability. K2P channels are highly regulated by phosphorylation and by G protein-mediated pathways. In this study, we show that protein kinase C (PKC) inhibits recombinant TASK3 channels. Inhibition by PKC is blocked by the PKC inhibitors bisindolylmaleimide 1 hydrochloride (BIM) and 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole (Gö6976). Gene-silencing experiments with a validated small interfering RNA sequence against PKCα ablates the effect of PKC. PKC acts directly on hTASK3 channels to phosphorylate an identified amino acid in the C terminus region (Thr341), thereby reducing channel current. PKC also inhibits mTASK3 channels despite their having a quite different C-terminal structure to hTASK3 channels. Activation of M3 muscarinic receptors inhibits both hTASK3 channels expressed in tsA-201 cells and standing outward potassium current (IKSO) in mouse cerebellar granule neurons through the activation of the G protein Gαq, because both effects are abolished by the selective Gαq antagonist YM-254890 (J Biol Chem 279:47438–47445, 2004). This inhibition is not directly transduced through activation of PKC because inhibition persists in mutated PKC-insensitive hTASK3 channels. Instead, inhibition seems to occur through a direct action of Gαq on the channel. Nevertheless, preactivation of PKC blocks muscarinic inhibition of both TASK3 channels and IKSO. Our results suggest that activation of PKC (via phospholipase C) has a role in opposing inhibition after M3 receptor activation rather than transducing it and may act as a negative regulator of G protein modulation to prevent prolonged current inhibition.

What are the roles of the many different types of potassium channel expressed in cerebellar granule cells

Potassium (K) channels have a key role in the regulation of neuronal excitability. Over a hundred different subunits encoding distinct K channel subtypes have been identified so far. A major challenge is to relate these many different channel subunits to the functional K currents observed in native neurons. In this review, we have concentrated on cerebellar granule neurons (CGNs). We have considered each of the three principal super families of K channels in turn, namely, the six transmembrane domain, voltage-gated super family, the two transmembrane domain, inward-rectifier super family and the four transmembrane domain, leak channel super family. For each super family, we have identified the subunits that are expressed in CGNs and related the properties of these expressed channel subunits to the functional currents seen in electrophysiological recordings from these neurons. In some cases, there are strong molecular candidates for proteins underlying observed currents. In other cases the correlation is less clear. We show that at least 26 potassium channel a subunits are moderately or strongly expressed in CGNs. Nevertheless, a good empirical model of CGN function has been obtained with just six distinct K conductances. The transient K current in CGNs, seems due to expression of Kv4.2 channels or Kv4.2/4.3 heteromers, while the Kca current is due to expression of large-conductance slo channels. The G-protein activated KIR current is probably due to heteromeric expression of KiR3.1 and KiR3.2. Perhaps KiR2.2 subunits underlie the KiR current when it is constitutively active. The leak conductance can be attributed to TASK-1 and or TASK-3 channels. With less certainty, the IK-slow current may be due to expression of one or more members of the KCNQ or EAG family. Lastly, the delayed-rectifier Kv current has as many as six different potential contributors from the extensive Kv family of α subunits. Since many of these subunits are highly regulated by neurotransmitters, physiological regulators and, often, auxiliary subunits, the resulting electrical properties of CGNs may be highly dynamic and subject to constant fine-tuning.

Gating of two pore domain potassium channels

Two‐pore‐domain potassium (K2P) channels are responsible for background leak currents which regulate the membrane potential and excitability of many cell types. Their activity is modulated by a variety of chemical and physical stimuli which act to increase or decrease the open probability of individual K2P channels. Crystallographic data and homology modelling suggest that all K+ channels possess a highly conserved structure for ion selectivity and gating mechanisms. Like other K+ channels, K2P channels are thought to have two primary conserved gating mechanisms: an inactivation (or C‐type) gate at the selectivity filter close to the extracellular side of the channel and an activation gate at the intracellular entrance to the channel involving key, identified, hinge glycine residues. Zinc and hydrogen ions regulate Drosophila KCNK0 and mammalian TASK channels, respectively, by interacting with the inactivation gate of these channels. In contrast, the voltage dependence of TASK3 channels is mediated through its activation gate. For KCNK0 it has been shown that the gates display positive cooperativity. It is of much interest to determine whether other K2P regulatory compounds interact with either the activation gate or the inactivation gate to alter channel activity or, indeed, whether additional regulatory gating pathways exist.

The M1P1 loop of TASK3 K2P channels apposes the selectivity filter and influences channel function.

Channels of the two-pore domain potassium (K2P) family contain two pore domains rather than one and an unusually long pre-pore extracellular linker called the M1P1 loop. The TASK (TASK1, TASK3, and TASK5) subfamily of K2P channels is regulated by a number of different pharmacological and physiological mediators. At pH 7.4 TASK3 channels are selectively blocked by zinc in a manner that is both pHo- and [K]o-dependent. Mutation of both the Glu-70 residue in the M1P1 loop and the His-98 residue in the pore region abolished block, suggesting the two residues may contribute to a zinc binding site. Mutation of one Glu-70 residue and one His-98 residue to cysteine in TASK3 fixed concatamer channels gave currents that were enhanced by dithiothreitol and then potently blocked by cadmium, suggesting that spontaneous disulfide bridges could be formed between these two residues. Swapping the M1P1 loops of TASK1 and TASK3 channels showed that the M1P1 loop is also involved in channel regulation by pH. Therefore, the TASK3 M1P1 loop lies close to the pore, regulating TASK3 channel activity.