TREK-1 Research Articles

Abstract 4043: Loss of imprinting of KCNK9 in breast cancer

Abstract KCNK9 (TASK-3) encodes a two-pore potassium channel that functions in tumor growth and survival. The potassium channel function is necessary for tumor proliferation and survival, by enhancing resistance to hypoxia and apoptosis. KCNK9 overexpression is observed in melanoma, colorectal cancer, and breast and brain tumors, but with no associated mutations found. Rather, the overexpression is often the apparent result of gene amplification of KCNK9, but any other mechanism that increases expression would presumably have a similar effect on tumorigenesis. This importance of gene dosage and expression levels is particularly relevant, as KCNK9 has been demonstrated to be a maternally expressed imprinted gene, which, in effect, reduces copy number. Monoallelic expression of KCNK9 was originally found in brain tissue, but our work has found that it is also imprinted in breast tissue. Thus, we hypothesized that loss of imprint control would be similar to a gene duplication, increasing the effective copy number, and therefore KCNK9 gene expression. To study the regulation of KCNK9 imprinting in tumors, we have screened for differentially methylated regions that may function as imprint control regions (ICRs), and examined the expression status of KCNK9 in breast tumor samples relative to the methylation state of the putative ICRs. Approximately 40-50% of breast tumor samples analyzed show loss of imprinting, seen as expression of both alleles of an exonic polymorphism. Additionally, loss of methylation at a putative ICR upstream of the transcription start site is observed in combination with loss of imprint regulation. This correlated loss of methylation and imprint control is seen most commonly in tumors of the “triple negative” subtype - tumors negative for expression of estrogen receptor, progesterone receptor, and HER2/neu. These novel findings regarding KCNK9 imprint regulation and its disruption in a subset of tumors may lead to new approaches in diagnosis and targeted therapy of breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4043. doi:1538-7445.AM2012-4043

Migraine: Role of the TRESK two-pore potassium channel

Migraine is a severe episodic headache disorder affecting one in five people. Genetic studies have identified mutations in the CACNA1, ATP1A2 and SCN1A genes in the rare familial hemiplegic migraine. Recently, a mutation in the KCNK18 gene, encoding the TRESK two-pore domain potassium channel, was described in a large family with migraine with aura. This review will elaborate on the possible role of the TRESK channel in regulating neuronal excitability, its role in migraine pathogenesis, and on promising therapeutic opportunities targeting this channel.

Functional Analysis of Mutations in the TRESK K2P Potassium Channel Associated with ‘migraine with Aura’

An inherited mutation in the KCNK18 gene has been shown to be associated with ‘migraine with aura’. This is a common, debilitating, recurrent headache disorder associated with transient and reversible focal neurological symptoms. KNCK18 encodes the TWIK-related spinal cord potassium channel (TRESK), a member of the K2P family of potassium channels. The F139WfsX24 mutation, segregates perfectly with typical migraine with aura in a large pedigree and functional characterization of this mutation demonstrates that it causes a complete loss of TRESK function and that the mutant subunit suppresses wild-type channel function through a dominant-negative effect, thus explaining the dominant penetrance of this allele (Lafreniere et al, doi:10.1038/nm.2216). This identifies a role for TRESK in the pathogenesis of typical migraine with aura and further supports the role of this channel as a potential therapeutic target. In this study we have examined the electrophysiological properties of other mutations identified in the human KCNK18 gene and find that several of these variants also produce a dramatic dominant-negative phenotype.

KCNK3 (Task-1) Potassium Leak Channels Internalize in a PKC- and 14-3-3beta-Dependent Manner

Ion channels encoded by the human KCNK3 gene give rise to the pH-sensitive outwardly rectifier potassium leak current in a variety of cell types, where they play a decisive role in determination of the hyperpolarized resting membrane potential, controlling excitability and hastening repolarization. These tandem-pore domain channels are regulated by various agents and stimuli, including neurotransmitters, hormones, volatile anesthetics, oxidases and protein kinases. Several studies have shown that activation of protein kinase C (PKC) inhibits KCNK3 (Task-1) currents; however, the cellular mechanism underlying this inhibition has not been elucidated. Using a combination of electrophysiological, biochemical and imaging approaches, we have determined that PKC-activation induces KCNK3 channel internalization. Moreover, we have identified specific residues in the KCNK3 C-terminus that contribute, but are not solely required, for PKC-mediated internalization. Since it was previously shown that the interaction with phosphoserine binding protein 14-3-3beta promotes ER exit of KCNK3, we examined the role of 14-3-3beta on KCNK3 internalization. Using overexpression and shRNA knockdown experiments of 14-3-3beta, we show that PKC-dependent KCNK3 internalization is also crucially dependent on the cellular levels of 14-3-3beta protein. Our results reveal a novel mechanism of KCNK3 current regulation by channel internalization in a PKC- and 14-3-3beta-dependent manner.

A dominant-negative mutation in the TRESK potassium channel is linked to familial migraine with aura

Migraine with aura is a common, debilitating, recurrent headache disorder associated with transient and reversible focal neurological symptoms. A role has been suggested for the two-pore domain (K2P) potassium channel, TWIK-related spinal cord potassium channel (TRESK, encoded by KCNK18), in pain pathways and general anaesthesia. We therefore examined whether TRESK is involved in migraine by screening the KCNK18 gene in subjects diagnosed with migraine. Here we report a frameshift mutation, F139WfsX24, which segregates perfectly with typical migraine with aura in a large pedigree. We also identified prominent TRESK expression in migraine-salient areas such as the trigeminal ganglion. Functional characterization of this mutation demonstrates that it causes a complete loss of TRESK function and that the mutant subunit suppresses wild-type channel function through a dominant-negative effect, thus explaining the dominant penetrance of this allele. These results therefore support a role for TRESK in the pathogenesis of typical migraine with aura and further support the role of this channel as a potential therapeutic target.

Effect of sevoflurane preconditioning on TREK-1 channel expression in hippocampus after focal cerebral ischemia-reperfusion in rats

Objective To investigate the effect of sevoflurane preconditioning on TREK-1 channel expression in hippocampus after focal cerebral ischemia-reperfusion (I/R)in rats and the mechanism.Methods Thirty-six male SD rats weighing 240-280 g were randomly divided into 3 groups(n=12 each):group Ⅰ sham operation (group S),group Ⅱ I/R and group Ⅲ sevoflurane preconditioning (group Sevo).Focal cerebral ischemia was produced by inserting a 4-0 nylon thread with rounded tip into right internal jugular vein.The nylon thread was the nylon thread Wag about 18-20 Innl.The right middle cerebral artery(MCA)Wag occluded for 2 h and then released for 24 h reperfusion.The Sevo group inhaled 2.4% sevoflurane for 30 min at l h before ischemia.Neurological deftcits were assessed and scored at the end of 24 h reperfusion (the higher was the score,the severer was the deficit).The cerebral infarct size was determined by TTC staining and the TREK-1 mRNA in hippocampus by RT-PCR.Results The cerebral infarct size was significantly smaller and the neurological deficit scores were significantly lower in Sevo group than in I/R group.The TREK-1 mBNA expression was significantly up-regulated in Sevo group as compared with I/R group.Conclusion Sevoflurane preconditioning Can protect the brain against I/R injury by activating TREK-1 in hippocampus. Key words: Inhalation anesthetics; Ischemic preconditioning; Infarction,middle cerebral artery; Potassium channels,tandem pore domain; Hippocampus

Pharmacogenetics Studies in STAR*D: Strengths, Limitations, and Results

Pharmacogenetics Studies in STAR*D: Strengths, Limitations, and Results

Support for the involvement of the KCNK2 gene in major depressive disorder and response to antidepressant treatment

The aim of this study was to examine the associations between genetic variations in the human KCNK2 gene and major depressive disorder (MDD) and response to antidepressant treatment. Four hundred and forty-nine patients with MDD and 421 normal controls were included in the study; among the MDD patients, 158 were further followed-up for 8 weeks to assess their response to antidepressant treatment. Five polymorphisms (rs12131478, rs6667764, rs10494994, rs11583745 and rs6686529) of the KCNK2 gene were investigated in terms of their association with MDD and antidepressant treatment efficacy. The genotype frequency of rs6686529 differed significantly between the MDD patients and controls (uncorrected P = 0.00052) and remained statistically significant after correction for multiple comparisons. Individuals with homozygous genotypes (CC or GG) showed greater susceptibility to MDD than those with heterozygous genotypes, indicating a possible heterosis effect of the polymorphism on MDD. In addition, this polymorphism also affected the efficacy of antidepressant treatment: the CC carriers had a greater probability of achieving remission after 8 weeks of treatment than the G-allele carriers [odds ratio = 2.55 (95% confidence interval = 1.11-5.88)]. Our findings are in line with those of animal studies, and show that KCNK2 is related to the susceptibility to MDD, and involved in antidepressant treatment response. However, the finding of heterosis association of rs6686529 and MDD may be mechanistic, and further replication studies will be essential.

Thrilling Moment of an Inhibitory Channel

Long thought to be potassium channels with calming influence quietly working away in the background, in this issue of Neuron, Thomas et al. demonstrate that K(2P)2.1 (TREK-1) channels have two endogenous isoforms due to a mechanism called alternative translation initiation (ATI). Hence, they can actually be excitable.

Sequence-based bioinformatic prediction and QUASEP identify genomic imprinting of the KCNK9 potassium channel gene in mouse and human

Genomic imprinting is the epigenetic marking of gene subsets resulting in monoallelic or predominant expression of one of the two parental alleles according to their parental origin. We describe the systematic experimental verification of a prioritized 16 candidate imprinted gene set predicted by sequence-based bioinformatic analyses. We used Quantification of Allele-Specific Expression by Pyrosequencing (QUASEP) and discovered maternal-specific imprinted expression of the Kcnk9 gene as well as strain-dependent preferential expression of the Rarres1 gene in E11.5 (C57BL/6 x Cast/Ei)F1 and informative (C57BL/6 x Cast/Ei) x C57BL/6 backcross mouse embryos. For the remaining 14 candidate imprinted genes, we observed biallelic expression. In adult mouse tissues, we found that Kcnk9 expression was restricted to the brain and also was maternal-specific. QUASEP analysis of informative human fetal brain samples further demonstrated maternal-specific imprinted expression of the human KCNK9 orthologue. The CpG islands associated with the mouse and human Kcnk9/KCNK9 genes were not differentially methylated, but strongly hypomethylated. Thus, we speculate that mouse Kcnk9 imprinting may be regulated by the maternal germline differentially methylated region in Peg13, an imprinted non-coding RNA gene in close proximity to Kcnk9 on distal mouse chromosome 15. Our data have major implications for the proposed role of Kcnk9 in neurodevelopment, apoptosis and tumourigenesis, as well as for the efficiency of sequence-based bioinformatic predictions of novel imprinted genes.

Melanoma cells exhibit strong intracellular TASK-3-specific immunopositivity in both tissue sections and cell culture.

Amplification of the kcnk9 gene and overexpression of the encoded channel protein (TASK-3) seems to be involved in carcinogenesis. In the present work, TASK-3 expression of melanoma cells has been studied. For the investigation of TASK-3-specific immunolabelling, a monoclonal antibody has been developed and applied along with two, commercially available polyclonal antibodies targeting different epitopes of the channel protein. Both primary and metastatic melanoma cells proved to be TASK-3 positive, showing prominent intracellular TASK-3-specific labelling; mostly concentrating around or in the proximity of the nuclei. The immunoreaction was associated with the nuclear envelope, and with the processes of the cells and it was also present in the cell surface membrane. Specificity of the immunolabelling was confirmed by Western blot and transfection experiments. As TASK-3 immunopositivity of benign melanocytes could also be demonstrated, the presence or absence of TASK-3 channels cannot differentiate between malignant and non-malignant melanocytic tumours.

A <I>TASK3</I> Channel (<I>KCNK9</I>) Mutation in a Genetic Model of Absence Epilepsy

Childhood absence epilepsy is an idiopathic, generalized, nonconvulsive epilepsy with a multifactorial genetic etiology. The KCNK9 gene coding for the TASK3 (Twik-like acid-sensitive K</U)+) channel is present on chromosome 8 at position 8q24, a locus that has shown positive linkage to the human absence epilepsy phenotype. Sequencing of the KCNK9 gene in the genetic absence epilepsy rats from Strasbourg (GAERS), a well established genetic model of this disease, reveals an additional alanine residue in a polyalanine tract within the C-terminal intracellular domain. This additional alanine is absent in the inbred nonepileptic control (NEC) strain, Wistar, and Wistar albino Glaxo strain bred in Rijswijk, another inbred rat model of absence epilepsy. Expression of the mutant channel in CHO cells produces a K+ current that is blocked by acidic pH and millimolar concentrations of barium or ruthenium red and is not different from the wild-type channel. In brain slices, thalamic neurons display a prominent pH-sensitive tonic K+ current, but no difference was observed between GAERS and NEC or Wistar rats. Ruthenium red had no effect in cortical, reticular thalamic, or sensory thalamic neurons in either GAERS or NEC, indicating that the TASK3 homodimer is not present in these structures. Twik-like acid-sensitive K+(TASK3) channels, therefore, are probably associated with TASK1 to form ruthenium red-insensitive heterodimers in these neurons. Finally, no difference was found between GAERS and NEC rats in the modulation of the leak K+ current following activation of muscarinic receptors. These studies describe the first mutation found in a genetic model of absence epilepsy. Although our experiments showed no difference in the leak K+ current between GAERS and NEC rats, further work is needed to ascertain whether this mutation contributes to the generation of absence seizures, possibly by mechanisms related to the expansion of the polyalanine run.

Altered expression of KCNK9 in colorectal cancers

K(+) channels have been reported to be involved in the proliferation of many types of cells, including some human carcinoma and tumor cell lines. KCNK9, a TASK channel, is amplified and overexpressed in several types of human cancer. In the present study, we examined the expression and somatic mutations of KCNK9 in 124 colorectal cancers by immunohistochemistry using tissue microarray and PCR-SSCP. Immunopositivity was observed in 57 (46.0%) of 124 colorectal cancers. Clinically, KCNK9 was immunopositive in 4 (30.7%) of 13 cases which were stage A, 26 (55.3%) of 47 which were stage B, 23 (41.1%) of 56 which were stage C, and 4 (50%) of 8 which were stage D. Statistically, KCNK9 protein expression was not related to tumor stage (Bartholomew test, p>0.05) and lymph node metastasis (Chi-Square test, p=0.8338). In the mutation study of the KCNK9 gene, we found only one sequence variation (ACG-->ACC, Thr-->Thr) at codon 170 both in corresponding normal and tumor DNAs. These results indicate that overexpression rather than mutation of the KCNK9 gene may contribute to the development of colorectal cancers and suggest that the development of KCNK9-targeted agents may provide new possibilities in the treatment of colorectal cancer.

Inhibition of Human Twik Related K+ Channel (TREK-1) by Bupivacaine and L-Bupivacaine

Inhibition of Human Twik Related K+ Channel (TREK-1) by Bupivacaine and L-Bupivacaine

KCNK2 Is Both Dependent and Independent

KCNK2 Is Both Dependent and Independent