Cloning, localisation and functional expression of a novel human, cerebellum specific, two pore domain potassium channel

Chapman, C.G.; Meadows, Helen; Godden, Rob J.; Campbell, David A.; Duckworth, Malcolm; Kelsell, Rosemary E.; Murdock, Paul R.; Randall, Andrew D.; Rennie, Gillian; Gloger, Israel

doi:10.1016/s0169-328x(00)00183-2

Cited by 82 publications

(53 citation statements)

References 19 publications

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“…Both functional and binding assays were used to confirm MCH as the cognate ligand for this receptor. Further characterization using an intracellular Ca 2ϩ assay demonstrated that salmon MCH and the synthetic analogue [Phe 13 ,Tyr 19 ]MCH were equipotent with MCH and were full agonists at MCH 2 . However, variant MCH, the putative product of a second, variant form of the MCH gene (21), was ϳ500-fold less potent than MCH.…”

Section: Discussionmentioning

confidence: 99%

“…TaqMan RT-PCR Tissue Localization-Quantitative RT-PCR was performed using gene-specific primers to MCH 2 and MCH 1 receptors on mRNA from 20 body tissues and 19 brain regions as described previously (19). The mRNA from each tissue and brain region analyzed by TaqMan quantitative RT-PCR was derived from four individuals, two male and two female (except prostate tissue).…”

Section: Reader (As Described Below)mentioning

confidence: 99%

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Molecular Cloning and Functional Characterization of MCH2, a Novel Human MCH Receptor

Hill¹,

Duckworth²,

Murdock³

et al. 2001

Journal of Biological Chemistry

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Melanin-concentrating hormone (MCH) is involved in the regulation of feeding and energy homeostasis. Recently, a 353-amino acid splice variant form of the human orphan receptor SLC-1 (1) (hereafter referred to as MCH 1 ) was identified as an MCH receptor. This report describes the cloning and functional characterization of a novel second human MCH receptor, which we designate MCH 2 , initially identified in a genomic survey sequence as being homologous to MCH 1 receptors. Using this sequence, a full-length cDNA was generated with an open reading frame of 1023 base pairs, encoding a polypeptide of 340 amino acids, with 38% identity to MCH 1 and with many of the structural features conserved in G protein-coupled receptors. This newly discovered receptor belongs to class 1 (rhodopsin-like) of the G protein-coupled receptor superfamily. HEK293 cells transfected with MCH 2 receptors responded to nanomolar concentrations of MCH with an increase in intracellular Ca 2؉ levels and increased cellular extrusion of protons. In addition, fluorescently labeled MCH bound with nanomolar affinity to these cells. The tissue localization of MCH 2 receptor mRNA, as determined by quantitative reverse transcription-polymerase chain reaction, was similar to that of MCH 1 in that both receptors are expressed predominantly in the brain. The discovery of a novel MCH receptor represents a new potential drug target and will allow the further elucidation of MCH-mediated responses. Melanin-concentrating hormone (MCH)1 is a cyclic neuropeptide that was first discovered in teleost fish, in which it acts as a skin color-regulating hormone (2). In rodents its tissue distribution in the perikarya of the lateral hypothalamus and the zona incerta suggests that MCH may be involved in a variety of behavioral responses (3). Similar tissue distributions have been reported in both bird (4) and monkey (5). Reports implicating MCH in the regulation of feeding behavior show that increased food intake occurs after direct administration of MCH into the brain (6) and that MCH is up-regulated after fasting and in obese leptin-deficient mice (7). There are also reports that suggest MCH may be involved in aggressive behavior, anxiety, and reproductive function (8, 9).Recently, several groups independently identified a 353-amino acid splice variant of the orphan G protein-coupled receptor (GPCR) SLC-1 (1) as an MCH receptor (10 -14). In view of the findings of the current study, we propose that this form of SLC-1 be hereafter referred to as MCH 1 . Southern blot and related studies have indicated the absence of additional MCH receptor subtypes that closely resemble MCH 1 at the DNA level (3, 10, 15). However, because degeneracy in receptor-ligand pairings throughout the GPCR superfamily is common, we reasoned that other MCH receptors with low homology to the MCH 1 receptor may exist. This suggestion is supported by reports of pharmacological differences between the MCH 1 receptor and MCH binding sites in various cell lines and tissues (10).Sequencing of the human...

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Section: Discussionmentioning

confidence: 99%

Section: Reader (As Described Below)mentioning

confidence: 99%

Molecular Cloning and Functional Characterization of MCH2, a Novel Human MCH Receptor

Hill¹,

Duckworth²,

Murdock³

et al. 2001

Journal of Biological Chemistry

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219

144

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“…These two channel subunits share a number of properties when expressed individually in heterologous expression systems. As their name suggests, they are sensitive to changes in extracellular pH; both are activated by alkalization and inhibited by acidification, although over different pH ranges (pK values for channel activity, ϳ7.5 and ϳ6.8 for TASK-1 and TASK-3, respectively) (Duprat et al, 1997;Kim et al, 1998Kim et al, , 2000Leonoudakis et al, 1998;Chapman et al, 2000;Rajan et al, 2000). In addition, each subunit produces whole-cell currents with voltage-dependent and kinetic properties of instantaneous open rectifiers, and both are activated by inhalation anesthetics and inhibited downstream of G-protein-coupled receptors (Patel et al, 1999;Czirjak et al, 2000;Sirois et al, 2000;Talley et al, 2000;Meadows and Randall, 2001;Talley and Bayliss, 2002;Chemin et al, 2003).…”

Section: Introductionmentioning

confidence: 99%

Motoneurons Express Heteromeric TWIK-Related Acid-Sensitive K⁺(TASK) Channels Containing TASK-1 (KCNK3) and TASK-3 (KCNK9) Subunits

Berg¹,

Talley²,

Manger³

et al. 2004

J. Neurosci.

188

244

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Background potassium currents carried by the KCNK family of two-pore-domain K ϩ channels are important determinants of resting membrane potential and cellular excitability. TWIK-related acid-sensitive K ϩ 1 (TASK-1, KCNK3) and TASK-3 (KCNK9) are pHsensitive subunits of the KCNK family that are closely related and coexpressed in many brain regions. There is accumulating evidence that these two subunits can form heterodimeric channels, but this evidence remains controversial. In addition, a substantial contribution of heterodimeric TASK channels to native currents has not been unequivocally established. In a heterologous expression system, we verified formation of heterodimeric TASK channels and characterized their properties; TASK-1 and TASK-3 were coimmunoprecipitated from membranes of mammalian cells transfected with the channel subunits, and a dominant negative TASK-1(Y191F) construct strongly diminished TASK-3 currents. Tandem-linked heterodimeric TASK channel constructs displayed a pH sensitivity (pK ϳ7.3) in the physiological range closer to that of TASK-1 (pK ϳ7.5) than TASK-3 (pK ϳ6.8). On the other hand, heteromeric TASK channels were like TASK-3 insofar as they were activated by high concentrations of isoflurane (0.8 mM), whereas TASK-1 channels were inhibited. The pH and isoflurane sensitivities of native TASK-like currents in hypoglossal motoneurons, which strongly express TASK-1 and TASK-3 mRNA, were best represented by TASK heterodimeric channels. Moreover, after blocking homomeric TASK-3 channels with ruthenium red, we found a major component of motoneuronal isoflurane-sensitive TASK-like current that could be attributed to heteromeric TASK channels. Together, these data indicate that TASK-1 and TASK-3 subunits coassociate in functional channels, and heteromeric TASK channels provide a substantial component of background K ϩ current in motoneurons with distinct modulatory properties.

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“…Like other two-pore domain members, these channels show little time or voltage dependence; thus, they have characteristics of leaky K ϩ channels, generating background currents that contribute to membrane potential and the regulation of cell excitability. The activity of TASK channels is modulated by volatile anesthetics (3,4), neurotransmitters (5,6), as well as extracellular pH in the physiological range (7)(8)(9)(10)(11). TASK3 is expressed at very low levels among the normal tissues except in the brain, where high levels expression of TASK3 were detected (7)(8)(9).…”

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confidence: 99%

“…The activity of TASK channels is modulated by volatile anesthetics (3,4), neurotransmitters (5,6), as well as extracellular pH in the physiological range (7)(8)(9)(10)(11). TASK3 is expressed at very low levels among the normal tissues except in the brain, where high levels expression of TASK3 were detected (7)(8)(9). The physiological functions of TASK channels are largely unknown, though their roles in the regulation of breathing (12,13), aldosterone secretion (5) and anesthetic-mediated neuronal activity (14) have been proposed.…”

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confidence: 99%

Oncogenic potential of TASK3 (Kcnk9) depends on K⁺channel function

Pei¹,

Wiser²,

Slavin³

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

161

124

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TASK3 gene (Kcnk9) is amplified and overexpressed in several types of human carcinomas. In this report, we demonstrate that a point mutation (G95E) within the consensus K ؉ filter of TASK3 not only abolished TASK3 potassium channel activity but also abrogated its oncogenic functions, including proliferation in low serum, resistance to apoptosis, and promotion of tumor growth. Furthermore, we provide evidence that TASK3 G95E is a dominant-negative mutation, because coexpression of the wild-type and the mutant TASK3 resulted in inhibition of K ؉ current of wild-type TASK3 and its tumorigenicity in nude mice. These results establish a direct link between the potassium channel activity of TASK3 and its oncogenic functions and imply that blockers for this potassium channel may have therapeutic potential for the treatment of cancers.T ASK (TWIK-related acid-sensitive K ϩ channels) channels are members of the two-pore domain family of potassium channels, whose structure consists of two-pore forming regions flanked by four transmembrane domains (1, 2). Like other two-pore domain members, these channels show little time or voltage dependence; thus, they have characteristics of leaky K ϩ channels, generating background currents that contribute to membrane potential and the regulation of cell excitability. The activity of TASK channels is modulated by volatile anesthetics (3, 4), neurotransmitters (5, 6), as well as extracellular pH in the physiological range (7-11). TASK3 is expressed at very low levels among the normal tissues except in the brain, where high levels expression of TASK3 were detected (7-9). The physiological functions of TASK channels are largely unknown, though their roles in the regulation of breathing (12, 13), aldosterone secretion (5) and anesthetic-mediated neuronal activity (14) have been proposed. Recently, we showed that TASK3 is amplified in 10% of breast cancers and is overexpressed at a higher frequency of breast, lung, colon, and metastatic prostate cancers (15), suggesting that TASK3 may play a role in pathogenesis of some human carcinomas.Is the dysregulated expression of TASK3 in tumor cells a consequence of their abnormal growth or is this K ϩ channel involved in promoting tumor growth? To begin to answer this question, we created an inactivating mutation of TASK3. We report here that TASK3 G95E is a dominant-negative mutation that abolishes not only TASK3 K ϩ channel activity but also its oncogenic functions. These results provide molecular basis for developing specific blockers for this K ϩ channel in the treatment of cancer. Materials and MethodsPlasmids and Mutagenesis. The coding region of TASK3 was cloned at BamHI and EcoRI sites of the pLPC retroviral expression vector (15) to generate pLPC-TASK3. Site-directed mutagenesis was performed to change Gly-95 to Glu to create pLPC-TASK3 G95E , by using QuickChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's protocol. pTracer-TASK 3 G95E was generated by excising TASK3 G95E from pLPC vector and cloned at KpnI...

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Cloning, localisation and functional expression of a novel human, cerebellum specific, two pore domain potassium channel

Cited by 82 publications

References 19 publications

Molecular Cloning and Functional Characterization of MCH2, a Novel Human MCH Receptor

Molecular Cloning and Functional Characterization of MCH2, a Novel Human MCH Receptor

Motoneurons Express Heteromeric TWIK-Related Acid-Sensitive K⁺(TASK) Channels Containing TASK-1 (KCNK3) and TASK-3 (KCNK9) Subunits

Oncogenic potential of TASK3 (Kcnk9) depends on K⁺channel function

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Cloning, localisation and functional expression of a novel human, cerebellum specific, two pore domain potassium channel

Cited by 82 publications

References 19 publications

Molecular Cloning and Functional Characterization of MCH2, a Novel Human MCH Receptor

Molecular Cloning and Functional Characterization of MCH2, a Novel Human MCH Receptor

Motoneurons Express Heteromeric TWIK-Related Acid-Sensitive K+(TASK) Channels Containing TASK-1 (KCNK3) and TASK-3 (KCNK9) Subunits

Oncogenic potential of TASK3 (Kcnk9) depends on K+channel function

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About

Motoneurons Express Heteromeric TWIK-Related Acid-Sensitive K⁺(TASK) Channels Containing TASK-1 (KCNK3) and TASK-3 (KCNK9) Subunits

Oncogenic potential of TASK3 (Kcnk9) depends on K⁺channel function