RESUMO
Coordinated expression of ion channels is crucial for cardiac rhythms, neural signaling, and cell cycle progression. Perturbation of this balance results in many disorders including cardiac arrhythmias. Prior work revealed association of mRNAs encoding cardiac NaV1.5 (SCN5A) and hERG1 (KCNH2), but the functional significance of this association was not established. Here, we provide a more comprehensive picture of KCNH2, SCN5A, CACNA1C, and KCNQ1 transcripts collectively copurifying with nascent hERG1, NaV1.5, CaV1.2, or KCNQ1 channel proteins. Single-molecule fluorescence in situ hybridization (smFISH) combined with immunofluorescence reveals that the channel proteins are synthesized predominantly as heterotypic pairs from discrete molecules of mRNA, not as larger cotranslational complexes. Puromycin disrupted colocalization of mRNA with its encoded protein, as expected, but remarkably also pairwise mRNA association, suggesting that transcript association relies on intact translational machinery or the presence of the nascent protein. Targeted depletion of KCHN2 by specific shRNA resulted in concomitant reduction of all associated mRNAs, with a corresponding reduction in the encoded channel currents. This co-knockdown effect, originally described for KCNH2 and SCN5A, thus appears to be a general phenomenon among transcripts encoding functionally related proteins. In multielectrode array recordings, proarrhythmic behavior arose when IKr was reduced by the selective blocker dofetilide at IC50 concentrations, but not when equivalent reductions were mediated by shRNA, suggesting that co-knockdown mitigates proarrhythmic behavior expected from the selective reduction of a single channel species. We propose that coordinated, cotranslational association of functionally related ion channel mRNAs confers electrical stability by co-regulating complementary ion channels in macromolecular complexes.
Assuntos
Arritmias Cardíacas , Canal de Potássio KCNQ1 , Humanos , Canal de Potássio KCNQ1/genética , Canal de Potássio ERG1/genética , Hibridização in Situ Fluorescente , Arritmias Cardíacas/genética , Arritmias Cardíacas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismoRESUMO
Slow deactivation is a critical property of voltage-gated K+ channels encoded by the human Ether-à-go-go-Related Gene 1 (hERG). hERG1 channel deactivation is modulated by interactions between intracellular N-terminal Per-Arnt-Sim (PAS) and C-terminal cyclic nucleotide-binding homology (CNBh) domains. The PAS domain is multipartite, comprising a globular domain (gPAS; residues 26-135) and an N-terminal PAS-cap that is further subdivided into an initial unstructured "tip" (residues 1-12) and an amphipathic α-helical region (residues 13-25). Although the PAS-cap tip has long been considered the effector of slow deactivation, how its position near the gating machinery is controlled has not been elucidated. Here, we show that a triad of hydrophobic interactions among the gPAS, PAS-cap α helix, and the CNBh domains is required to support slow deactivation in hERG1. The primary sequence of this "hydrophobic nexus" is highly conserved among mammalian ERG channels but shows key differences to fast-deactivating Ether-à-go-go 1 (EAG1) channels. Combining sequence analysis, structure-directed mutagenesis, electrophysiology, and molecular dynamics simulations, we demonstrate that polar serine substitutions uncover an intermediate deactivation mode that is also mimicked by deletion of the PAS-cap α helix. Molecular dynamics simulation analyses of the serine-substituted channels show an increase in distance among the residues of the hydrophobic nexus, a rotation of the intracellular gating ring, and a retraction of the PAS-cap tip from its receptor site near the voltage sensor domain and channel gate. These findings provide compelling evidence that the hydrophobic nexus coordinates the respective components of the intracellular gating ring and positions the PAS-cap tip to control hERG1 deactivation gating.
Assuntos
Interações Hidrofóbicas e Hidrofílicas , Animais , Humanos , Sequência de Aminoácidos , Canal de Potássio ERG1/metabolismo , Canal de Potássio ERG1/química , Canal de Potássio ERG1/genética , Canais de Potássio Éter-A-Go-Go/química , Canais de Potássio Éter-A-Go-Go/metabolismo , Canais de Potássio Éter-A-Go-Go/genética , Células HEK293 , Espaço Intracelular/metabolismo , Ativação do Canal Iônico , Domínios ProteicosRESUMO
The human ERG (hERG) K+ channel has a crucial function in cardiac repolarization, and mutations or channel block can give rise to long QT syndrome and catastrophic ventricular arrhythmias. The cytosolic assembly formed by the Per-Arnt-Sim (PAS) and cyclic nucleotide binding homology (CNBh) domains is the defining structural feature of hERG and related KCNH channels. However, the molecular role of these two domains in channel gating remains unclear. We have previously shown that single-chain variable fragment (scFv) antibodies can modulate hERG function by binding to the PAS domain. Here, we mapped the scFv2.12 epitope to a site overlapping with the PAS/CNBh domain interface using NMR spectroscopy and mutagenesis and show that scFv binding in vitro and in the cell is incompatible with the PAS interaction with CNBh. By generating a fluorescently labeled scFv2.12, we demonstrate that association with the full-length hERG channel is state dependent. We detect Förster resonance energy transfer (FRET) with scFv2.12 when the channel gate is open but not when it is closed. In addition, state dependence of scFv2.12 FRET signal disappears when the R56Q mutation, known to destabilize the PAS-CNBh interaction, is introduced in the channel. Altogether, these data are consistent with an extensive structural alteration of the PAS/CNBh assembly when the cytosolic gate opens, likely favoring PAS domain dissociation from the CNBh domain.
Assuntos
Canal de Potássio ERG1/metabolismo , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Citosol/metabolismo , Canal de Potássio ERG1/genética , Canal de Potássio ERG1/imunologia , Canais de Potássio Éter-A-Go-Go/imunologia , Canais de Potássio Éter-A-Go-Go/metabolismo , Transferência Ressonante de Energia de Fluorescência , Células HEK293 , Humanos , Ativação do Canal Iônico , Síndrome do QT Longo/genética , Conformação Molecular , Mutação , Conformação Proteica , Domínios Proteicos/genética , Domínios Proteicos/imunologia , Proteínas Serina-Treonina Quinases/metabolismo , Relação Estrutura-AtividadeRESUMO
The human ether-a-go-go-related gene1 (hERG) ion channel has been the subject of fascination since it was identified as a target of long QT syndrome more than 20 years ago. In this Biophysical Perspective, we look at what makes hERG intriguing and vexingly unique. By probing recent high-resolution structures in the context of functional and biochemical data, we attempt to summarize new insights into hERG-specific function and articulate important unanswered questions. X-ray crystallography and cryo-electron microscopy have revealed features not previously on the radar-the "nonswapped" transmembrane architecture, an "intrinsic ligand," and hydrophobic pockets off a pore cavity that is surprisingly small. Advances in our understanding of drug block and inactivation mechanisms are noted, but a full picture will require more investigation.
Assuntos
Canal de Potássio ERG1 , Síndrome do QT Longo , Microscopia Crioeletrônica , Cristalografia por Raios X , HumanosRESUMO
Reduced levels of the cardiac human (h)ERG ion channel protein and the corresponding repolarizing current IKr can cause arrhythmia and sudden cardiac death, but the underlying cellular mechanisms controlling hERG surface expression are not well understood. Here, we identified TRIOBP-1, an F-actin-binding protein previously associated with actin polymerization, as a putative hERG-interacting protein in a yeast-two hybrid screen of a cardiac library. We corroborated this interaction by performing Förster resonance energy transfer (FRET) in HEK293 cells and co-immunoprecipitation in HEK293 cells and native cardiac tissue. TRIOBP-1 overexpression reduced hERG surface expression and current density, whereas reducing TRIOBP-1 expression via shRNA knockdown resulted in increased hERG protein levels. Immunolabeling in rat cardiomyocytes showed that native TRIOBP-1 colocalized predominantly with myosin-binding protein C and secondarily with rat ERG. In human stem cell-derived cardiomyocytes, TRIOBP-1 overexpression caused intracellular co-sequestration of hERG signal, reduced native IKr and disrupted action potential repolarization. Ca2+ currents were also somewhat reduced and cell capacitance was increased. These findings establish that TRIOBP-1 interacts directly with hERG and can affect protein levels, IKr magnitude and cardiac membrane excitability.
Assuntos
Proteínas dos Microfilamentos/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Cálcio/metabolismo , Células HEK293 , Humanos , Masculino , Proteínas dos Microfilamentos/genética , Ligação Proteica , Transporte Proteico , Ratos , Regulador Transcricional ERG/genética , Regulador Transcricional ERG/metabolismoRESUMO
Oligomers of homomeric voltage-gated potassium channels associate early in biogenesis as the nascent proteins emerge from the polysome. Less is known about how proteins emerging from different polysomes associate to form hetero-oligomeric channels. Here, we report that alternate mRNA transcripts encoding human ether-à-go-go-related gene (hERG) 1a and 1b subunits, which assemble to produce ion channels mediating cardiac repolarization, are physically associated during translation. We show that shRNA specifically targeting either hERG 1a or 1b transcripts reduced levels of both transcripts, but only when they were coexpressed heterologously. Both transcripts could be copurified with an Ab against the nascent hERG 1a N terminus. This interaction occurred even when translation of 1b was prevented, indicating the transcripts associate independent of their encoded proteins. The association was also demonstrated in cardiomyocytes, where levels of both hERG transcripts were reduced by either 1a or 1b shRNA, but native KCNE1 and ryanodine receptor 2 (RYR2) transcripts were unaffected. Changes in protein levels and membrane currents mirrored changes in transcript levels, indicating the targeted transcripts were undergoing translation. The physical association of transcripts encoding different subunits provides the spatial proximity required for nascent proteins to interact during biogenesis, and may represent a general mechanism facilitating assembly of heteromeric protein complexes involved in a range of biological processes.
Assuntos
Canais de Potássio Éter-A-Go-Go/genética , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Células Cultivadas , Células HEK293 , Humanos , Imunoprecipitação , Células-Tronco Pluripotentes Induzidas/citologia , Potenciais da Membrana , Miócitos Cardíacos/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Subunidades Proteicas , RNA Mensageiro/genética , RNA Interferente Pequeno/metabolismo , RNA Interferente Pequeno/farmacologia , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , TransfecçãoRESUMO
The human human ether-à-go-go-related gene (hERG) potassium channel plays a critical role in the repolarization of the cardiac action potential. Changes in hERG channel function underlie long QT syndrome (LQTS) and are associated with cardiac arrhythmias and sudden death. A striking feature of this channel and KCNH channels in general is the presence of an N-terminal Per-Arnt-Sim (PAS) domain. In other proteins, PAS domains bind ligands and modulate effector domains. However, the PAS domains of KCNH channels are orphan receptors. We have uncovered a family of positive modulators of hERG that specifically bind to the PAS domain. We generated two single-chain variable fragments (scFvs) that recognize different epitopes on the PAS domain. Both antibodies increase the rate of deactivation but have different effects on channel activation and inactivation. Importantly, we show that both antibodies, on binding to the PAS domain, increase the total amount of current that permeates the channel during a ventricular action potential and significantly reduce the action potential duration recorded in human cardiomyocytes. Overall, these molecules constitute a previously unidentified class of positive modulators and establish that allosteric modulation of hERG channel function through ligand binding to the PAS domain can be attained.
Assuntos
Potenciais de Ação/efeitos dos fármacos , Canais de Potássio Éter-A-Go-Go/fisiologia , Ativação do Canal Iônico/efeitos dos fármacos , Anticorpos de Cadeia Única/farmacologia , Animais , Sítios de Ligação/genética , Sítios de Ligação/imunologia , Células Cultivadas , Galinhas , Estimulação Elétrica/métodos , Epitopos/genética , Epitopos/imunologia , Canais de Potássio Éter-A-Go-Go/genética , Canais de Potássio Éter-A-Go-Go/imunologia , Células HEK293 , Humanos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/fisiologia , Técnicas de Patch-Clamp , Anticorpos de Cadeia Única/imunologiaRESUMO
The human ether-à-go-go-related gene (hERG; or KCNH2) encodes the voltage-gated potassium channel underlying IKr, a repolarizing current in the heart. Mutations in KCNH2 or pharmacological agents that reduce IKr slow action potential (AP) repolarization and can trigger cardiac arrhythmias associated with long QT syndrome. Two channel-forming subunits encoded by KCNH2 (hERG 1a and 1b) are expressed in cardiac tissue. In heterologous expression systems, these subunits avidly coassemble and exhibit biophysical and pharmacological properties distinct from those of homomeric hERG 1a channels. Despite these findings, adoption of hERG 1a/1b heteromeric channels as a model for cardiac IKr has been hampered by the lack of evidence for a direct functional role for the 1b subunit in native tissue. In this study, we measured IKr and APs at physiological temperature in cardiomyocytes derived from human induced pluripotent stem cells (iPSC-CMs). We found that specific knockdown of the 1b subunit using shRNA caused reductions in 1b mRNA, 1b protein levels, and IKr magnitude by roughly one-half. AP duration was increased and AP variability was enhanced relative to controls. Early afterdepolarizations, considered cellular substrates for arrhythmia, were also observed in cells with reduced 1b expression. Similar behavior was elicited when channels were effectively converted from heteromers to 1a homomers by expressing a fragment corresponding to the 1a-specific N-terminal Per-Arnt-Sim domain, which is omitted from hERG 1b by alternate transcription. These findings establish that hERG 1b is critical for normal repolarization and that loss of 1b is proarrhythmic in human cardiac cells.
Assuntos
Canais de Potássio Éter-A-Go-Go/metabolismo , Potenciais da Membrana/fisiologia , Miócitos Cardíacos/fisiologia , Função Ventricular/fisiologia , Potenciais de Ação/fisiologia , Análise de Variância , Polaridade Celular/fisiologia , Canal de Potássio ERG1 , Técnicas de Silenciamento de Genes , Humanos , Imuno-Histoquímica , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo RealRESUMO
The authors have withdrawn their manuscript owing to technical concerns merged during peer review. Therefore, the authors do not wish this work to be cited as a reference. If you have any questions, please contact the corresponding author.
RESUMO
To signal properly, excitable cells must establish and maintain the correct balance of various types of ion channels that increase or decrease membrane excitability. The mechanisms by which this balance is regulated remain largely unknown. Here, we describe a regulatory mechanism uncovered by a Drosophila behavioral mutant, down and out (dao). At elevated temperatures, dao loss-of-function mutants exhibit seizures associated with spontaneous bursts of neural activity. This phenotype closely resembles that of seizure mutations, which impair activity of ether-a-go-go-related gene (Erg)-type potassium channels. Conversely, neural over-expression of wild-type Dao confers dominant temperature-sensitive paralysis with kinetics reminiscent of paralytic sodium-channel mutants. The over-expression phenotype of dao is suppressed in a seizure mutant background, suggesting that Dao acts by an effect on Erg channels. In support of this hypothesis, functional expression of Erg channels in a heterologous system is dependent on the presence of Dao. These results indicate that Dao has an important role in establishing the proper level of neuronal membrane excitability by regulating functional expression of Erg channels.
Assuntos
Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiologia , Drosophila/genética , Drosophila/fisiologia , Canais de Potássio Éter-A-Go-Go/genética , Canais de Potássio Éter-A-Go-Go/fisiologia , Animais , Animais Geneticamente Modificados , Comportamento Animal , Mapeamento Cromossômico , Feminino , Expressão Gênica , Genes de Insetos , Técnicas In Vitro , Mutação , Oócitos/metabolismo , Fenótipo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Xenopus laevisRESUMO
Heterologously expressed hERG channels represent a mainstay of in vitro drug safety screens intended to mitigate risk of cardiac IKr block and sudden cardiac death. This is true even as more channel types are adopted as part of the Comprehensive in vitro Proarrhythmia Assay (CiPA) intended to elevate specificity and thus enhance throughput of promising lead drugs. Until now, hERG1a homomeric channels have been used as a proxy for IKr despite a wealth of evidence showing that hERG1a/1b heteromers better represent native channels in terms of protein abundance and channel biophysical and pharmacological properties. Past efforts to create a stable hERG1a/1b cell line were met with unpredictable silencing of hERG1b expression despite stable integration of the gene into the HEK293 cell genome. Here we report a new cell line stably expressing hERG1a, with hERG1b reliably controlled by an inducible promoter sensitive to doxycycline. Co-immunoprecipitation, Western blot analysis and patch-clamp electrophysiology confirm the heteromeric composition of the expressed channels. Association with hERG1b was found to promote hERG1a protein levels and enhance membrane current levels. Optimal conditions for drug screening and experimental investigation were achieved at 24 h exposure to 100 ng/ml doxycycline. Differences in pharmacological sensitivity between homomeric and heteromeric channels were observed for dofetilide and ebastine, but not fluoxetine, as evaluated by their IC50 values. Using these values in the O'Hara-Rudy-CiPA in silico model revealed discrepancies in pro-arrhythmia risk, implying the hERG1a homomeric platform overestimates risk for these two drugs. Dofetilide block was use-dependent and faster for hERG1a/1b than hERG1a channels, whereas ebastine showed considerable block at rest and had a slower progression for hERG1a/1b channels. The hERG1a/1b cell line thus represents an advanced model for contemporary drug safety screening assays such as CiPA that employ IC50 values to estimate risk of proarrhythmia in computational models of ventricular cardiomyocytes. This novel technology fulfills an unmet need to enhance specificity and foster a safe yet expanded drug development pipeline.
Assuntos
Arritmias Cardíacas , Canais de Potássio Éter-A-Go-Go , Canal de Potássio ERG1/genética , Canais de Potássio Éter-A-Go-Go/genética , Células HEK293 , Humanos , Miócitos CardíacosRESUMO
[Figure: see text].
Assuntos
Potenciais de Ação , Canais de Potássio Éter-A-Go-Go/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Síndrome do QT Longo/metabolismo , Miócitos Cardíacos/metabolismo , Adulto , Canais de Potássio Éter-A-Go-Go/genética , Feminino , Predisposição Genética para Doença , Variação Genética , Glicosilação , Células HEK293 , Humanos , Ativação do Canal Iônico , Cinética , Síndrome do QT Longo/genética , Fenótipo , Transporte ProteicoRESUMO
Cardiac I Kr is a critical repolarizing current in the heart and a target for inherited and acquired long-QT syndrome (LQTS). Biochemical and functional studies have demonstrated that I Kr channels are heteromers composed of both hERG 1a and 1b subunits, yet our current understanding of I Kr functional properties derives primarily from studies of homooligomers of the original hERG 1a isolate. Here, we examine currents produced by hERG 1a and 1a/1b channels expressed in HEK-293 cells at near-physiological temperatures. We find that heteromeric hERG 1a/1b currents are much larger than hERG 1a currents and conduct 80% more charge during an action potential. This surprising difference corresponds to a 2-fold increase in the apparent rates of activation and recovery from inactivation, thus reducing rectification and facilitating current rebound during repolarization. Kinetic modeling shows these gating differences account quantitatively for the differences in current amplitude between the 2 channel types. Drug sensitivity was also different. Compared to homomeric 1a channels, heteromeric 1a/1b channels were inhibited by E-4031 with a slower time course and a corresponding 4-fold shift in the IC50. The importance of hERG 1b in vivo is supported by the identification of a 1b-specific A8V missense mutation in 1/269 unrelated genotype-negative LQTS patients that was absent in 400 control alleles. Mutant 1bA8V expressed alone or with hERG 1a in HEK-293 cells dramatically reduced 1b protein levels. Thus, mutations specifically disrupting hERG 1b function are expected to reduce cardiac I Kr and enhance drug sensitivity, and represent a potential mechanism underlying inherited or acquired LQTS.
Assuntos
Substituição de Aminoácidos , Canais de Potássio Éter-A-Go-Go/genética , Canais de Potássio Éter-A-Go-Go/metabolismo , Síndrome do QT Longo/genética , Síndrome do QT Longo/metabolismo , Mutação de Sentido Incorreto , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/genética , Antiarrítmicos/farmacologia , Linhagem Celular , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/genética , Cinética , Modelos Biológicos , Piperidinas , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , PiridinasRESUMO
Catastrophic arrhythmias and sudden cardiac death can occur with even a small imbalance between inward sodium currents and outward potassium currents, but mechanisms establishing this critical balance are not understood. Here, we show that mRNA transcripts encoding INa and IKr channels (SCN5A and hERG, respectively) are associated in defined complexes during protein translation. Using biochemical, electrophysiological and single-molecule fluorescence localization approaches, we find that roughly half the hERG translational complexes contain SCN5A transcripts. Moreover, the transcripts are regulated in a way that alters functional expression of both channels at the membrane. Association and coordinate regulation of transcripts in discrete 'microtranslatomes' represents a new paradigm controlling electrical activity in heart and other excitable tissues.
Assuntos
Canal de Potássio ERG1/metabolismo , Regulação da Expressão Gênica , Coração/fisiologia , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Potássio/metabolismo , Sódio/metabolismo , Canal de Potássio ERG1/genética , Células HEK293 , Humanos , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , TransfecçãoRESUMO
The Drosophila EAG (dEAG) potassium channel is the founding member of the superfamily of KNCH channels, which are involved in cardiac repolarization, neuronal excitability and cellular proliferation. In flies, dEAG is involved in regulation of neuron firing and assembles with CaMKII to form a complex implicated in memory formation. We have characterized the interaction between the kinase domain of CaMKII and a 53-residue fragment of the dEAG channel that includes a canonical CaMKII recognition sequence. Crystal structures together with biochemical/biophysical analysis show a substrate-kinase complex with an unusually tight and extensive interface that appears to be strengthened by phosphorylation of the channel fragment. Electrophysiological recordings show that catalytically active CaMKII is required to observe active dEAG channels. A previously identified phosphorylation site in the recognition sequence is not the substrate for this crucial kinase activity, but rather contributes importantly to the tight interaction of the kinase with the channel. The available data suggest that the dEAG channel is a docking platform for the kinase and that phosphorylation of the channel's kinase recognition sequence modulates the strength of the interaction between the channel and the kinase.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/química , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Canais de Potássio Éter-A-Go-Go/química , Canais de Potássio Éter-A-Go-Go/metabolismo , Animais , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Drosophila melanogaster/química , Fenômenos Eletrofisiológicos , Modelos Moleculares , Simulação de Acoplamento Molecular , Fosforilação , Ligação Proteica , Conformação ProteicaRESUMO
PURPOSE: Glioblastoma is the most malignant primary brain tumor, with a median survival of less than 2 years. More effective therapeutic approaches are needed to improve clinical outcomes. EXPERIMENTAL DESIGN: Glioblastoma patient-derived cells (GPDC) were isolated from patient glioblastomas and implanted in mice to form xenografts. IHC was performed for human Ether-à-go-go-Related Gene (hERG) expression and tumor proliferation. Sphere-forming assays with the hERG blocker E-4031 were performed on a high and low hERG-expressing lines. A glioblastoma tissue microarray (TMA; 115 patients) was used to correlate hERG expression with patient survival. Clinical data were analyzed to determine whether patient survival was affected by incidental administration of hERG inhibitory drugs and the correlative effect of patient glioblastoma hERG expression levels. RESULTS: hERG expression was upregulated in glioblastoma xenografts with higher proliferative indices. High hERG-expressing GPDCs showed a reduction in sphere formation when treated with hERG inhibitors compared with low hERG-expressing GPDCs. Glioblastoma TMA analysis showed worse survival for glioblastoma patients with high hERG expression versus low expression-43.5 weeks versus 60.9 weeks, respectively (P = 0.022). Furthermore, patients who received at least one hERG blocker had a better survival rate compared with patients who did not (P = 0.0015). Subgroup analysis showed that glioblastoma patients with high hERG expression who received hERG blockers had improved survival (P = 0.0458). There was no difference in survival for low hERG-expressing glioblastoma patients who received hERG blockers (P = 0.4136). CONCLUSIONS: Our findings suggest that hERG is a potential glioblastoma survival marker, and that already approved drugs with non-torsadogenic hERG inhibitory activity may potentially be repurposed as adjuvant glioblastoma therapy in high hERG-expressing glioblastoma patients. Clin Cancer Res; 23(1); 73-80. ©2016 AACRSee related commentary by Arcangeli and Becchetti, p. 3.
Assuntos
Antineoplásicos/administração & dosagem , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/mortalidade , Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Canais de Potássio Éter-A-Go-Go/metabolismo , Glioblastoma/metabolismo , Glioblastoma/mortalidade , Piperidinas/administração & dosagem , Piridinas/administração & dosagem , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Canais de Potássio Éter-A-Go-Go/genética , Expressão Gênica , Glioblastoma/diagnóstico , Glioblastoma/tratamento farmacológico , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Camundongos , Terapia de Alvo Molecular , Esferoides Celulares , Análise Serial de Tecidos , Carga Tumoral/efeitos dos fármacos , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Channels in the ether-à-go-go or KCNH family of potassium channels are characterized by a conserved, C-terminal domain with homology to cyclic nucleotide-binding homology domains (CNBhDs). Instead of cyclic nucleotides, two amino acid residues, Y699 and L701, occupy the binding pocket, forming an "intrinsic ligand." The role of the CNBhD in KCNH channel gating is still unclear, however, and a detailed characterization of the intrinsic ligand is lacking. In this study, we show that mutating both Y699 and L701 to alanine, serine, aspartate, or glycine impairs human EAG1 channel function. These mutants slow channel activation and shift the conductance-voltage (G-V) relation to more depolarized potentials. The mutations affect activation and the G-V relation progressively, indicating that the gating machinery is sensitive to multiple conformations of the CNBhD. Substitution with glycine at both sites (GG), which eliminates the side chains that interact with the binding pocket, also reduces the ability of voltage prepulses to populate more preactivated states along the activation pathway (i.e., the Cole-Moore effect), as if stabilizing the voltage sensor in deep resting states. Notably, deletion of the entire CNBhD (577-708, ΔCNBhD) phenocopies the GG mutant, suggesting that GG is a loss-of-function mutation and the CNBhD requires an intrinsic ligand to exert its functional effects. We developed a kinetic model for both wild-type and ΔCNBhD mutant channels that describes all our observations on activation kinetics, the Cole-Moore shift, and G-V relations. These findings support a model in which the CNBhD both promotes voltage sensor activation and stabilizes the open pore. The intrinsic ligand is critical for these functional effects.