231 results on '"Olaf Pongs"'
Search Results
2. NCS-1 Deficiency Affects mRNA Levels of Genes Involved in Regulation of ATP Synthesis and Mitochondrial Stress in Highly Vulnerable Substantia nigra Dopaminergic Neurons
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Carsten Simons, Julia Benkert, Nora Deuter, Christina Poetschke, Olaf Pongs, Toni Schneider, Johanna Duda, and Birgit Liss
- Subjects
ND1 ,ENO2 ,Parkinson’s disease ,mitochondrial uncoupling proteins ,voltage-gated calcium channels ,Cav2.3 ,Neurosciences. Biological psychiatry. Neuropsychiatry ,RC321-571 - Abstract
Neuronal Ca2+ sensor proteins (NCS) transduce changes in Ca2+ homeostasis into altered signaling and neuronal function. NCS-1 activity has emerged as important for neuronal viability and pathophysiology. The progressive degeneration of dopaminergic (DA) neurons, particularly within the Substantia nigra (SN), is the hallmark of Parkinson’s disease (PD), causing its motor symptoms. The activity-related Ca2+ homeostasis of SN DA neurons, mitochondrial dysfunction, and metabolic stress promote neurodegeneration and PD. In contrast, NCS-1 in general has neuroprotective effects. The underlying mechanisms are unclear. We analyzed transcriptional changes in SN DA neurons upon NCS-1 loss by combining UV-laser microdissection and RT-qPCR-approaches to compare expression levels of a panel of PD and/or Ca2+-stress related genes from wildtype and NCS-1 KO mice. In NCS-1 KO, we detected significantly lower mRNA levels of mitochondrially coded ND1, a subunit of the respiratory chain, and of the neuron-specific enolase ENO2, a glycolytic enzyme. We also detected lower levels of the mitochondrial uncoupling proteins UCP4 and UCP5, the PARK7 gene product DJ-1, and the voltage-gated Ca2+ channel Cav2.3 in SN DA neurons from NCS-1 KO. Transcripts of other analyzed uncoupling proteins (UCPs), mitochondrial Ca2+ transporters, PARK genes, and ion channels were not altered. As Cav channels are linked to regulation of gene expression, metabolic stress and degeneration of SN DA neurons in PD, we analyzed Cav2.3 KO mice, to address if the transcriptional changes in NCS-1 KO were also present in Cav.2.3 KO, and thus probably correlated with lower Cav2.3 transcripts. However, in SN DA neurons from Cav2.3 KO mice, ND1 mRNA as well as genomic DNA levels were elevated, while ENO2, UCP4, UCP5, and DJ-1 transcript levels were not altered. In conclusion, our data indicate a possible novel function of NCS-1 in regulating gene transcription or stabilization of mRNAs in SN DA neurons. Although we do not provide functional data, our findings at the transcript level could point to impaired ATP production (lower ND1 and ENO2) and elevated metabolic stress (lower UCP4, UCP5, and DJ-1 levels) in SN DA neurons from NCS-1 KO mice. We speculate that NCS-1 is involved in stimulating ATP synthesis, while at the same time controlling mitochondrial metabolic stress, and in this way could protect SN DA neurons from degeneration.
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- 2019
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3. NCS-1 Deficiency Is Associated With Obesity and Diabetes Type 2 in Mice
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Olga Ratai, Joanna Hermainski, Keerthana Ravichandran, and Olaf Pongs
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neuronal calcium sensor-1 ,obesity ,adipocyte ,insulin receptor ,insulin resistance ,depression ,Neurosciences. Biological psychiatry. Neuropsychiatry ,RC321-571 - Abstract
Neuronal calcium sensor-1 (NCS-1) knockout (KO) in mice (NCS-1−/− mice) evokes behavioral phenotypes ranging from learning deficits to avolition and depressive-like behaviors. Here, we showed that with the onset of adulthood NCS-1−/− mice gain considerable weight. Adult NCS-1−/− mice are obese, especially when fed a high-fat diet (HFD), are hyperglycemic and hyperinsulinemic and thus develop a diabetes type 2 phenotype. In comparison to wild type (WT) NCS-1−/− mice display a significant increase in adipose tissue mass. NCS-1−/− adipocytes produce insufficient serum concentrations of resistin and adiponectin. In contrast to WT littermates, adipocytes of NCS-1−/− mice are incapable of up-regulating insulin receptor (IR) concentration in response to HFD. Thus, HFD-fed NCS-1−/− mice exhibit in comparison to WT littermates a significantly reduced IR expression, which may explain the pronounced insulin resistance observed especially with HFD-fed NCS-1−/− mice. We observed a direct correlation between NCS-1 and IR concentrations in the adipocyte membrane and that NCS-1 can be co-immunoprecipitated with IR indicating a direct interplay between NCS-1 and IR. We propose that NCS-1 plays an important role in adipocyte function and that NCS-1 deficiency gives rise to obesity and diabetes type 2 in adult mice. Given the association of altered NCS-1 expression with behaviorial abnormalities, NCS-1−/− mice may offer an interesting perspective for studying in a mouse model a potential genetic link between some psychiatric disorders and the risk of being obese.
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- 2019
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4. The Low-Threshold Calcium Channel Cav3.2 Determines Low-Threshold Mechanoreceptor Function
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Amaury François, Niklas Schüetter, Sophie Laffray, Juan Sanguesa, Anne Pizzoccaro, Stefan Dubel, Annabelle Mantilleri, Joel Nargeot, Jacques Noël, John N. Wood, Aziz Moqrich, Olaf Pongs, and Emmanuel Bourinet
- Subjects
Biology (General) ,QH301-705.5 - Abstract
The T-type calcium channel Cav3.2 emerges as a key regulator of sensory functions, but its expression pattern within primary afferent neurons and its contribution to modality-specific signaling remain obscure. Here, we elucidate this issue using a unique knockin/flox mouse strain wherein Cav3.2 is replaced by a functional Cav3.2-surface-ecliptic GFP fusion. We demonstrate that Cav3.2 is a selective marker of two major low-threshold mechanoreceptors (LTMRs), Aδ- and C-LTMRs, innervating the most abundant skin hair follicles. The presence of Cav3.2 along LTMR-fiber trajectories is consistent with critical roles at multiple sites, setting their strong excitability. Strikingly, the C-LTMR-specific knockout uncovers that Cav3.2 regulates light-touch perception and noxious mechanical cold and chemical sensations and is essential to build up that debilitates allodynic symptoms of neuropathic pain, a mechanism thought to be entirely A-LTMR specific. Collectively, our findings support a fundamental role for Cav3.2 in touch/pain pathophysiology, validating their critic pharmacological relevance to relieve mechanical and cold allodynia.
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- 2015
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5. Inhibition of Kv2.1 Potassium Channels by MiDCA1, A Pre-Synaptically Active PLA2-Type Toxin from Micrurus dumerilii carinicauda Coral Snake Venom
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Niklas Schütter, Yuri Correia Barreto, Vitya Vardanyan, Sönke Hornig, Stephen Hyslop, Sérgio Marangoni, Léa Rodrigues-Simioni, Olaf Pongs, and Cháriston André Dal Belo
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Micrurus dumerilii carinicauda venom ,phospholipase A2 neurotoxin ,mouse dorsal root ganglion neurons ,Kv2 selective inhibition ,Medicine - Abstract
MiDCA1, a phospholipase A2 (PLA2) neurotoxin isolated from Micrurus dumerilii carinicauda coral snake venom, inhibited a major component of voltage-activated potassium (Kv) currents (41 ± 3% inhibition with 1 μM toxin) in mouse cultured dorsal root ganglion (DRG) neurons. In addition, the selective Kv2.1 channel blocker guangxitoxin (GxTx-1E) and MiDCA1 competitively inhibited the outward potassium current in DRG neurons. MiDCA1 (1 µM) reversibly inhibited the Kv2.1 current by 55 ± 8.9% in a Xenopus oocyte heterologous system. The toxin showed selectivity for Kv2.1 channels over all the other Kv channels tested in this study. We propose that Kv2.1 channel blockade by MiDCA1 underlies the toxin’s action on acetylcholine release at mammalian neuromuscular junctions.
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- 2019
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6. A truncation variant of the cation channel P2RX5 is upregulated during T cell activation.
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Pierre Abramowski, Christoph Ogrodowczyk, Roland Martin, and Olaf Pongs
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Medicine ,Science - Abstract
Members of the P2X family of ligand-gated cation channels (P2RX) are expressed by various cell types including neurons, smooth- and cardiac muscle cells, and leukocytes. The channels mediate signalling in response to extracellular ATP. Seven subunit isoforms (P2RX1-P2RX7) have been identified and these can assemble as homo- and heterotrimeric molecules. In humans, P2RX5 exists as a natural deletion mutant lacking amino acids 328-349 of exon 10, which are part of transmembrane (TM) 2 and pre-TM2 regions in other organisms like rat, chicken and zebrafish. We show that P2RX5 gene expression of human T lymphocytes is upregulated during activation. P2RX5 is recruited to the cell surface. P2RX5-siRNA-transfected CD4+ T cells produced twofold more IL-10 than controls. Surface and intracellular P2RX5 expression was upregulated in activated antigen-specific CD4+ T cell clones. These data indicate a functional role of the human P2RX5 splice variant in T cell activation and immunoregulation.
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- 2014
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7. A molecular switch driving inactivation in the cardiac K+ channel HERG.
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David A Köpfer, Ulrike Hahn, Iris Ohmert, Gert Vriend, Olaf Pongs, Bert L de Groot, and Ulrich Zachariae
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Medicine ,Science - Abstract
K(+) channels control transmembrane action potentials by gating open or closed in response to external stimuli. Inactivation gating, involving a conformational change at the K(+) selectivity filter, has recently been recognized as a major K(+) channel regulatory mechanism. In the K(+) channel hERG, inactivation controls the length of the human cardiac action potential. Mutations impairing hERG inactivation cause life-threatening cardiac arrhythmia, which also occur as undesired side effects of drugs. In this paper, we report atomistic molecular dynamics simulations, complemented by mutational and electrophysiological studies, which suggest that the selectivity filter adopts a collapsed conformation in the inactivated state of hERG. The selectivity filter is gated by an intricate hydrogen bond network around residues S620 and N629. Mutations of this hydrogen bond network are shown to cause inactivation deficiency in electrophysiological measurements. In addition, drug-related conformational changes around the central cavity and pore helix provide a functional mechanism for newly discovered hERG activators.
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- 2012
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8. High-resolution 3D structure determination of kaliotoxin by solid-state NMR spectroscopy.
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Jegannath Korukottu, Robert Schneider, Vinesh Vijayan, Adam Lange, Olaf Pongs, Stefan Becker, Marc Baldus, and Markus Zweckstetter
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Medicine ,Science - Abstract
High-resolution solid-state NMR spectroscopy can provide structural information of proteins that cannot be studied by X-ray crystallography or solution NMR spectroscopy. Here we demonstrate that it is possible to determine a protein structure by solid-state NMR to a resolution comparable to that by solution NMR. Using an iterative assignment and structure calculation protocol, a large number of distance restraints was extracted from (1)H/(1)H mixing experiments recorded on a single uniformly labeled sample under magic angle spinning conditions. The calculated structure has a coordinate precision of 0.6 A and 1.3 A for the backbone and side chain heavy atoms, respectively, and deviates from the structure observed in solution. The approach is expected to be applicable to larger systems enabling the determination of high-resolution structures of amyloid or membrane proteins.
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- 2008
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9. Myristoylated Neuronal Calcium Sensor-1 captures the ciliary vesicle at distal appendages
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Tomoharu Kanie, Roy Ng, Keene L. Abbott, Olaf Pongs, and Peter K. Jackson
- Abstract
SummaryThe primary cilium is a microtubule-based organelle that cycles through assembly and disassembly. In many cell types, formation of the cilium is initiated by recruitment of ciliary vesicles to the distal appendage of the mother centriole. However, the distal appendage mechanism that directly captures ciliary vesicles is yet to be identified. In an accompanying paper, we show that the distal appendage protein, CEP89, is important for thef ciliary vesicle recruitment, but not for other steps of cilium formation (Tomoharu Kanie, Love, Fisher, Gustavsson, & Jackson, 2023). The lack of a membrane binding motif in CEP89 suggests that it may indirectly recruit ciliary vesicles via another binding partner. Here, we identify Neuronal Calcium Sensor-1 (NCS1) as a stoichiometric interactor of CEP89. NCS1 localizes to the position between CEP89 and a ciliary vesicle marker, RAB34, at the distal appendage. This localization was completely abolished in CEP89 knockouts, suggesting that CEP89 recruits NCS1 to the distal appendage. Similarly to CEP89 knockouts, ciliary vesicle recruitment as well as subsequent cilium formation was perturbed in NCS1 knockout cells. The ability of NCS1 to recruit the ciliary vesicle is dependent on its myristoylation motif and NCS1 knockout cells expressing myristoylation defective mutant failed to rescue the vesicle recruitment defect despite localizing proper localization to the centriole. In sum, our analysis reveals the first known mechanism for how the distal appendage recruits the ciliary vesicles.
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- 2023
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10. Voltage-Gated K+ Channels
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Olaf Pongs
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- 2021
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11. NCS-1 Deficiency Is Associated With Obesity and Diabetes Type 2 in Mice
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Keerthana Ravichandran, Olga Ratai, Olaf Pongs, and Joanna Hermainski
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0301 basic medicine ,obesity ,medicine.medical_specialty ,Adipose tissue ,adipocyte ,behavioral disciplines and activities ,lcsh:RC321-571 ,neuronal calcium sensor-1 ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,chemistry.chemical_compound ,0302 clinical medicine ,Insulin resistance ,insulin resistance ,Diabetes mellitus ,Internal medicine ,Adipocyte ,mental disorders ,medicine ,insulin receptor ,lcsh:Neurosciences. Biological psychiatry. Neuropsychiatry ,Molecular Biology ,Original Research ,Adiponectin ,biology ,Wild type ,medicine.disease ,Insulin receptor ,030104 developmental biology ,Endocrinology ,chemistry ,depression ,biology.protein ,Resistin ,030217 neurology & neurosurgery ,Neuroscience - Abstract
Neuronal calcium sensor-1 (NCS-1) knockout (KO) in mice (NCS-1−/− mice) evokes behavioral phenotypes ranging from learning deficits to avolition and depressive-like behaviors. Here, we showed that with the onset of adulthood NCS-1−/− mice gain considerable weight. Adult NCS-1−/− mice are obese, especially when fed a high-fat diet (HFD), are hyperglycemic and hyperinsulinemic and thus develop a diabetes type 2 phenotype. In comparison to wild type (WT) NCS-1−/− mice display a significant increase in adipose tissue mass. NCS-1−/− adipocytes produce insufficient serum concentrations of resistin and adiponectin. In contrast to WT littermates, adipocytes of NCS-1−/− mice are incapable of up-regulating insulin receptor (IR) concentration in response to HFD. Thus, HFD-fed NCS-1−/− mice exhibit in comparison to WT littermates a significantly reduced IR expression, which may explain the pronounced insulin resistance observed especially with HFD-fed NCS-1−/− mice. We observed a direct correlation between NCS-1 and IR concentrations in the adipocyte membrane and that NCS-1 can be co-immunoprecipitated with IR indicating a direct interplay between NCS-1 and IR. We propose that NCS-1 plays an important role in adipocyte function and that NCS-1 deficiency gives rise to obesity and diabetes type 2 in adult mice. Given the association of altered NCS-1 expression with behaviorial abnormalities, NCS-1−/− mice may offer an interesting perspective for studying in a mouse model a potential genetic link between some psychiatric disorders and the risk of being obese.
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- 2019
- Full Text
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12. Neuronal calcium sensor-1 deletion in the mouse decreases motivation and dopamine release in the nucleus accumbens
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John C. Roder, Ping Su, Rafael K. Varaschin, Albert H.C. Wong, Bernard Le Foll, Louis-Eric Trudeau, Caleb J. Browne, Fang Liu, Enoch Ng, Olaf Pongs, and Joanna Hermainski
- Subjects
Male ,0301 basic medicine ,Dopamine ,Conditioning, Classical ,Neuronal Calcium-Sensor Proteins ,Striatum ,Motor Activity ,Nucleus accumbens ,Biology ,Satiety Response ,behavioral disciplines and activities ,Nucleus Accumbens ,Tissue Culture Techniques ,Food Preferences ,03 medical and health sciences ,Behavioral Neuroscience ,0302 clinical medicine ,Dopamine receptor D1 ,Dopamine receptor D3 ,Dopamine receptor D2 ,mental disorders ,medicine ,Animals ,Mice, Knockout ,Motivation ,Neuropeptides ,Dopaminergic ,Feeding Behavior ,Mice, Inbred C57BL ,Amphetamine ,030104 developmental biology ,Neuronal calcium sensor-1 ,biology.protein ,Central Nervous System Stimulants ,Neuroscience ,Locomotion ,030217 neurology & neurosurgery ,medicine.drug - Abstract
Calcium sensors detect intracellular calcium changes and interact with downstream targets to regulate many functions. Neuronal Calcium Sensor-1 (NCS-1) or Frequenin is widely expressed in the nervous system, and involved in neurotransmission, synaptic plasticity and learning. NCS-1 interacts with and regulates dopamine D2 receptor (D2R) internalization and is implicated in disorders like schizophrenia and substance abuse. However, the role of NCS-1 in behaviors dependent on dopamine signaling in the striatum, where D2R is most highly expressed, is unknown. We show that Ncs-1 deletion in the mouse decreases willingness to work for food. Moreover, Ncs-1 knockout mice have significantly lower activity-dependent dopamine release in the nucleus accumbens core in acute slice recordings. In contrast, food preference, responding for conditioned reinforcement, ability to represent changes in reward value, and locomotor response to amphetamine are not impaired. These studies identify novel roles for NCS-1 in regulating activity-dependent striatal dopamine release and aspects of motivated behavior.
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- 2016
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13. NCS-1 Deficiency Affects mRNA Levels of Genes Involved in Regulation of ATP Synthesis and Mitochondrial Stress in Highly Vulnerable
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Carsten, Simons, Julia, Benkert, Nora, Deuter, Christina, Poetschke, Olaf, Pongs, Toni, Schneider, Johanna, Duda, and Birgit, Liss
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Cav2.3 ,mitochondrial uncoupling proteins ,voltage-gated calcium channels ,DJ-1/PARK7 ,ND1 ,ENO2 ,Parkinson’s disease ,KChip3/DREAM/Calsenilin ,Neuroscience ,Original Research - Abstract
Neuronal Ca2+ sensor proteins (NCS) transduce changes in Ca2+ homeostasis into altered signaling and neuronal function. NCS-1 activity has emerged as important for neuronal viability and pathophysiology. The progressive degeneration of dopaminergic (DA) neurons, particularly within the Substantia nigra (SN), is the hallmark of Parkinson’s disease (PD), causing its motor symptoms. The activity-related Ca2+ homeostasis of SN DA neurons, mitochondrial dysfunction, and metabolic stress promote neurodegeneration and PD. In contrast, NCS-1 in general has neuroprotective effects. The underlying mechanisms are unclear. We analyzed transcriptional changes in SN DA neurons upon NCS-1 loss by combining UV-laser microdissection and RT-qPCR-approaches to compare expression levels of a panel of PD and/or Ca2+-stress related genes from wildtype and NCS-1 KO mice. In NCS-1 KO, we detected significantly lower mRNA levels of mitochondrially coded ND1, a subunit of the respiratory chain, and of the neuron-specific enolase ENO2, a glycolytic enzyme. We also detected lower levels of the mitochondrial uncoupling proteins UCP4 and UCP5, the PARK7 gene product DJ-1, and the voltage-gated Ca2+ channel Cav2.3 in SN DA neurons from NCS-1 KO. Transcripts of other analyzed uncoupling proteins (UCPs), mitochondrial Ca2+ transporters, PARK genes, and ion channels were not altered. As Cav channels are linked to regulation of gene expression, metabolic stress and degeneration of SN DA neurons in PD, we analyzed Cav2.3 KO mice, to address if the transcriptional changes in NCS-1 KO were also present in Cav.2.3 KO, and thus probably correlated with lower Cav2.3 transcripts. However, in SN DA neurons from Cav2.3 KO mice, ND1 mRNA as well as genomic DNA levels were elevated, while ENO2, UCP4, UCP5, and DJ-1 transcript levels were not altered. In conclusion, our data indicate a possible novel function of NCS-1 in regulating gene transcription or stabilization of mRNAs in SN DA neurons. Although we do not provide functional data, our findings at the transcript level could point to impaired ATP production (lower ND1 and ENO2) and elevated metabolic stress (lower UCP4, UCP5, and DJ-1 levels) in SN DA neurons from NCS-1 KO mice. We speculate that NCS-1 is involved in stimulating ATP synthesis, while at the same time controlling mitochondrial metabolic stress, and in this way could protect SN DA neurons from degeneration.
- Published
- 2018
14. Structural Basis of a Kv7.1 Potassium Channel Gating Module: Studies of the Intracellular C-Terminal Domain in Complex with Calmodulin
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Asher Peretz, Dmitri I. Svergun, Bernard Attali, Giancarlo Tria, William Sam Tobelaim, Roi Strulovich, Meidan Dvir, Olaf Pongs, Joel A. Hirsch, and Dana Sachyani
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Models, Molecular ,Calmodulin ,Context (language use) ,Gating ,Protomer ,Crystallography, X-Ray ,Protein Structure, Secondary ,03 medical and health sciences ,0302 clinical medicine ,Protein structure ,Structural Biology ,ddc:570 ,Scattering, Small Angle ,Humans ,Molecular Biology ,030304 developmental biology ,0303 health sciences ,Binding Sites ,biology ,Chemistry ,C-terminus ,Potassium channel ,Crystallography ,HEK293 Cells ,KCNQ1 Potassium Channel ,Mutation ,biology.protein ,Biophysics ,Protein Multimerization ,030217 neurology & neurosurgery ,Intracellular - Abstract
SummaryKv7 channels tune neuronal and cardiomyocyte excitability. In addition to the channel membrane domain, they also have a unique intracellular C-terminal (CT) domain, bound constitutively to calmodulin (CaM). This CT domain regulates gating and tetramerization. We investigated the structure of the membrane proximal CT module in complex with CaM by X-ray crystallography. The results show how the CaM intimately hugs a two-helical bundle, explaining many channelopathic mutations. Structure-based mutagenesis of this module in the context of concatemeric tetramer channels and functional analysis along with in vitro data lead us to propose that one CaM binds to one individual protomer, without crosslinking subunits and that this configuration is required for proper channel expression and function. Molecular modeling of the CT/CaM complex in conjunction with small-angle X-ray scattering suggests that the membrane proximal region, having a rigid lever arm, is a critical gating regulator.
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- 2014
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15. Expression of voltage-gated K+ channels in insulin-producing cells. Analysis by polymerase chain reaction
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Stephen J.H. Ashcroft, Olaf Pongs, A. Baumann, Andrew Grupe, Patrik Rorsman, Frances M. Ashcroft, Susan Kenna, John Sandblom, Christer Betsholtz, Per Olof Berggren, and Michael J. Welsh
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Potassium Channels ,Sequence analysis ,medicine.medical_treatment ,Molecular Sequence Data ,Northern blot analysis ,Biophysics ,Mice, Obese ,Biology ,Insulin-producing cell ,Biochemistry ,Cell Line ,law.invention ,Islets of Langerhans ,Mice ,Structural Biology ,law ,Insulin Secretion ,Genetics ,medicine ,Animals ,Insulin ,RNA, Messenger ,Potassium channel ,Northern blot ,Molecular Biology ,Ion transporter ,Polymerase chain reaction ,Base Sequence ,Membrane Proteins ,RNA ,Cell Biology ,Molecular biology ,Pancreatic Neoplasms ,Cell culture ,Hyperglycemia ,Insulinoma ,Oligonucleotide Probes ,Poly A - Abstract
We have used the polymerase chain reaction (PCR) with primers against the S5 and S6 regions of voltage-gated K + channels to identify 8 different specific amplification products using poly(A) + RNA isolated from islets of Langerhans from obese hyperglycemic ( ob/ob ) mice and from the two insulin-producing cell lines HIT T15 and RINmSF. Sequence analysis suggests that they derive from mRNAs coding for a family of voltage-gated K + channels; 5 of these have been recently identified in mammalian brain and 3 are novel. These hybridize in classes to different mRNAs which distribute differently to a number of tissues and cell lines including insulin-producing cells.
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- 2016
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16. The C-Terminal Tail of Human Neuronal Calcium Sensor 1 Regulates the Conformational Stability of the Ca2+-Activated State
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Ida J. Bjerrum-Bohr, Birthe B. Kragelund, Gitte A. Jensen, Flemming M. Poulsen, Olaf Pongs, Bryan E. Finn, and Pétur O. Heidarsson
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Mutation ,biology ,Chemistry ,Autistic spectrum disorder ,Nuclear magnetic resonance spectroscopy ,medicine.disease_cause ,Ligand (biochemistry) ,Yeast ,chemistry.chemical_compound ,Neuronal calcium sensor-1 ,Biochemistry ,Structural Biology ,biology.protein ,medicine ,Biophysics ,Conformational stability ,Neurotransmitter ,Molecular Biology - Abstract
Neuronal calcium sensor 1 (NCS-1) and orthologs are expressed in all organisms from yeast to humans. In the latter, NCS-1 plays an important role in neurotransmitter release and interacts with a plethora of binding partners mostly through a large solvent-exposed hydrophobic crevice. The structural basis behind the multispecific binding profile is not understood. To begin to address this, we applied NMR spectroscopy to determine the solution structure of calcium-bound human NCS-1. The structure in solution demonstrates interdomain flexibility and, in the absence of a binding partner, the C-terminal tail residues occupy the hydrophobic crevice as a ligand mimic. A variant with a C-terminal tail deletion shows lack of a defined structure but maintained cooperative unfolding and dramatically reduced global stability. The results suggest that the C-terminal tail is important for regulating the conformational stability of the Ca 2+ -activated state. Furthermore, a single amino acid mutation that was recently diagnosed in a patient with autistic spectrum disorder was seen to affect the C-terminal tail and binding crevice in NCS-1.
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- 2012
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17. Gain-of-Function Mutations in TRPM4 Cause Autosomal Dominant Isolated Cardiac Conduction Disease
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Olaf Pongs, Gerard Blaysat, Romain Guinamard, André Mégarbané, Andre Bozio, Hui Liu, Elisabeth Villain, Martin Kruse, Alf Beckmann, Gueven Kurtbay, Loubna El Zein, Iris Ohmert, and Patrice Bouvagnet
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Male ,medicine.medical_specialty ,Genetic Linkage ,Heart block ,TRPM Cation Channels ,CHO Cells ,Disease ,Biology ,Transfection ,Sudden death ,Cricetulus ,Cardiac Conduction System Disease ,Genetic linkage ,Cricetinae ,Internal medicine ,Chlorocebus aethiops ,Cardiac conduction ,Genetics ,medicine ,Animals ,Humans ,Family ,Cells, Cultured ,Genetics (clinical) ,Genes, Dominant ,Bundle branch block ,medicine.disease ,Pedigree ,Heart Block ,Gain of function ,COS Cells ,Mutation ,Cardiology ,Female ,Cardiology and Cardiovascular Medicine - Abstract
Background— Isolated cardiac conduction block is a relatively common condition in young and elderly populations. Genetic predisposing factors have long been suspected because of numerous familial case reports. Deciphering genetic predisposing factors of conduction blocks may give a hint at stratifying conduction block carriers in a more efficient way. Methods and Results— One Lebanese family and 2 French families with autosomal dominant isolated cardiac conduction blocks were used for linkage analysis. A maximum combined multipoint lod score of 10.5 was obtained on a genomic interval including more than 300 genes. After screening 12 genes of this interval for mutation, we found a heterozygous missense mutation of the TRPM4 gene in each family (p.Arg164Trp, p.Ala432Thr, and p.Gly844Asp). This gene encodes the TRPM4 channel, a calcium-activated nonselective cation channel of the transient receptor potential melastatin (TRPM) ion channel family. All 3 mutations result in an increased current density. This gain of function is due to an elevated TRPM4 channel density at the cell surface secondary to impaired endocytosis and deregulation of Small Ubiquitin MOdifier conjugation (SUMOylation). Furthermore, we showed by immunohistochemistry that TRPM4 channel signal level is higher in atrial cardiomyocytes than in common ventricular cells, but is highest in Purkinje fibers. Small bundles of highly TRPM4-positive cells were found in the subendocardium and in rare intramural bundles. Conclusions— the TRPM4 gene is a causative gene in isolated cardiac conduction disease with mutations resulting in a gain of function and TRPM4 channel being highly expressed in cardiac Purkinje fibers.
- Published
- 2010
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18. Ins and outs of cardiac voltage-gated potassium channels
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Olaf Pongs
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Pharmacology ,Controlling (action) ,Chemistry ,Myocardium ,Lipid composition ,Cell Membrane ,Cardiac muscle ,Gating ,Voltage-gated potassium channel ,Anatomy ,Heart muscles ,Lipids ,Endocytosis ,Kv channel ,Protein Transport ,medicine.anatomical_structure ,Potassium Channels, Voltage-Gated ,Drug Discovery ,medicine ,Biophysics ,Animals ,Humans ,Myocytes, Cardiac ,Lipid bilayer - Abstract
Voltage-gated potassium (Kv) channels play an important role in regulating cardiac muscle excitability by controlling action potential duration and frequency. Essential for this activity is proper localization and organization of the cardiac Kv channels in specific microdomains of the plasma membrane. The underlying processes involve tight control of anterograde and retrograde Kv channel trafficking into and out of the plasma membrane. Thus, cardiac Kv channel density at the cell surface is regulated by a dynamic interplay of endocytotic and recycling pathways, the mechanisms of which are mostly unknown. Recent studies have indicated that the lipid composition in the Kv channel surround profoundly influences these processes. Local differences in lipid composition altering the mechanic state of the lipid bilayer or a specific interaction with an important domain of the Kv channel markedly affect voltage-sensitive gating, clustering, and mobility of cardiac Kv channels and, thereby, the excitability in the healthy and diseased heart muscles.
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- 2009
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19. The Molecular Mechanism of Toxin-Induced Conformational Changes in a Potassium Channel: Relation to C-Type Inactivation
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Ulrich Zachariae, Adam Lange, Phanindra Velisetty, Robert Schneider, Soren Wacker, Bert L. de Groot, Stefan Becker, Marc Baldus, Olaf Pongs, Yasmin Karimi-Nejad, Gert Vriend, and Daniel Seeliger
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Chemical and physical biology [NCMLS 7] ,Models, Molecular ,Patch-Clamp Techniques ,Microinjections ,Stereochemistry ,Bioinformatics ,Xenopus ,Static Electricity ,Molecular Conformation ,Kaliotoxin ,Scorpion Venoms ,Protein Structure, Secondary ,RNA, Complementary ,Scorpions ,Molecular dynamics ,Structural Biology ,Static electricity ,medicine ,Potassium Channel Blockers ,Animals ,Computer Simulation ,Patch clamp ,Nuclear Magnetic Resonance, Biomolecular ,Molecular Biology ,Scorpion toxin ,Kv1.3 Potassium Channel ,biology ,Chemistry ,Potassium channel blocker ,biology.organism_classification ,Potassium channel ,Electrophysiology ,Growth and differentiation [NCMLS 3] ,SIGNALING ,Potassium Channels, Voltage-Gated ,Mutation ,Oocytes ,CELLBIO ,Female ,medicine.drug - Abstract
Contains fulltext : 70715.pdf (Publisher’s version ) (Closed access) Recently, a solid-state NMR study revealed that scorpion toxin binding leads to conformational changes in the selectivity filter of potassium channels. The exact nature of the conformational changes, however, remained elusive. We carried out all-atom molecular dynamics simulations that enabled us to cover the complete pathway of toxin approach and binding, and we validated our simulation results by using solid-state NMR data and electrophysiological measurements. Our structural model revealed a mechanism of cooperative toxin-induced conformational changes that accounts both for the signal changes observed in solid-state NMR and for the tight interaction between KcsA-Kv1.3 and Kaliotoxin. We show that this mechanism is structurally and functionally closely related to recovery from C-type inactivation. Furthermore, our simulations indicate heterogeneity in the binding modes of Kaliotoxin, which might serve to enhance its affinity for KcsA-Kv1.3 further by entropic stabilization.
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- 2008
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20. C-terminal HERG (LQT2) mutations disrupt IKr channel regulation through 14-3-3ϵ
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Colleen E. Clancy, Chi un Choe, Gerold Mönnig, Zheng I. Zhu, Eric Schulze-Bahr, Jun Xu, Olaf Pongs, Pascale Guicheney, Dirk Isbrandt, Silvia G. Priori, Carlo Neapolitano, Axel Neu, Robert Bähring, Jan Heidemann, and Kathrin Sauter
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Male ,ERG1 Potassium Channel ,congenital, hereditary, and neonatal diseases and abnormalities ,Potassium Channels ,Mutant ,hERG ,Gene Expression ,Stimulation ,CHO Cells ,Pharmacology ,Biology ,Gene mutation ,medicine.disease_cause ,Ventricular action potential ,Cricetinae ,Genetics ,medicine ,Animals ,Humans ,Myocytes, Cardiac ,cardiovascular diseases ,Protein kinase A ,Molecular Biology ,Genetics (clinical) ,Mutation ,Models, Cardiovascular ,Signal transducing adaptor protein ,General Medicine ,Ether-A-Go-Go Potassium Channels ,Recombinant Proteins ,Pedigree ,Cell biology ,Long QT Syndrome ,Protein Subunits ,14-3-3 Proteins ,biology.protein ,Female - Abstract
Beta-adrenergic receptor-mediated cAMP or protein kinase A (PKA)-dependent modulation of cardiac potassium currents controls ventricular action potential duration (APD) at faster heart rates. HERG (KCNH2) gene mutations are associated with congenital long-QT syndrome (LQT2) and affect IKr activity, a key determinant in ventricular repolarization. Physical activity or emotional stress often triggers lethal arrhythmias in LQT2 patients. Beta-adrenergic stimulation of HERG channel activity is amplified and prolonged in vitro by the adaptor protein 14-3-3epsilon. In LQT2 families, we identified three novel heterozygous HERG mutations (G965X, R1014PfsX39, V1038AfsX21) in the C-terminus that led to protein truncation and loss of a PKA phosphorylation site required for binding of 14-3-3epsilon. When expressed in CHO cells, the mutants produced functional HERG channels with normal kinetic properties. We now provide evidence that HERG channel regulation by 14-3-3epsilon is of physiological significance in humans. Upon co-expression with 14-3-3epsilon, mutant channels still bound 14-3-3epsilon but did not respond with a hyperpolarizing shift in voltage dependence as seen in wild-type channels. Co-expression experiments of wild-type and mutant channels revealed dominant-negative behavior of all three HERG mutations. Simulations of the effects of sympathetic stimulation of HERG channel activity on the whole-cell action potential suggested a role in rate-dependent control of APD and an impaired ability of mutant cardiac myocytes to respond to a triggered event or an ectopic beat. In summary, the attenuated functional effects of 14-3-3epsilon on C-terminally truncated HERG channels demonstrate the physiological importance of coupling beta-adrenergic stimulation and HERG channel activity.
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- 2006
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21. NADPH Oxidase Accounts for Enhanced Superoxide Production and Impaired Endothelium-Dependent Smooth Muscle Relaxation in BKβ1 −/− Mice
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Olaf Pongs, Meike Coldewey, Jörg Faulhaber, Ascan Warnholtz, Ingrid Fleming, Matthias Oelze, Andrei L. Kleschyov, Thomas Münzel, Philip Wenzel, Heimo Ehmke, Ulrich Hink, Andreas Daiber, Thomas Meinertz, and Sven Wassmann
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medicine.medical_specialty ,Nitric Oxide Synthase Type III ,Endothelium ,Aorta, Thoracic ,Nitric Oxide ,Muscle, Smooth, Vascular ,Nitric oxide ,Mice ,chemistry.chemical_compound ,Superoxides ,Internal medicine ,Cyclic GMP-Dependent Protein Kinases ,medicine ,Animals ,Humans ,Protein Isoforms ,NADH, NADPH Oxidoreductases ,Large-Conductance Calcium-Activated Potassium Channels ,Mice, Knockout ,NADPH oxidase ,biology ,Superoxide ,Microfilament Proteins ,NADPH Oxidases ,Phosphoproteins ,Molecular biology ,Vasodilation ,Endocrinology ,medicine.anatomical_structure ,chemistry ,Guanylate Cyclase ,NAD(P)H oxidase ,NOX1 ,Apocynin ,NADPH Oxidase 1 ,biology.protein ,Endothelium, Vascular ,Cardiology and Cardiovascular Medicine ,Soluble guanylyl cyclase ,Cell Adhesion Molecules ,Signal Transduction - Abstract
Objective— Nitric oxide (NO)-induced vasorelaxation involves activation of large conductance Ca 2+ -activated K + channels (BK). A regulatory BKβ1 subunit confers Ca 2+ , voltage, and NO/cGMP sensitivity to the BK channel. We investigated whether endothelial function and NO/cGMP signaling is affected by a deletion of the β1-subunit. Methods and Results— Vascular superoxide in BKβ1 −/− was measured using the fluorescent dye hydroethidine and lucigenin-enhanced chemiluminescence. Vascular NO formation was analyzed using electron paramagnetic resonance (EPR), expression of NADPH oxidase subunits, the endothelial NO synthase (eNOS), the soluble guanylyl cyclase (sGC), as well as the activity and expression of the cyclic GMP-dependent kinase I (cGK-I) were assessed by Western blotting technique. eNOS, sGC, cGK-I expression and acetylcholine-induced NO production were unaltered in Bkβ1 −/− animals, whereas endothelial function was impaired and the activity of the cGK-I was reduced. Vascular O 2 − and expression of the NADPH oxidase subunits p67 phox and Nox1 were increased. Endothelial dysfunction was normalized by the NADPH oxidase inhibitor apocynin. Potassium chloride- and iberiotoxin-induced depolarization mimicked the effect of BKβ1-deletion by increasing vascular O 2 − in an NADPH-dependent fashion. Conclusions— The deletion of BKβ1 causes endothelial dysfunction by increasing O 2 − formation via increasing activity and expression of the vascular NADPH oxidase.
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- 2006
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22. Toxin-induced conformational changes in a potassium channel revealed by solid-state NMR
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Stefan Becker, Marie-France Martin-Eauclaire, Adam Lange, Olaf Pongs, Karin Giller, Sönke Hornig, and Marc Baldus
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Models, Molecular ,Potassium Channels ,Protein Conformation ,Stereochemistry ,Recombinant Fusion Proteins ,Xenopus ,Molecular Sequence Data ,KcsA potassium channel ,Scorpion Venoms ,Kaliotoxin ,complex mixtures ,Scorpions ,Bacterial Proteins ,Potassium Channel Blockers ,Animals ,Amino Acid Sequence ,Binding site ,Nuclear Magnetic Resonance, Biomolecular ,Kv1.3 Potassium Channel ,Multidisciplinary ,Scorpion toxin ,biology ,Chemistry ,Active site ,Nuclear magnetic resonance spectroscopy ,Ligand (biochemistry) ,Solid-state nuclear magnetic resonance ,Mutation ,Oocytes ,biology.protein - Abstract
The active site of potassium (K+) channels catalyses the transport of K+ ions across the plasma membrane--similar to the catalytic function of the active site of an enzyme--and is inhibited by toxins from scorpion venom. On the basis of the conserved structures of K+ pore regions and scorpion toxins, detailed structures for the K+ channel-scorpion toxin binding interface have been proposed. In these models and in previous solution-state nuclear magnetic resonance (NMR) studies using detergent-solubilized membrane proteins, scorpion toxins were docked to the extracellular entrance of the K+ channel pore assuming rigid, preformed binding sites. Using high-resolution solid-state NMR spectroscopy, here we show that high-affinity binding of the scorpion toxin kaliotoxin to a chimaeric K+ channel (KcsA-Kv1.3) is associated with significant structural rearrangements in both molecules. Our approach involves a combined analysis of chemical shifts and proton-proton distances and demonstrates that solid-state NMR is a sensitive method for analysing the structure of a membrane protein-inhibitor complex. We propose that structural flexibility of the K+ channel and the toxin represents an important determinant for the high specificity of toxin-K+ channel interactions.
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- 2006
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23. Contribution of N- and C-terminal channel domains to Kv channel interacting proteins in a mammalian cell line
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Robert Bähring, Olaf Pongs, L. Sven Hartmann, Kathrin Sauter, Britta Callsen, and Dirk Isbrandt
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Gene isoform ,chemistry.chemical_classification ,Physiology ,Immunoprecipitation ,Mutant ,Mutagenesis ,Gating ,Biology ,Amino acid ,Biochemistry ,chemistry ,Cytoplasm ,cardiovascular system ,Biophysics ,Patch clamp - Abstract
Association of Shal gene-related voltage-gated potassium (Kv4) channels with cytoplasmic Kv channel interacting proteins (KChIPs) influences inactivation gating and surface expression. We investigated both functional and biochemical consequences of mutations in cytoplasmic N and C-terminal Kv4.2 domains to characterize structural determinants for KChIP interaction. We performed a lysine-scanning mutagenesis within the proximal 40 amino acid portion and a structure-based mutagenesis in the tetramerization 1 (T1) domain of Kv4.2. In addition, the cytoplasmic Kv4.2 C-terminus was truncated at various positions. Wild-type and mutant Kv4.2 channels were coexpressed with KChIP2 isoforms in mammalian cell lines. The KChIP2-induced modulation of Kv4.2 currents was studied with whole-cell patch clamp and the binding of KChIP2 isoforms to Kv4.2 channels with coimmunoprecipitation experiments. Our results define one major interaction site for KChIPs, including amino acids in the proximal N-terminus between residues 11 and 23, where binding and functional modulation are essentially equivalent. A further interaction site includes residues in the T1 domain. Notably, C-terminal deletions also had marked effects on KChIP2-dependent gating modulation and KChIP2 binding, revealing a previously unknown involvement of domains within the cytoplasmic Kv4.2 C-terminus in KChIP interaction. Less coincidence of binding and functional modulation indicates a more loose ‘anchoring’ at T1- and C-terminal interaction sites. Our results refine and extend previously proposed structural models for Kv4.2/KChIP complex formation.
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- 2005
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24. Ein Konzept zur schnellen Bestimmung von Proteinstrukturen durch Festkörper-NMR-Spektroskopie
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Olaf Pongs, Adam Lange, Stefan Becker, Karin Giller, Karsten Seidel, and Marc Baldus
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Chemistry ,General Medicine - Published
- 2005
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25. Conditional transgenic suppression of M channels in mouse brain reveals functions in neuronal excitability, resonance and behavior
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Olaf Pongs, Hua Hu, Johan F. Storm, H. Christian Peters, and Dirk Isbrandt
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Systems neuroscience ,Cerebellum ,General Neuroscience ,Hippocampus ,Afterhyperpolarization ,Hippocampal formation ,Biology ,Neuroscientist ,medicine.anatomical_structure ,nervous system ,M current ,medicine ,Neuron ,Neuroscience - Abstract
In humans, mutations in the KCNQ2 or KCNQ3 potassium-channel genes are associated with an inherited epilepsy syndrome. We have studied the contribution of KCNQ/M-channels to the control of neuronal excitability by using transgenic mice that conditionally express dominant-negative KCNQ2 subunits in brain. We show that suppression of the neuronal M current in mice is associated with spontaneous seizures, behavioral hyperactivity and morphological changes in the hippocampus. Restriction of transgene expression to defined developmental periods revealed that M-channel activity is critical to the development of normal hippocampal morphology during the first postnatal weeks. Suppression of the M current after this critical period resulted in mice with signs of increased neuronal excitability and deficits in hippocampus-dependent spatial memory. M-current-deficient hippocampal CA1 pyramidal neurons showed increased excitability, reduced spike-frequency adaptation, attenuated medium afterhyperpolarization and reduced intrinsic subthreshold theta resonance. M channels are thus critical determinants of cellular and neuronal network excitability, postnatal brain development and cognitive performance.
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- 2004
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26. Clinical and electrophysiological characterization of a novel mutation R863X in HERG C-terminus associated with long QT syndrome
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Robert Bähring, Lijuan Ma, Yingxue Dong, Yanzong Yang, Jue Ye, Olaf Pongs, Rutai Hui, Chunxia Lin, Zhi-hu Lin, Vitya Vardanyan, and Siyong Teng
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Male ,Patch-Clamp Techniques ,Potassium Channels ,Time Factors ,DNA Mutational Analysis ,Mutant ,Pharmacology ,medicine.disease_cause ,Cricetinae ,Drug Discovery ,Cloning, Molecular ,Cation Transport Proteins ,Genetics (clinical) ,Mutation ,Microscopy, Confocal ,biology ,Stop codon ,Pedigree ,Electrophysiology ,Long QT Syndrome ,Phenotype ,Potassium Channels, Voltage-Gated ,Molecular Medicine ,Female ,congenital, hereditary, and neonatal diseases and abnormalities ,medicine.medical_specialty ,Long QT syndrome ,Molecular Sequence Data ,hERG ,Nonsense mutation ,Torsades de pointes ,CHO Cells ,QT interval ,Structure-Activity Relationship ,Internal medicine ,medicine ,Animals ,Humans ,cardiovascular diseases ,Base Sequence ,Models, Genetic ,business.industry ,Cell Membrane ,medicine.disease ,Ether-A-Go-Go Potassium Channels ,Protein Structure, Tertiary ,Endocrinology ,Haplotypes ,biology.protein ,business ,Gene Deletion - Abstract
We have found a novel nonsense mutation in the C-terminus of HERG in a four-generation Chinese family with long QT syndrome and investigated the molecular mechanism of this mutation in vitro. Six family members, including the proband, were clinically affected. Syncope and ventricular tachycardia of torsades de pointes were triggered by startling or emotional stress, and beta-adrenergic blockade treatment was ineffective. Haplotype analysis showed that only LQT2 markers cosegregated with the disease, and sequence analysis revealed a substitution of T with C at nucleotide position 2770 of the HERG gene (U04270), which creates a stop codon at amino acid position 863 (R863X) of the HERG protein, leading to a deletion of 296 amino acids. Whole cell patch clamp studies showed that the R863X HERG could not induce time-dependent current. Coexpression of R863X with wild-type HERG showed reduced current densities and accelerated voltage-dependent inactivation of HERG channels. Subcellular localization of R863X-EGFP revealed that the mutant did not traffic to the cell surface. These data suggest that R863X failed to form functional HERG channels, contributing to a prolongation of the QT interval and long QT syndrome with a dominant phenotype. These findings provide new insights into the structure-function relationships of the HERG C-terminus.
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- 2004
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27. Effective long-term control of cardiac events with β-blockers in a family with a common LQT1 mutation
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T Wülfing, Olaf Pongs, H. Wedekind, H Djonlagic, Eric Schulze-Bahr, S Hauenschild, G. Breithardt, Martin Schwarz, Wilhelm Haverkamp, and Dirk Isbrandt
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medicine.medical_specialty ,Heart disease ,Heart block ,business.industry ,Long QT syndrome ,medicine.disease ,QT interval ,Sudden death ,Asymptomatic ,Sudden cardiac death ,Romano–Ward syndrome ,Endocrinology ,Internal medicine ,Genetics ,medicine ,medicine.symptom ,business ,Genetics (clinical) - Abstract
The congenital long QT syndrome (LQTS) is characterized by a prolonged QT interval on the surface electrocardiogram and an increased risk of recurrent syncope and sudden cardiac death. Mutations in seven genes have been identified as the molecular basis of LQTS. beta-blockers are the treatment of choice to reduce cardiac symptoms. However, long-term follow-up of genotyped families with LQTS has been rarely reported. We have clinically followed a four-generation family with LQTS being treated with beta-blocker therapy over a period of 23 years. Seven family members were carriers of two amino acid alterations in cis (V254M-V417M) in the cardiac potassium channel gene KCNQ1. Voltage-clamp recordings of mutant KCNQ1 protein in Xenopus oocytes showed that only the V254M mutation reduced the IKs current and that the effect of the V417M variant was negligible. The family exhibited the complete clinical spectrum of the disease, from asymptomatic patients to victims of sudden death before beta-blocker therapy. There was no significant reduction in QTc (556 +/- 40 ms(1/2) before therapy, 494 +/- 20 ms(1/2) during 17 years of treatment; n = 5 individuals). Of nine family members, one female died suddenly before treatment, three females of the second generation were asymptomatic, and four individuals of the third and fourth generation were symptomatic. All mutation carriers were treated with beta-blockers and remained asymptomatic for a follow-up up to 23 years. Long-term follow-up of a LQT1 family with a common mutation (V254M) being on beta-blocker therapy was effective and safe. This study underscores the importance of long-term follow-up in families with specific LQT mutations to provide valuable information for clinicians for an appropriate antiarrhythmic treatment.
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- 2004
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28. N-type Inactivation Features of Kv4.2 Channel Gating
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Andreas Nolting, Robert Bähring, Dirk Isbrandt, Britta Callsen, Kathrin Sauter, Manuel Gebauer, and Olaf Pongs
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Potassium Channels ,Molecular Sequence Data ,Kinetics ,Biophysics ,Peptide ,CHO Cells ,Kidney ,Cell Line ,Structure-Activity Relationship ,chemistry.chemical_compound ,Cricetulus ,Channels, Receptors, and Transporters ,Cricetinae ,Animals ,Humans ,Amino Acid Sequence ,Shaker ,chemistry.chemical_classification ,Tetraethylammonium ,Shal Potassium Channels ,Voltage-gated ion channel ,Molecular biology ,Potassium channel ,Amino Acid Substitution ,chemistry ,Potassium Channels, Voltage-Gated ,Mutagenesis, Site-Directed ,cardiovascular system ,Ion Channel Gating ,Intracellular - Abstract
We examined whether the N-terminus of Kv4.2 A-type channels (4.2NT) possesses an autoinhibitory N-terminal peptide domain, which, similar to the one of Shaker, mediates inactivation of the open state. We found that chimeric Kv2.1(4.2NT) channels, where the cytoplasmic Kv2.1 N-terminus had been replaced by corresponding Kv4.2 domains, inactivated relatively fast, with a mean time constant of 120 ms as compared to 3.4 s in Kv2.1 wild-type. Notably, Kv2.1(4.2NT) showed features typically observed for Shaker N-type inactivation: fast inactivation of Kv2.1(4.2NT) channels was slowed by intracellular tetraethylammonium and removed by N-terminal truncation (Delta40). Kv2.1(4.2NT) channels reopened during recovery from inactivation, and recovery was accelerated in high external K+. Moreover, the application of synthetic N-terminal Kv4.2 and ShB peptides to inside-out patches containing slowly inactivating Kv2.1 channels mimicked N-type inactivation. Kv4.2 channels, after fractional inactivation, mediated tail currents with biphasic decay, indicative of passage through the open state during recovery from inactivation. Biphasic tail current kinetics were less prominent in Kv4.2/KChIP2.1 channel complexes and virtually absent in Kv4.2Delta40 channels. N-type inactivation features of Kv4.2 open-state inactivation, which may be suppressed by KChIP association, were also revealed by the finding that application of Kv4.2 N-terminal peptide accelerated the decay kinetics of both Kv4.2Delta40 and Kv4.2/KChIP2.1 patch currents. However, double mutant cycle analysis of N-terminal inactivating and pore domains indicated differences in the energetics and structural determinants between Kv4.2 and Shaker N-type inactivation.
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- 2004
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29. Differential modulation of Kv1 channel-mediated currents by co-expression of Kvβ3 subunit in a mammalian cell-line
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Vitya Vardanyan, Olaf Pongs, and Robert Bähring
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Patch-Clamp Techniques ,Potassium Channels ,Protein subunit ,Xenopus ,CHO Cells ,complex mixtures ,Membrane Potentials ,Structure-Activity Relationship ,Cricetinae ,medicine ,Animals ,Humans ,natural sciences ,Patch clamp ,Cloning, Molecular ,Receptor ,Molecular Biology ,Membrane potential ,Ion Transport ,biology ,urogenital system ,Chinese hamster ovary cell ,Electric Conductivity ,Cell Biology ,Oocyte ,biology.organism_classification ,Molecular biology ,Recombinant Proteins ,Kinetics ,Protein Subunits ,medicine.anatomical_structure ,nervous system ,Biophysics ,Heterologous expression ,biological phenomena, cell phenomena, and immunity ,Ion Channel Gating - Abstract
The effect of Kvbeta3 subunit co-expression on currents mediated by the Shaker-related channels Kv1.1 to Kv1.6 in Chinese hamster ovary (CHO) cells was studied with patch-clamp techniques. In the presence of Kvbeta3, differences in the voltage dependence of activation for Kv1.1, Kv1.3 and Kv1.6 were detected, but not for Kv1.2- and Kv1.4-mediated currents. Co-expression of Kvbeta3 did not cause a significant increase in current density for any of the tested channels. In contrast to previous studies in Xenopus oocyte expression system, Kvbeta3 confered a rapid inactivation to all except Kv1.3 channels. Also, Kv1.6 channels that possess an N-type inactivation prevention (NIP) domain for Kvbeta1.1, inactivated rapidly when co-expressed with Kvbeta3. Onset and recovery kinetics of channel inactivation distinctly differed for the various Kv1alpha/Kvbeta3 subunit combinations investigated in this study. The results indicate that the choice of expression system may critically determine Kvbeta3 inactivating activity. This suggests that the presence of an inactivating domain and a receptor in a channel pore, although necessary, may not be sufficient for an effective rapid N-type inactivation of Kv1 channels in heterologous expression systems.
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- 2004
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30. Conservation of Regulatory Function in Calcium-binding Proteins
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Olaf Pongs, Birgit Grafelmann, Thomas Strahl, Jens Dannenberg, and Jeremy Thorner
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Gene isoform ,biology ,Kinase ,Saccharomyces cerevisiae ,chemistry.chemical_element ,Cell Biology ,Calcium ,biology.organism_classification ,Biochemistry ,Yeast ,chemistry.chemical_compound ,chemistry ,Neuronal calcium sensor-1 ,Calcium-binding protein ,biology.protein ,Phosphatidylinositol ,Molecular Biology - Abstract
Frequenin, also known as neuronal calcium sensor-1 (NCS-1), is an N-myristoylated Ca2+-binding protein that has been conserved in both sequence and three-dimensional fold during evolution. We demonstrate using both genetic and biochemical approaches that the observed structural conservation between Saccharomyces cerevisiae frequenin (Frq1) and human NCS-1 is also reflected at the functional level. In yeast, the sole essential target of Frq1 is the phosphatidylinositol 4-kinase isoform, Pik1; both FRQ1 and PIK1 are indispensable for cell viability. Expression of human NCS-1 in yeast, but not a close relative (human KChIP2), rescues the inviability of frq1 cells. Furthermore, in vitro, Frq1 and NCS-1 (either N-myristoylated or unmyristoylated) compete for binding to a small 28-residue motif near the N terminus of Pik1. Site-directed mutagenesis indicates that the binding determinant in Pik1 is a hydrophobic α-helix and that frequenins bind to one side of this α-helix. We propose, therefore, that the function of NCS-1 in mammals may closely resemble that of Frq1 in S. cerevisiae and, hence, that frequenins in general may serve as regulators of certain isoforms of phosphatidylinositol 4-kinase.
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- 2003
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31. Characterization of a novel Long QT syndrome mutation G52R-KCNE1 in a Chinese family
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Lijuan Ma, Liang Li, Olaf Pongs, Rutai Hui, Chunxia Lin, Vitya Vardanyan, Robert Bähring, Siyong Teng, and Yongping Chai
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Adult ,Male ,China ,congenital, hereditary, and neonatal diseases and abnormalities ,Potassium Channels ,Adolescent ,Physiology ,Long QT syndrome ,DNA Mutational Analysis ,Mutant ,Biology ,Gene mutation ,Transfection ,medicine.disease_cause ,QT interval ,Sudden death ,Xenopus laevis ,Physiology (medical) ,medicine ,Animals ,Humans ,Missense mutation ,KvLQT1 ,Child ,Polymorphism, Single-Stranded Conformational ,Genetics ,Mutation ,Sequence Analysis, DNA ,Middle Aged ,medicine.disease ,Pedigree ,Long QT Syndrome ,Potassium Channels, Voltage-Gated ,Oocytes ,cardiovascular system ,biology.protein ,Female ,Cardiology and Cardiovascular Medicine - Abstract
Objectives: To identify the underlying genetic basis of a Chinese pedigree with Long QT syndrome, the causally related genes were screened in a family and the functional consequence of the identified gene mutation was evaluated in vitro. Methods: Mutations in the five defined Long QT syndrome related genes were screened with polymerase chain reaction and single-strand conformation polymorphism methods and direct sequencing. The electrophysiological properties of the identified mutation were characterized in the Xenopus oocyte heterologous expression system. Results: A novel missense mutation, G to A at position 154 in the KCNE1 gene was identified in a Chinese Long QT syndrome family, which leads to an amino acid substitution of arginine (R) for glycine (G) at position 52 (G52R-KCNE1). Of 26 family members (one DNA was not available), seven were mutation carriers and two of them with normal electrocardiogram. Compared with wild-type KCNE1/KCNQ1 channels, coexpression of G52R-KCNE1 with KCNQ1 in Xenopus oocytes did not amplify the KCNQ1 current amplitudes and slightly changed the activation kinetics of the KCNQ1 channels. Coexpression of KCNQ1 together with wild type KCNE1 and G52R-KCNE1 reduced the wild-type I ks current amplitude by 50%, whereas other biophysical properties of the I ks were not altered. Conclusions: Our findings indicate that glycine52 in the transmembrane domain is critical for KCNE1 function. The mutant G52R-KCNE1 has a dominant negative effect on I ks current, which reduces the I ks current amplitude and leads to a prolongation of the cardiac action potential. This could underlie the molecular mechanism of ventricular arrhythmias and sudden death in those patients.
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- 2003
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32. The pore region of the Kv1.2α subunit is an important component of recombinant Kv1.2 channel oxygen sensitivity
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David E. Millhorn, Laura Conforti, Olaf Pongs, Koichi Takimoto, and Milan Petrovic
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Potassium Channels ,Xenopus ,Protein subunit ,Molecular Sequence Data ,Biophysics ,Biology ,complex mixtures ,Biochemistry ,RNA, Complementary ,law.invention ,chemistry.chemical_compound ,Protein structure ,law ,Kv1.2 Potassium Channel ,Animals ,Point Mutation ,Amino Acid Sequence ,Hypoxia ,Molecular Biology ,chemistry.chemical_classification ,Methionine ,Sequence Homology, Amino Acid ,Voltage-gated ion channel ,Cell Biology ,biology.organism_classification ,Recombinant Proteins ,Potassium channel ,Protein Structure, Tertiary ,Amino acid ,Electrophysiology ,Oxygen ,chemistry ,Potassium Channels, Voltage-Gated ,Oocytes ,Potassium ,Recombinant DNA ,Peptides ,Oxidation-Reduction - Abstract
Oxygen-sensitive K(+) channels are important elements in the cellular response to hypoxia. Although much progress has been made in identifying their molecular composition, the structural components associated to their O(2)-sensitivity are not yet understood. Recombinant Kv1.2 currents expressed in Xenopus oocytes are inhibited by a decrease in O(2) availability. On the contrary, heterologous Kv2.1 channels are O(2)-insensitive. To elucidate the protein segment responsible for the O(2)-sensitivity of Kv1.2 channels, we analyzed the response to anoxia of Kv1.2/Kv2.1 chimeric channels. Expression of chimeric Kv2.1 channels each containing the S4, the S1-S3 or the S6-COOH segments of Kv1.2 polypeptide resulted in a K(+) current insensitive to anoxia. In contrast, transferring the S5-S6 segment of Kv1.2 into Kv2.1 produced an O(2)-sensitive K(+) current. Finally, mutating a redox-sensitive methionine residue (M380) of Kv1.2 polypeptide did not affect O(2)-sensitivity. Thus, the pore and its surrounding regions of Kv1.2 polypeptide confer its hypoxic inhibition. This response is independent on the redox modulation of methionine residues in this protein segment.
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- 2003
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33. Novel gene hKCNE4 slows the activation of the KCNQ1 channel
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Chunxia Lin, Yisong Zhen, Robert Bähring, Vitya Vardanyan, Lijuan Ma, Olaf Pongs, Rutai Hui, and Siyong Teng
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ERG1 Potassium Channel ,DNA, Complementary ,Potassium Channels ,Xenopus ,Molecular Sequence Data ,Biophysics ,CHO Cells ,Biochemistry ,Mice ,KCNN4 ,Transcriptional Regulator ERG ,Pregnancy ,Cricetinae ,Animals ,Humans ,Gene family ,Tissue Distribution ,Amino Acid Sequence ,RNA, Messenger ,Northern blot ,Cation Transport Proteins ,Molecular Biology ,Gene ,KCNN2 ,Base Sequence ,KCNQ Potassium Channels ,Sequence Homology, Amino Acid ,biology ,Membrane Proteins ,Cell Biology ,KCNE4 ,Molecular biology ,Ether-A-Go-Go Potassium Channels ,Recombinant Proteins ,Potassium channel ,DNA-Binding Proteins ,Kinetics ,Transmembrane domain ,Potassium Channels, Voltage-Gated ,KCNQ1 Potassium Channel ,Oocytes ,Trans-Activators ,biology.protein ,Female ,Carrier Proteins - Abstract
The KCNE genes encode small, single transmembrane domain peptides that associate with pore-forming potassium channel subunits to form mixed complexes with unique characteristics. We have identified a novel member of the human KCNE gene family, hKCNE4. The hKCNE4 gene encodes 170 amino acid protein and is localized to chromosome 2q35-36. The protein sequence shows 90% homology to mouse KCNE4 and 38% identity to human KCNE1. Northern blot analysis revealed that hKCNE4 is expressed strongly in heart, skeletal muscle, and kidney, less in placenta, lung, and liver, and weakly in brain and blood cells. Electrophysiological study showed that hKCNE4 modulates the activation of the KCNQ1 channel.
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- 2003
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34. Biophysical Properties of Kv3.1 Channels in SH-SY5Y Human Neuroblastoma Cells
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James P. Dilger, Patrick Friederich, Olaf Pongs, Bernd W. Urban, Dirk Isbrandt, and Kathrin Sauter
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DNA, Complementary ,Patch-Clamp Techniques ,Potassium Channels ,Time Factors ,SH-SY5Y ,Clinical Biochemistry ,Biophysics ,Analytical chemistry ,Gating ,Polymerase Chain Reaction ,Biophysical Phenomena ,Cell Line ,Endocrinology ,Cell Line, Tumor ,Neuroblastoma ,medicine ,Animals ,Humans ,Patch clamp ,4-Aminopyridine ,Pharmacology ,Membrane potential ,Genome ,Dose-Response Relationship, Drug ,Reverse Transcriptase Polymerase Chain Reaction ,Chemistry ,Neuropeptides ,Time constant ,Conductance ,Cell Biology ,medicine.disease ,Potassium channel ,Rats ,Electrophysiology ,Kinetics ,Shaw Potassium Channels ,Potassium Channels, Voltage-Gated ,Potassium - Abstract
Biophysical properties of delayed rectifier K channels in the human neuroblastoma SH-SY5Y were established using patch clamp recordings. The whole cell K+ conductance activated at membrane potentials positive to -20 mV. The midpoint of current activation was 9.6 +/- 5.1 mV, the equivalent charge was 3.7 +/-.6. Whole-cell currents inactivated slightly with time constants of 700 ms and 5 s. The K+ currents were sensitive to micromolar concentrations of TEA and 4-aminopyridine. RT-PCR experiments amplified a cDNA fragment specific for human Kv3.1 channels. Activation gating parameters in outside-out patches were shifted by approximately 14 mV in the hyperpolarizing direction.
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- 2003
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35. A Truncation Variant of the Cation Channel P2RX5 Is Upregulated during T Cell Activation
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Roland Martin, Olaf Pongs, Christoph Ogrodowczyk, Pierre Abramowski, and University of Zurich
- Subjects
CD4-Positive T-Lymphocytes ,lcsh:Medicine ,Lymphocyte Activation ,Cell membrane ,Cell-Mediated Immunity ,Heterotrimeric G protein ,Medicine and Health Sciences ,Cytotoxic T cell ,lcsh:Science ,Multidisciplinary ,Cell Polarity ,Cell biology ,Interleukin-10 ,Up-Regulation ,Protein Transport ,medicine.anatomical_structure ,Gene Knockdown Techniques ,Intracellular ,Research Article ,T cell ,Immunology ,610 Medicine & health ,1100 General Agricultural and Biological Sciences ,Biology ,TCIRG1 ,Immune Activation ,Immunomodulation ,1300 General Biochemistry, Genetics and Molecular Biology ,Extracellular ,medicine ,Genetics ,Animals ,Humans ,RNA, Messenger ,1000 Multidisciplinary ,Gene Expression Profiling ,HEK 293 cells ,lcsh:R ,Cell Membrane ,Immunity ,Biology and Life Sciences ,Immunoregulation ,Molecular biology ,Lymphocyte Subsets ,10040 Clinic for Neurology ,Clone Cells ,Alternative Splicing ,Protein Subunits ,HEK293 Cells ,lcsh:Q ,Mutant Proteins ,Gene Function ,Receptors, Purinergic P2X5 - Abstract
Members of the P2X family of ligand-gated cation channels (P2RX) are expressed by various cell types including neurons, smooth- and cardiac muscle cells, and leukocytes. The channels mediate signalling in response to extracellular ATP. Seven subunit isoforms (P2RX1-P2RX7) have been identified and these can assemble as homo- and heterotrimeric molecules. In humans, P2RX5 exists as a natural deletion mutant lacking amino acids 328–349 of exon 10, which are part of transmembrane (TM) 2 and pre-TM2 regions in other organisms like rat, chicken and zebrafish. We show that P2RX5 gene expression of human T lymphocytes is upregulated during activation. P2RX5 is recruited to the cell surface. P2RX5-siRNA-transfected CD4+ T cells produced twofold more IL-10 than controls. Surface and intracellular P2RX5 expression was upregulated in activated antigen-specific CD4+ T cell clones. These data indicate a functional role of the human P2RX5 splice variant in T cell activation and immunoregulation.
- Published
- 2014
36. Smooth muscle BK channel activity influences blood pressure independent of vascular tone in mice
- Author
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Gregor, Sachse, Jörg, Faulhaber, Anika, Seniuk, Heimo, Ehmke, and Olaf, Pongs
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Muscle Cells ,Large-Conductance Calcium-Activated Potassium Channel beta Subunits ,Xenopus ,Oocytes ,Animals ,Aorta, Thoracic ,Blood Pressure ,Mice, Transgenic ,Muscle, Smooth ,In Vitro Techniques ,Kidney ,Cardiovascular ,Aldosterone - Abstract
The large conductance voltage- and Ca(2+)-activated K(+) (BK) channel is an important determinant of vascular tone and contributes to blood pressure regulation. Both activities depend on the ancillary BKβ1 subunit. To determine the significance of smooth muscle BK channel activity for blood pressure regulation, we investigated the potential link between changes in arterial tone and altered blood pressure in BKβ1 knockout (BKβ1(-/-)) mice from three different genetically defined strains. While vascular tone was consistently increased in all BKβ1(-/-) mice independent of genetic background, BKβ1(-/-) strains exhibited increased (strain A), unaltered (strain B) or decreased (strain C) mean arterial blood pressures compared to their corresponding BKβ1(+/+) controls. In agreement with previous data on aldosterone regulation by renal/adrenal BK channel function, BKβ1(-/-) strain A mice have increased plasma aldosterone and increased blood pressure. Consistently, blockade of mineralocorticoid receptors by spironolactone treatment reversibly restored the elevated blood pressure to the BKβ1(+/+) strain A level. In contrast, loss of BKβ1 did not affect plasma aldosterone in strain C mice. Smooth muscle-restricted restoration of BKβ1 expression increased blood pressure in BKβ1(-/-) strain C mice, implying that impaired smooth muscle BK channel activity lowers blood pressure in these animals. We conclude that BK channel activity directly affects vascular tone but influences blood pressure independent of this effect via different pathways.
- Published
- 2014
37. Editorial overview: Cardiovascular and renal: Novel therapeutic strategies and approaches for targeting unmet cardiovascular needs
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Olaf Pongs and Gregory J. Kaczorowski
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Pharmacology ,medicine.medical_specialty ,business.industry ,Nanotechnology ,Kidney ,Cardiovascular System ,Ion Channels ,Cardiovascular Diseases ,Drug Discovery ,medicine ,Animals ,Humans ,Kidney Diseases ,Molecular Targeted Therapy ,Intensive care medicine ,business - Published
- 2014
38. Long QT mutations disrupt IKS regulation by PKA and PIP2 at the same KCNQ1 helix C-KCNE1 interface
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Meidan Dvir, Inbal Ben Tal Cohen, Bernard Attali, Yoni Haitin, Olaf Pongs, Carmen W. Dessauer, Dana Sachyani, Roi Strulovich, Joel A. Hirsch, and Robert S. Kass
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medicine.medical_specialty ,Mutation ,endocrine system diseases ,Long QT syndrome ,Mutant ,Depolarization ,Cardiac action potential ,Cell Biology ,Biology ,medicine.disease ,medicine.disease_cause ,Potassium channel ,Cell biology ,Endocrinology ,Internal medicine ,cardiovascular system ,medicine ,Phosphorylation ,Repolarization - Abstract
KCNQ1 and KCNE1 co-assembly generates the I(KS) K(+) current, which is crucial to the cardiac action potential repolarization. Mutations in their corresponding genes cause long QT syndrome (LQT) and atrial fibrillation. The A-kinase anchor protein, yotiao (also known as AKAP9), brings the I(KS) channel complex together with signaling proteins to achieve regulation upon β1-adrenergic stimulation. Recently, we have shown that KCNQ1 helix C interacts with the KCNE1 distal C-terminus. We postulated that this interface is crucial for I(KS) channel modulation. Here, we examined the yet unknown molecular mechanisms of LQT mutations located at this intracellular intersubunit interface. All LQT mutations disrupted the internal KCNQ1-KCNE1 intersubunit interaction. LQT mutants in KCNQ1 helix C led to a decreased current density and a depolarizing shift of channel activation, mainly arising from impaired phosphatidylinositol-4,5-bisphosphate (PIP2) modulation. In the KCNE1 distal C-terminus, the LQT mutation P127T suppressed yotiao-dependent cAMP-mediated upregulation of the I(KS) current, which was caused by reduced KCNQ1 phosphorylation at S27. Thus, KCNQ1 helix C is important for channel modulation by PIP2, whereas the KCNE1 distal C-terminus appears essential for the regulation of IKS by yotiao-mediated PKA phosphorylation.
- Published
- 2014
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39. β1-subunit of BK channels regulates arterial wall [Ca2+] and diameter in mouse cerebral arteries
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Olaf Pongs, Matthias Löhn, Birgit Lauterbach, Friedrich C. Luft, Maik Gollasch, and Hermann Haller
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medicine.medical_specialty ,BK channel ,Potassium Channels ,Physiology ,Cerebral arteries ,Cardiomegaly ,Mice ,Potassium Channels, Calcium-Activated ,Physiology (medical) ,Internal medicine ,medicine ,Animals ,Large-Conductance Calcium-Activated Potassium Channels ,Mice, Knockout ,biology ,Ryanodine ,Chemistry ,Ryanodine receptor ,Anatomy ,Cerebral Arteries ,Iberiotoxin ,Hyperpolarization (biology) ,Calcium-activated potassium channel ,Potassium channel ,Mice, Inbred C57BL ,Endocrinology ,Vasoconstriction ,Cerebrovascular Circulation ,Hypertension ,Circulatory system ,biology.protein ,Calcium ,Peptides - Abstract
Mice with a disrupted β1(BKβ1)-subunit of the large-conductance Ca2+-activated K+ (BK) channel gene develop systemic hypertension and cardiac hypertrophy, which is likely caused by uncoupling of Ca2+ sparks to BK channels in arterial smooth muscle cells. However, little is known about the physiological levels of global intracellular Ca2+ concentration ([Ca2+]i) and its regulation by Ca2+ sparks and BK channel subunits. We utilized a BKβ1 knockout C57BL/6 mouse model and studied the effects of inhibitors of ryanodine receptor and BK channels on the global [Ca2+]i and diameter of small cerebral arteries pressurized to 60 mmHg. Ryanodine (10 μM) or iberiotoxin (100 nM) increased [Ca2+]i by ∼75 nM and constricted +/+ BKβ1 wild-type arteries (pressurized to 60 mmHg) with myogenic tone by ∼10 μm. In contrast, ryanodine (10 μM) or iberiotoxin (100 nM) had no significant effect on [Ca2+]i and diameter of −/− BKβ1-pressurized (60 mmHg) arteries. These results are consistent with the idea that Ca2+ sparks in arterial smooth muscle cells limit myogenic tone through activation of BK channels. The activation of BK channels by Ca2+ sparks reduces the voltage-dependent Ca2+ influx and [Ca2+]i through tonic hyperpolarization. Deletion of BKβ1 disrupts this negative feedback mechanism, leading to increased arterial tone through an increase in global [Ca2+]i.
- Published
- 2001
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40. Role of the S2 and S3 Segment in Determining the Activation Kinetics in Kv2.1 Channels
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Olaf Pongs, Klaus Benndorf, Rolf Koopmann, A. Scholle, Thorsten Leicher, Thomas Zimmer, and J. Ludwig
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Patch-Clamp Techniques ,Potassium Channels ,Microinjections ,Physiology ,Recombinant Fusion Proteins ,Molecular Sequence Data ,Kinetics ,Biophysics ,Xenopus ,Gating ,In Vitro Techniques ,Polymerase Chain Reaction ,Structure-Activity Relationship ,Xenopus laevis ,Shab Potassium Channels ,Kv1.2 Potassium Channel ,Animals ,Humans ,Amino Acid Sequence ,Cloning, Molecular ,biology ,Chemistry ,Substitution (logic) ,Cell Biology ,Human physiology ,biology.organism_classification ,Rats ,Crystallography ,Mutagenesis ,Potassium Channels, Voltage-Gated ,Time course ,Oocytes ,Female ,Ion Channel Gating ,Sequence Alignment ,Delayed Rectifier Potassium Channels - Abstract
We constructed chimeras between the rapidly activating Kv1.2 channel and the slowly activating Kv2.1 channel in order to study to what extent sequence differences within the S1-S4 region contribute to the difference in activation kinetics. The channels were expressed in Xenopus oocytes and the currents were measured with a two-microelectrode voltage-clamp technique. Substitution of the S1-S4 region of Kv2.1 subunits by the ones of Kv1.2 resulted in chimeric channels which activated more rapidly than Kv2.1. Furthermore, activation kinetics were nearly voltage-independent in contrast to the pronounced voltage-dependent activation kinetics of both parent channels. Systematic screening of the S1-S4 region by the replacement of smaller protein parts resolved that the main functional changes generated by the S1-S4 substitution were generated by the S2 and the S3 segment. However, the effects of these segments were different: The S3 substitution reduced the effective gating charge and accelerated both a voltage-dependent and a voltage-independent component of the activation time course. In contrast, the S2 substitution accelerated predominantly the voltage-dependent component of the activation time course thereby leaving the effective gating charge unchanged. It is concluded that the S2 and the S3 segment determine the activation kinetics in a specific manner.
- Published
- 2001
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41. Screening lead compounds for QT interval prolongation
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Ulrike Bischoff, Rainer Netzer, Olaf Pongs, and Andreas Ebneth
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Pharmacology ,biology ,Heart disease ,Drug discovery ,business.industry ,hERG ,KCNE2 ,medicine.disease ,QT interval ,Drug development ,In vivo ,Drug Discovery ,biology.protein ,medicine ,Pharmaceutical sciences ,business - Abstract
The late detection of cardiotoxic side effects, such as QT prolongation, induced by compounds of pharmacological interest can dramatically impede drug discovery and development projects, and consequently increase their cost. The launch of new drugs with undetected cardiotoxic side effects could have hazardous consequences and could trigger lethal cardiac dysrhythmias in patients. It is desirable, therefore, to test for the potential cardiotoxic side effects of compounds at an early stage of drug development. Electrophysiological test systems and cellular-based fluorometric high-throughput assays are now available for cloned human cardiac ion channels. These test systems are important tools in the preclinical safety evaluation of drugs and newly developed compounds.
- Published
- 2001
- Full Text
- View/download PDF
42. Generating a High Affinity Scorpion Toxin Receptor in KcsA-Kv1.3 Chimeric Potassium Channels
- Author
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Verena Pollmann, Marie-France Martin-Eauclaire, Angela M. Farrell, Hervé Darbon, Christian Legros, Olaf Pongs, Hans-Günther Knaus, and Pierre E. Bougis
- Subjects
Kv1.3 Potassium Channel ,Potassium Channels ,Scorpion toxin ,Sequence Homology, Amino Acid ,Voltage-gated ion channel ,Stereochemistry ,Recombinant Fusion Proteins ,Molecular Sequence Data ,KcsA potassium channel ,Scorpion Venoms ,Kaliotoxin ,Cell Biology ,Plasma protein binding ,Biology ,Biochemistry ,Sodium Channels ,Potassium channel ,Bacterial Proteins ,Potassium Channels, Voltage-Gated ,Humans ,Amino Acid Sequence ,Molecular Biology ,Linker ,Ion channel ,Protein Binding - Abstract
The crystal structure of the bacterial K(+) channel, KcsA (Doyle, D. A., Morais, C. J., Pfuetzner, R. A., Kuo, A., Gulbis, J. M., Cohen, S. L., Chait, B. T., and MacKinnon, R. (1998) Science 280, 69-77), and subsequent mutagenesis have revealed a high structural conservation from bacteria to human (MacKinnon, R., Cohen, S. L., Kuo, A., Lee, A., and Chait, B. T. (1998) Science 280, 106-109). We have explored this conservation by swapping subregions of the M1-M2 linker of KcsA with those of the S5-S6 linker of the human Kv-channel Kv1.3. The chimeric K(+) channel constructs were expressed in Escherichia coli, and their multimeric state was analyzed after purification. We used two scorpion toxins, kaliotoxin and hongotoxin 1, which bind specifically to Kv1.3, to analyze the pharmacological properties of the KcsA-Kv1.3 chimeras. The results demonstrate that the high affinity scorpion toxin receptor of Kv1.3 could be transferred to KcsA. Our biochemical studies with purified KcsA-Kv1.3 chimeras provide direct chemical evidence that a tetrameric channel structure is necessary for forming a functional scorpion toxin receptor. We have obtained KcsA-Kv1.3 chimeras with kaliotoxin affinities (IC(50) values of approximately 4 pm) like native Kv1.3 channels. Furthermore, we show that a subregion of the S5-S6 linker may be an important determinant of the pharmacological profile of K(+) channels. Using available structural information on KcsA and kaliotoxin, we have developed a structural model for the complex between KcsA-Kv1.3 chimeras and kaliotoxin to aid future pharmacological studies of K(+) channels.
- Published
- 2000
- Full Text
- View/download PDF
43. Gene Structures and Expression Profiles of Three Human KCND (Kv4) Potassium Channels Mediating A-Type Currents ITO and ISA
- Author
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Xin-Ran Zhu, Olaf Pongs, Dirk Isbrandt, Kathrin Sauter, Ulrich F O Luhmann, Thorsten Leicher, Ralph Waldschütz, and Uwe Michel
- Subjects
DNA, Complementary ,Potassium Channels ,Transcription, Genetic ,Molecular Sequence Data ,Gene Expression ,Biology ,Polymerase Chain Reaction ,Homology (biology) ,03 medical and health sciences ,Exon ,0302 clinical medicine ,Complementary DNA ,Gene expression ,Genetics ,Humans ,Tissue Distribution ,RNA, Messenger ,Northern blot ,Cloning, Molecular ,Gene ,Gene Library ,030304 developmental biology ,0303 health sciences ,Genome ,Sequence Homology, Amino Acid ,Electric Conductivity ,Nucleic acid sequence ,Chromosome Mapping ,Exons ,Molecular biology ,Introns ,Open reading frame ,Shal Potassium Channels ,Potassium Channels, Voltage-Gated ,cardiovascular system ,Ion Channel Gating ,Sequence Alignment ,030217 neurology & neurosurgery - Abstract
The four known members of the KCND/Kv4 channel family encode voltage-gated potassium channels. Recent studies provide evidence that members of the Kv4 channel family are responsible for native, rapidly inactivating (A-type) currents described in heart (I(TO)) and neurons (I(SA)). In this study, we cloned the human KCND1 cDNA, localized the KCND1 gene to chromosome Xp11.23-p11.3, and determined the genomic structure and tissue-specific expression of the KCND1, KCND2, and KCND3 genes, respectively. The open reading frame of Kv4. 1 is 1941 nucleotides long, predicting a protein of 647 amino acids. The deduced protein sequence of Kv4.1 shows an overall identity of 60% with Kv4.2 and Kv4.3L and corresponds to the common structure of voltage-gated potassium channels. KCND1-specific transcripts were detectable in human brain, heart, liver, kidney, thyroid gland, and pancreas, as revealed by Northern blot and RT-PCR experiments. The comparison of the expression patterns of the known Kv4 family members shows subtype specificity with significant overlaps. The KCND gene structures exhibit an evolutionarily conserved exon pattern with a large first exon containing the intracellular N-terminus and the putative membrane-spanning regions S1 to S5, as well as part of the pore region. The KCND3 gene contains an additional exon of 57 bp, which is not present in the other two KCND genes and gives rise to the C-terminal splice KCND3L variant with an insertion of 19 amino acids.
- Published
- 2000
- Full Text
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44. A recessive C-terminal Jervell and Lange-Nielsen mutation of the KCNQ1 channel impairs subunit assembly
- Author
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Ilane Abitbol, Olaf Pongs, Asher Peretz, Nicole Schmitt, Martin Schwarz, and Bernard Attali
- Subjects
Potassium Channels ,Microinjections ,Xenopus ,Long QT syndrome ,Protein subunit ,Molecular Sequence Data ,Mutant ,Genes, Recessive ,Locus (genetics) ,CHO Cells ,Transfection ,Sudden death ,General Biochemistry, Genetics and Molecular Biology ,RNA, Complementary ,Cricetinae ,medicine ,Animals ,Humans ,Amino Acid Sequence ,KvLQT1 ,Cloning, Molecular ,Molecular Biology ,Genetics ,KCNQ Potassium Channels ,General Immunology and Microbiology ,biology ,General Neuroscience ,Articles ,medicine.disease ,Molecular biology ,Potassium channel ,Electrophysiology ,Long QT Syndrome ,Jervell and Lange-Nielsen syndrome ,Potassium Channels, Voltage-Gated ,KCNQ1 Potassium Channel ,Mutation ,Oocytes ,biology.protein ,Sequence Alignment - Abstract
The LQT1 locus (KCNQ1) has been correlated with the most common form of inherited long QT (LQT) syndrome. LQT patients suffer from syncopal episodes and high risk of sudden death. The KCNQ1 gene encodes KvLQT1 alpha-subunits, which together with auxiliary IsK (KCNE1, minK) subunits form IK(s) K(+) channels. Mutant KvLQT1 subunits may be associated either with an autosomal dominant form of inherited LQT, Romano-Ward syndrome, or an autosomal recessive form, Jervell and Lange-Nielsen syndrome (JLNS). We have identified a small domain between residues 589 and 620 in the KvLQT1 C-terminus, which may function as an assembly domain for KvLQT1 subunits. KvLQT1 C-termini do not assemble and KvLQT1 subunits do not express functional K(+) channels without this domain. We showed that a JLN deletion-insertion mutation at KvLQT1 residue 544 eliminates important parts of the C-terminal assembly domain. Therefore, JLN mutants may be defective in KvLQT1 subunit assembly. The results provide a molecular basis for the clinical observation that heterozygous JLN carriers show slight cardiac dysfunctions and that the severe JLNS phenotype is characterized by the absence of KvLQT1 channel.
- Published
- 2000
- Full Text
- View/download PDF
45. Voltage-gated potassium channels: from hyperexcitability to excitement
- Author
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Olaf Pongs
- Subjects
Nervous system ,Potassium Channels ,Biophysics ,Hyperexcitability ,Biochemistry ,SK channel ,chemistry.chemical_compound ,Epilepsy ,Voltage-activated potassium channel ,Structural Biology ,Genetics ,medicine ,Animals ,Humans ,Molecular Biology ,Action-potential ,Episodic ataxia ,Muscarine ,Hearing loss ,Cell Biology ,Anatomy ,Voltage-gated potassium channel ,medicine.disease ,Potassium channel ,medicine.anatomical_structure ,chemistry ,Neuron ,Ion Channel Gating ,Neuroscience - Abstract
The superfamily of voltage-activated potassium channels may express structurally and functionally diverse voltage-activated potassium channels in the nervous system. The roles of some voltage-activated potassium channel types, e.g. rapidly inactivating (transiently active type) channels and muscarine sensitive muscarine sensitive channels, are beginning to be understood. They may significantly influence dendritic action-potential back-propagation, signal to noise ratios in presynaptic excitability or the responsiveness of a neuron to synaptic input. Inherited disorders related to changes in excitability (episodic ataxia, epilepsy, heart arrhythmia) or to defects in sensory perception (hearing loss) have been associated with mutations in a few voltage-activated potassium channel genes. Most likely, more voltage-activated potassium channel genes will be linked to related disorders in the near future.
- Published
- 1999
- Full Text
- View/download PDF
46. Functional and Molecular Aspects of Voltage-Gated K+ Channel beta Subunits
- Author
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Michaela Berger, Robert Bähring, Karl Peter Giese, Alcino J. Silva, Thorsten Leicher, Olaf Pongs, Johan F. Storm, Jochen Roeper, and Dennis Wray
- Subjects
Patch-Clamp Techniques ,Potassium Channels ,Xenopus ,Gene Expression ,Alpha (ethology) ,Bioinformatics ,General Biochemistry, Genetics and Molecular Biology ,Mice ,History and Philosophy of Science ,Animals ,Humans ,RNA, Messenger ,Patch clamp ,Beta (finance) ,Mice, Knockout ,Neurons ,Voltage-gated ion channel ,biology ,General Neuroscience ,Alternative splicing ,biology.organism_classification ,Potassium channel ,Alternative Splicing ,Oocytes ,Shaker Superfamily of Potassium Channels ,Biophysics ,Heterologous expression ,Ion Channel Gating - Abstract
Voltage-gated potassium channels (Kv) of the Shaker-related superfamily are assembled from membrane-integrated alpha subunits and auxiliary beta subunits. The beta subunits may increase Kv channel surface expression and/or confer A-type behavior to noninactivating Kv channels in heterologous expression systems. The interaction of Kv alpha and Kv beta subunits depends on the presence or absence of several domains including the amino-terminal N-type inactivating and NIP domains and the Kv alpha and Kv beta binding domains. Loss of function of Kv beta 1.1 subunits leads to a reduction of A-type Kv channel activity in hippocampal and striatal neurons of knock-out mice. This reduction may be correlated with altered cognition and motor control in the knock-out mice.
- Published
- 1999
- Full Text
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47. Reduced K+ Channel Inactivation, Spike Broadening, and After-Hyperpolarization in Kvβ1.1-Deficient Mice with Impaired Learning
- Author
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Olaf Pongs, Li-Rong Shao, Karl Peter Giese, Fedorov Nikolai, Alcino J. Silva, Thorsten Leicher, Johan F. Storm, and Dirk Reuter
- Subjects
Voltage-gated ion channel ,Chemistry ,Cognitive Neuroscience ,Hippocampus ,Water maze ,Hippocampal formation ,Potassium channel ,Synapse ,Cellular and Molecular Neuroscience ,Neuropsychology and Physiological Psychology ,Slow afterhyperpolarization ,Synaptic plasticity ,Biophysics ,Neuroscience - Abstract
A-type K+ channels are known to regulate neuronal firing, but their role in repetitive firing and learning in mammals is not well characterized. To determine the contribution of the auxiliary K+ channel subunit Kvβ1.1 to A-type K+ currents and to study the physiological role of A-type K+ channels in repetitive firing and learning, we deleted the Kvβ1.1 gene in mice. The loss of Kvβ1.1 resulted in a reduced K+ current inactivation in hippocampal CA1 pyramidal neurons. Furthermore, in the mutant neurons, frequency-dependent spike broadening and the slow afterhyperpolarization (sAHP) were reduced. This suggests that Kvβ1.1-dependent A-type K+ channels contribute to frequency-dependent spike broadening and may regulate the sAHP by controlling Ca2+ influx during action potentials. The Kvβ1.1-deficient mice showed normal synaptic plasticity but were impaired in the learning of a water maze test and in the social transmission of food preference task, indicating that the Kvβ1.1 subunit contributes to certain types of learning and memory.
- Published
- 1998
- Full Text
- View/download PDF
48. NIP domain prevents N-type inactivation in voltage-gated potassium channels
- Author
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Sabine Sewing, Ying Zhang, Jochen Roeper, Olaf Pongs, Siegmund G. Wanner, and Tobias Sommer
- Subjects
Potassium Channels ,Recombinant Fusion Proteins ,Protein subunit ,Molecular Sequence Data ,CHO Cells ,Gating ,Cricetinae ,Electrochemistry ,Escherichia coli ,Potassium Channel Blockers ,Animals ,Amino Acid Sequence ,Cloning, Molecular ,Integral membrane protein ,Binding Sites ,Multidisciplinary ,Chemistry ,Voltage-gated potassium channel ,Electrophysiology ,Biochemistry ,Structural biology ,Mutation ,Shaker Superfamily of Potassium Channels ,Biophysics ,NIP ,Signal transduction ,Ion Channel Gating - Abstract
Shaker-related voltage-gated K+ (Kv) channels are assembled from ion-conducting K(v)alpha subunits, which are integral membrane proteins, and auxiliary K(v)beta subunits. This leads to the formation of highly diverse heteromultimeric Kv channels that mediate outward currents with a wide range of time courses for inactivation. Two principal inactivation mechanisms have been recognized: C-type inactivation correlated with carboxy-terminal K(v)alpha-subunit structures, and N-type inactivation conferred by 'ball' domains in the amino termini of certain K(v)alpha and K(v)beta subunits. Assembly of heteromultimers with one or more K(v)alpha- and/or K(v)beta ball domains appears to be an essential principle of the generation of A-type Kv channel diversity. Here we show that, unexpectedly, the presence of K(v)alpha- or K(v)beta-ball domains does not dominate the gating phenotype in heteromultimers containing Kv1.6alpha subunits. These heteromultimers mediate non-inactivating currents because of the dominant-negative activity of a new type of N-type inactivation-prevention (NIP) domain present in the Kv1.6 amino terminus. Mutations in the NIP domain lead to loss of function, and its transfer to another K(v)alpha subunit leads to gain of function. Our discovery of the NIP domain, which neutralizes the activity of K(v)alpha- and K(v)beta-inactivation gates, establishes a new determinant for the gating behaviour of heteromultimeric Kv channels.
- Published
- 1998
- Full Text
- View/download PDF
49. Carboxy-terminal domain mediates assembly of the voltage-gated rat ether-a-go-go potassium channel
- Author
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David R. Owen, Jost Ludwig, and Olaf Pongs
- Subjects
Protein Folding ,Patch-Clamp Techniques ,Potassium Channels ,Protein Conformation ,Potassium ,Protein subunit ,Molecular Sequence Data ,chemistry.chemical_element ,CHO Cells ,Biology ,General Biochemistry, Genetics and Molecular Biology ,SK channel ,Cricetulus ,Cricetinae ,Animals ,Amino Acid Sequence ,RNA, Messenger ,Molecular Biology ,Ion transporter ,Ion Transport ,General Immunology and Microbiology ,Voltage-gated ion channel ,Inward-rectifier potassium ion channel ,General Neuroscience ,Ether-A-Go-Go Potassium Channels ,Potassium channel ,Rats ,chemistry ,Biochemistry ,Mutagenesis, Site-Directed ,Shaker Superfamily of Potassium Channels ,Biophysics ,Research Article - Abstract
The specific assembly of subunits to oligomers is an important prerequisite for producing functional potassium channels. We have studied the assembly of voltage-gated rat ether-à-go-go (r-eag) potassium channels with two complementary assays. In protein overlay binding experiments it was shown that a 41-amino-acid domain, close to the r-eag subunit carboxy-terminus, is important for r-eag subunit interaction. In an in vitro expression system it was demonstrated that r-eag subunits lacking this assembly domain cannot form functional potassium channels. Also, a approximately 10-fold molar excess of the r-eag carboxy-terminus inhibited in co-expression experiments the formation of functional r-eag channels. When the r-eag carboxy-terminal assembly domain had been mutated, the dominant-negative effect of the r-eag carboxy-terminus on r-eag channel expression was abolished. The results demonstrate that a carboxy-terminal assembly domain is essential for functional r-eag potassium channel expression, in contrast to the one of Shaker-related potassium channels, which is directed by an amino-terminal assembly domain.
- Published
- 1997
- Full Text
- View/download PDF
50. TRPM4 channels in the cardiovascular system
- Author
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Martin Kruse and Olaf Pongs
- Subjects
Pharmacology ,Bradycardia ,medicine.medical_specialty ,Cardiac rhythmicity ,Mutant ,TRPM Cation Channels ,Biology ,Right bundle branch block ,medicine.disease ,Cardiovascular System ,Cardiovascular Physiological Phenomena ,Transient receptor potential channel ,Internal medicine ,Drug Discovery ,Cardiac conduction ,cardiovascular system ,medicine ,Cardiology ,Repolarization ,Animals ,Humans ,medicine.symptom ,Brugada syndrome - Abstract
The non-selective Transient Receptor Potential Melastatin 4 (TRPM4) cation channel is abundantly expressed in cardiac cells, being involved in several aspects of cardiac rhythmicity, including cardiac conduction, pace making and action-potential repolarization. Dominantly inherited mutations in the TRPM4 gene are associated with the cardiac bundle-branch disorder progressive familial heart block type I (PFHBI) and isolated cardiac conduction disease (ICCD) giving rise to atrio-ventricular conduction block (AVB), right bundle branch block, bradycardia, and the Brugada syndrome. The mutant phenotypes closely resemble those associated with mutations in the SCN5A gene, encoding the voltage-gated Na(+) channel NaV1.5. These observations and the unexpected partnership with sulfonylurea-receptors (SURs) makes the TRPM4 channel a promising novel target for treatment of cardiac disorders.
- Published
- 2013
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