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Covarrubias M, Liang Q, Nguyen-Phuong L, Kennedy KJ, Alexander TD, Sam A. Structural insights into the function, dysfunction and modulation of Kv3 channels. Neuropharmacology 2025; 275:110483. [PMID: 40288604 DOI: 10.1016/j.neuropharm.2025.110483] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2025] [Revised: 04/03/2025] [Accepted: 04/24/2025] [Indexed: 04/29/2025]
Abstract
The third subfamily of voltage-gated K+ (Kv) channels includes four members, Kv3.1, Kv3.2, Kv3.3 and Kv3.4. Fast gating and activation at relatively depolarized membrane potentials allows Kv3 channels to be major drivers of fast action potential repolarization in the nervous system. Consequently, they help determine the fast-spiking phenotype of inhibitory interneurons and regulate fast synaptic transmission at glutamatergic synapses and the neuromuscular junction. Recent studies from our group and a team of collaborators have used cryo-EM to demonstrate the surprising gating role of the Kv3.1 cytoplasmic T1 domain, the structural basis of a developmental epileptic encephalopathy caused by the Kv3.2-C125Y variant and the mechanism of action of positive allosteric modulators involving unexpected interactions and conformational changes in Kv3.1 and Kv3.2. Furthermore, our recent work has shown that Kv3.4 regulates use-dependent spike broadening in a manner that depends on gating modulation by phosphorylation of the channel's N-terminal inactivation domain, which can impact activity-dependent synaptic facilitation. Here, we review and integrate these studies to provide a perspective on our current understanding of Kv3 channel function, dysfunction and pain modulation in the nervous system.
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Affiliation(s)
- Manuel Covarrubias
- Department of Neuroscience, Sidney Kimmel Medical College of Thomas Jefferson University, Bluemle Life Science Building, 233 South 10th Street, Room 231, Philadelphia, PA, 19107, USA; Vickie and Jack Farber Institute for Neuroscience, USA; Jefferson Synaptic Biology Center, USA.
| | - Qiansheng Liang
- Department of Neuroscience, Sidney Kimmel Medical College of Thomas Jefferson University, Bluemle Life Science Building, 233 South 10th Street, Room 231, Philadelphia, PA, 19107, USA; Vickie and Jack Farber Institute for Neuroscience, USA; Jefferson Synaptic Biology Center, USA
| | - Linh Nguyen-Phuong
- Department of Neuroscience, Sidney Kimmel Medical College of Thomas Jefferson University, Bluemle Life Science Building, 233 South 10th Street, Room 231, Philadelphia, PA, 19107, USA; Vickie and Jack Farber Institute for Neuroscience, USA; Jefferson Synaptic Biology Center, USA
| | - Kyle J Kennedy
- Department of Neuroscience, Sidney Kimmel Medical College of Thomas Jefferson University, Bluemle Life Science Building, 233 South 10th Street, Room 231, Philadelphia, PA, 19107, USA; Vickie and Jack Farber Institute for Neuroscience, USA; Jefferson Synaptic Biology Center, USA
| | - Tyler D Alexander
- Department of Neuroscience, Sidney Kimmel Medical College of Thomas Jefferson University, Bluemle Life Science Building, 233 South 10th Street, Room 231, Philadelphia, PA, 19107, USA; Vickie and Jack Farber Institute for Neuroscience, USA; Jefferson Synaptic Biology Center, USA
| | - Andrew Sam
- Department of Neuroscience, Sidney Kimmel Medical College of Thomas Jefferson University, Bluemle Life Science Building, 233 South 10th Street, Room 231, Philadelphia, PA, 19107, USA; Vickie and Jack Farber Institute for Neuroscience, USA; Jefferson Synaptic Biology Center, USA
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Simonson BT, Jiang Z, Ryan JF, Jegla T. Ctenophores and parahoxozoans independently evolved functionally diverse voltage-gated K+ channels. J Gen Physiol 2025; 157:e202413740. [PMID: 40100064 PMCID: PMC11917167 DOI: 10.1085/jgp.202413740] [Citation(s) in RCA: 2] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/11/2024] [Revised: 01/29/2025] [Accepted: 02/26/2025] [Indexed: 03/20/2025] Open
Abstract
The ctenophore species Mnemiopsis leidyi is known to have a large set of voltage-gated K+ channels, but little is known about the functional diversity of these channels or their evolutionary history in other ctenophore species. Here, we searched the genomes of two additional ctenophore species, Beroe ovata and Hormiphora californensis, for voltage-gated K+ channels and functionally expressed a subset of M. leidyi channels. We found that the last common ancestor of these three disparate ctenophore lineages probably had at least 33 voltage-gated K+ channels. Two of these genes belong to the EAG family, and the remaining 31 belong to the Shaker family and form a single clade within the animal/choanoflagellate Shaker phylogeny. We additionally found evidence for 10 of these Shaker channels in a transcriptome of the early branching ctenophore lineage Euplokamis dunlapae, suggesting that the diversification of these channels was already underway early in ctenophore evolution. We functionally expressed 16 Mnemiopsis Shakers and found that they encode a diverse array of voltage-gated K+ conductances with functional orthologs for many classic Shaker family subtypes found in cnidarians and bilaterians. Analysis of Mnemiopsis transcriptome data show these 16 Shaker channels are expressed in a wide variety of cell types, including neurons, muscle, comb cells, and colloblasts. Ctenophores therefore appear to have independently evolved much of the voltage-gated K+ channel diversity that is shared between cnidarians and bilaterians.
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Affiliation(s)
- Benjamin T. Simonson
- Department of Biology and Huck Institutes of the Life Sciences, Penn State University, University Park, PA, USA
| | - Zhaoyang Jiang
- Department of Biology and Huck Institutes of the Life Sciences, Penn State University, University Park, PA, USA
| | - Joseph F. Ryan
- Whitney Laboratory for Marine Bioscience, University of Florida, St. Augustine, FL, USA
- Department of Biology, University of Florida, Gainesville, FL, USA
| | - Timothy Jegla
- Department of Biology and Huck Institutes of the Life Sciences, Penn State University, University Park, PA, USA
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Xie C, Kessi M, Yin F, Peng J. Roles of KCNA2 in Neurological Diseases: from Physiology to Pathology. Mol Neurobiol 2024; 61:8491-8517. [PMID: 38517617 DOI: 10.1007/s12035-024-04120-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2023] [Accepted: 03/10/2024] [Indexed: 03/24/2024]
Abstract
Potassium voltage-gated channel subfamily a member 2 (Kv1.2, encoded by KCNA2) is highly expressed in the central and peripheral nervous systems. Based on the patch clamp studies, gain-of function (GOF), loss-of-function (LOF), and a mixed type (GOF/LOF) variants can cause different conditions/disorders. KCNA2-related neurological diseases include epilepsy, intellectual disability (ID), attention deficit/hyperactive disorder (ADHD), autism spectrum disorder (ASD), pain as well as autoimmune and movement disorders. Currently, the molecular mechanisms for the reported variants in causing diverse disorders are unknown. Consequently, this review brings up to date the related information regarding the structure and function of Kv1.2 channel, expression patterns, neuronal localizations, and tetramerization as well as important cell and animal models. In addition, it provides updates on human genetic variants, genotype-phenotype correlations especially highlighting the deep insight into clinical prognosis of KCNA2-related developmental and epileptic encephalopathy, mechanisms, and the potential treatment targets for all KCNA2-related neurological disorders.
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Affiliation(s)
- Changning Xie
- Department of Pediatrics, Xiangya Hospital, Central South University, Xiangya Road 87, Hunan, Changsha, 410008, China
| | - Miriam Kessi
- Department of Pediatrics, Xiangya Hospital, Central South University, Xiangya Road 87, Hunan, Changsha, 410008, China
| | - Fei Yin
- Department of Pediatrics, Xiangya Hospital, Central South University, Xiangya Road 87, Hunan, Changsha, 410008, China
| | - Jing Peng
- Department of Pediatrics, Xiangya Hospital, Central South University, Xiangya Road 87, Hunan, Changsha, 410008, China.
- Hunan Intellectual and Development Disabilities Research Center, Hunan, Changsha, 410008, China.
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4
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Jegla T, Simonson BT, Spafford JD. A broad survey of choanoflagellates revises the evolutionary history of the Shaker family of voltage-gated K + channels in animals. Proc Natl Acad Sci U S A 2024; 121:e2407461121. [PMID: 39018191 PMCID: PMC11287247 DOI: 10.1073/pnas.2407461121] [Citation(s) in RCA: 3] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/16/2024] [Accepted: 06/21/2024] [Indexed: 07/19/2024] Open
Abstract
The Shaker family of voltage-gated K+ channels has been thought of as an animal-specific ion channel family that diversified in concert with nervous systems. It comprises four functionally independent gene subfamilies (Kv1-4) that encode diverse neuronal K+ currents. Comparison of animal genomes predicts that only the Kv1 subfamily was present in the animal common ancestor. Here, we show that some choanoflagellates, the closest protozoan sister lineage to animals, also have Shaker family K+ channels. Choanoflagellate Shaker family channels are surprisingly most closely related to the animal Kv2-4 subfamilies which were believed to have evolved only after the divergence of ctenophores and sponges from cnidarians and bilaterians. Structural modeling predicts that the choanoflagellate channels share a T1 Zn2+ binding site with Kv2-4 channels that is absent in Kv1 channels. We functionally expressed three Shakers from Salpingoeca helianthica (SheliKvT1.1-3) in Xenopus oocytes. SheliKvT1.1-3 function only in two heteromultimeric combinations (SheliKvT1.1/1.2 and SheliKvT1.1/1.3) and encode fast N-type inactivating K+ channels with distinct voltage dependence that are most similar to the widespread animal Kv1-encoded A-type Shakers. Structural modeling of the T1 assembly domain supports a preference for heteromeric assembly in a 2:2 stoichiometry. These results push the origin of the Shaker family back into a common ancestor of metazoans and choanoflagellates. They also suggest that the animal common ancestor had at least two distinct molecular lineages of Shaker channels, a Kv1 subfamily lineage predicted from comparison of animal genomes and a Kv2-4 lineage predicted from comparison of animals and choanoflagellates.
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Affiliation(s)
- Timothy Jegla
- Department of Biology, Eberly College of Sciences and Huck Institutes of the Life Sciences, Penn State University, University Park, PA16802
| | - Benjamin T. Simonson
- Department of Biology, Eberly College of Sciences and Huck Institutes of the Life Sciences, Penn State University, University Park, PA16802
| | - J. David Spafford
- Department of Biology, University of Waterloo, Waterloo, ONN2L 3G1, Canada
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Simonson BT, Jegla M, Ryan JF, Jegla T. Functional analysis of ctenophore Shaker K + channels: N-type inactivation in the animal roots. Biophys J 2024; 123:2038-2049. [PMID: 38291751 PMCID: PMC11309979 DOI: 10.1016/j.bpj.2024.01.027] [Citation(s) in RCA: 3] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/26/2023] [Revised: 11/16/2023] [Accepted: 01/24/2024] [Indexed: 02/01/2024] Open
Abstract
Here we explore the evolutionary origins of fast N-type ball-and-chain inactivation in Shaker (Kv1) K+ channels by functionally characterizing Shaker channels from the ctenophore (comb jelly) Mnemiopsis leidyi. Ctenophores are the sister lineage to other animals and Mnemiopsis has >40 Shaker-like K+ channels, but they have not been functionally characterized. We identified three Mnemiopsis channels (MlShak3-5) with N-type inactivation ball-like sequences at their N termini and functionally expressed them in Xenopus oocytes. Two of the channels, MlShak4 and MlShak5, showed rapid inactivation similar to cnidarian and bilaterian Shakers with rapid N-type inactivation, whereas MlShak3 inactivated ∼100-fold more slowly. Fast inactivation in MlShak4 and MlShak5 required the putative N-terminal inactivation ball sequences. Furthermore, the rate of fast inactivation in these channels depended on the number of inactivation balls/channel, but the rate of recovery from inactivation did not. These findings closely match the mechanism of N-type inactivation first described for Drosophila Shaker in which 1) inactivation balls on the N termini of each subunit can independently block the pore, and 2) only one inactivation ball occupies the pore binding site at a time. These findings suggest classical N-type activation evolved in Shaker channels at the very base of the animal phylogeny in a common ancestor of ctenophores, cnidarians, and bilaterians and that fast-inactivating Shakers are therefore a fundamental type of animal K+ channel. Interestingly, we find evidence from functional co-expression experiments and molecular dynamics that MlShak4 and MlShak5 do not co-assemble, suggesting that Mnemiopsis has at least two functionally independent N-type Shaker channels.
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Affiliation(s)
- Benjamin T Simonson
- Department of Biology and Huck Institutes of the Life Sciences, Penn State University, University Park, Pennsylvania
| | - Max Jegla
- Department of Biology and Huck Institutes of the Life Sciences, Penn State University, University Park, Pennsylvania
| | - Joseph F Ryan
- Whitney Laboratory for Marine Bioscience, University of Florida, St. Augustine, FL; Department of Biology, University of Florida, Gainesville, FL
| | - Timothy Jegla
- Department of Biology and Huck Institutes of the Life Sciences, Penn State University, University Park, Pennsylvania.
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Kalm T, Schob C, Völler H, Gardeitchik T, Gilissen C, Pfundt R, Klöckner C, Platzer K, Klabunde-Cherwon A, Ries M, Syrbe S, Beccaria F, Madia F, Scala M, Zara F, Hofstede F, Simon MEH, van Jaarsveld RH, Oegema R, van Gassen KLI, Holwerda SJB, Barakat TS, Bouman A, van Slegtenhorst M, Álvarez S, Fernández-Jaén A, Porta J, Accogli A, Mancardi MM, Striano P, Iacomino M, Chae JH, Jang S, Kim SY, Chitayat D, Mercimek-Andrews S, Depienne C, Kampmeier A, Kuechler A, Surowy H, Bertini ES, Radio FC, Mancini C, Pizzi S, Tartaglia M, Gauthier L, Genevieve D, Tharreau M, Azoulay N, Zaks-Hoffer G, Gilad NK, Orenstein N, Bernard G, Thiffault I, Denecke J, Herget T, Kortüm F, Kubisch C, Bähring R, Kindler S. Etiological involvement of KCND1 variants in an X-linked neurodevelopmental disorder with variable expressivity. Am J Hum Genet 2024; 111:1206-1221. [PMID: 38772379 PMCID: PMC11179411 DOI: 10.1016/j.ajhg.2024.04.019] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2023] [Revised: 04/25/2024] [Accepted: 04/25/2024] [Indexed: 05/23/2024] Open
Abstract
Utilizing trio whole-exome sequencing and a gene matching approach, we identified a cohort of 18 male individuals from 17 families with hemizygous variants in KCND1, including two de novo missense variants, three maternally inherited protein-truncating variants, and 12 maternally inherited missense variants. Affected subjects present with a neurodevelopmental disorder characterized by diverse neurological abnormalities, mostly delays in different developmental domains, but also distinct neuropsychiatric signs and epilepsy. Heterozygous carrier mothers are clinically unaffected. KCND1 encodes the α-subunit of Kv4.1 voltage-gated potassium channels. All variant-associated amino acid substitutions affect either the cytoplasmic N- or C-terminus of the channel protein except for two occurring in transmembrane segments 1 and 4. Kv4.1 channels were functionally characterized in the absence and presence of auxiliary β subunits. Variant-specific alterations of biophysical channel properties were diverse and varied in magnitude. Genetic data analysis in combination with our functional assessment shows that Kv4.1 channel dysfunction is involved in the pathogenesis of an X-linked neurodevelopmental disorder frequently associated with a variable neuropsychiatric clinical phenotype.
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Affiliation(s)
- Tassja Kalm
- Institute for Cellular and Integrative Physiology, Center for Experimental Medicine, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany
| | - Claudia Schob
- Institute of Human Genetics, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany
| | - Hanna Völler
- Institute of Human Genetics, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany
| | - Thatjana Gardeitchik
- Department of Human Genetics, Radboud University Medical Center, Nijmegen 6525 GA, the Netherlands
| | - Christian Gilissen
- Department of Human Genetics, Radboud University Medical Center, Nijmegen 6525 GA, the Netherlands
| | - Rolph Pfundt
- Department of Human Genetics, Radboud University Medical Center, Nijmegen 6525 GA, the Netherlands
| | - Chiara Klöckner
- Institute of Human Genetics, University of Leipzig Medical Center, 04103 Leipzig, Germany
| | - Konrad Platzer
- Institute of Human Genetics, University of Leipzig Medical Center, 04103 Leipzig, Germany
| | - Annick Klabunde-Cherwon
- Division of Pediatric Epileptology, Centre for Paediatric and Adolescent Medicine, University Hospital Heidelberg, 69120 Heidelberg, Germany
| | - Markus Ries
- Division of Pediatric Epileptology, Centre for Paediatric and Adolescent Medicine, University Hospital Heidelberg, 69120 Heidelberg, Germany
| | - Steffen Syrbe
- Division of Pediatric Epileptology, Centre for Paediatric and Adolescent Medicine, University Hospital Heidelberg, 69120 Heidelberg, Germany
| | - Francesca Beccaria
- Epilepsy Center, Department of Child Neuropsychiatry, Territorial Social-Health Agency, 46100 Mantova, Italy
| | - Francesca Madia
- Medical Genetics Unit, IRCCS Istituto Giannina Gaslini, 16147 Genoa, Italy
| | - Marcello Scala
- Medical Genetics Unit, IRCCS Istituto Giannina Gaslini, 16147 Genoa, Italy; Department of Neurosciences, Rehabilitation, Ophthalmology, Genetics, Maternal and Child Health, University of Genoa, 16145 Genoa, Italy
| | - Federico Zara
- Medical Genetics Unit, IRCCS Istituto Giannina Gaslini, 16147 Genoa, Italy; Department of Neurosciences, Rehabilitation, Ophthalmology, Genetics, Maternal and Child Health, University of Genoa, 16145 Genoa, Italy
| | - Floris Hofstede
- Department of General Pediatrics, Wilhelmina Children's Hospital, University Medical Centre Utrecht, Utrecht, the Netherlands
| | - Marleen E H Simon
- Department of Clinical Genetics, University Medical Center Utrecht, Utrecht 3584 EA, the Netherlands
| | - Richard H van Jaarsveld
- Department of Clinical Genetics, University Medical Center Utrecht, Utrecht 3584 EA, the Netherlands
| | - Renske Oegema
- Department of Clinical Genetics, University Medical Center Utrecht, Utrecht 3584 EA, the Netherlands
| | - Koen L I van Gassen
- Department of Clinical Genetics, University Medical Center Utrecht, Utrecht 3584 EA, the Netherlands
| | - Sjoerd J B Holwerda
- Department of Clinical Genetics, University Medical Center Utrecht, Utrecht 3584 EA, the Netherlands
| | - Tahsin Stefan Barakat
- Department of Clinical Genetics, Erasmus MC University Medical Center, Rotterdam 3000 CA, the Netherlands; ENCORE Expertise Center for Neurodevelopmental Disorders, Erasmus MC University Medical Center, Rotterdam 3000 CA, the Netherlands; Discovery Unit, Department of Clinical Genetics, Erasmus MC University Medical Center, Rotterdam 3000 CA, the Netherlands
| | - Arjan Bouman
- Department of Clinical Genetics, Erasmus MC University Medical Center, Rotterdam 3000 CA, the Netherlands
| | - Marjon van Slegtenhorst
- Department of Clinical Genetics, Erasmus MC University Medical Center, Rotterdam 3000 CA, the Netherlands
| | - Sara Álvarez
- Genomics and Medicine, NIMGenetics, 28108 Madrid, Spain
| | - Alberto Fernández-Jaén
- Pediatric Neurology Department, Quironsalud University Hospital Madrid, School of Medicine, European University of Madrid, 28224 Madrid, Spain
| | - Javier Porta
- Genomics, Genologica Medica, 29016 Málaga, Spain
| | - Andrea Accogli
- Division of Medical Genetics, Department of Specialized Medicine, Montreal Children's Hospital, McGill University Health Centre, QC H4A 3J1 Montreal, Canada; Department of Human Genetics, McGill University, QC H4A 3J1 Montreal, Canada
| | | | - Pasquale Striano
- Department of Neurosciences, Rehabilitation, Ophthalmology, Genetics, Maternal and Child Health, University of Genoa, 16145 Genoa, Italy; Pediatric Neurology and Neuromuscular Diseases Unit, IRCCS Istituto Giannina Gaslini, 16147 Genoa, Italy
| | - Michele Iacomino
- Medical Genetics Unit, IRCCS Istituto Giannina Gaslini, 16147 Genoa, Italy
| | - Jong-Hee Chae
- Department of Pediatrics, Seoul National University College of Medicine, Seoul 110-744, Republic of Korea; Department of Genomic Medicine, Rare Disease Center, Seoul National University Hospital, Seoul 03080, Republic of Korea
| | - SeSong Jang
- Department of Pediatrics, Seoul National University College of Medicine, Seoul 110-744, Republic of Korea
| | - Soo Y Kim
- Department of Genomic Medicine, Rare Disease Center, Seoul National University Hospital, Seoul 03080, Republic of Korea
| | - David Chitayat
- The Prenatal Diagnosis and Medical Genetics Program, Department of Obstetrics and Gynecology, Mount Sinai Hospital, University of Toronto ON M5G 1E2 Toronto, Canada; Division of Clinical and Metabolic Genetics, Department of Pediatrics, The Hospital for SickKids, University of Toronto, M5G 1X8 Toronto, Canada
| | - Saadet Mercimek-Andrews
- Division of Clinical and Metabolic Genetics, Department of Pediatrics, The Hospital for SickKids, University of Toronto, M5G 1X8 Toronto, Canada; Department of Medical Genetics, Faculty of Medicine and Dentistry, University of Alberta, AB T6G 2H7 Edmonton, Canada
| | - Christel Depienne
- Institute of Human Genetics, University Hospital Essen, University Duisburg-Essen, 45122 Essen, Germany
| | - Antje Kampmeier
- Institute of Human Genetics, University Hospital Essen, University Duisburg-Essen, 45122 Essen, Germany
| | - Alma Kuechler
- Institute of Human Genetics, University Hospital Essen, University Duisburg-Essen, 45122 Essen, Germany
| | - Harald Surowy
- Institute of Human Genetics, University Hospital Essen, University Duisburg-Essen, 45122 Essen, Germany
| | | | | | - Cecilia Mancini
- Molecular Genetics and Functional Genomics, Bambino Gesù Children's Hospital, IRCCS, 00146 Rome, Italy
| | - Simone Pizzi
- Molecular Genetics and Functional Genomics, Bambino Gesù Children's Hospital, IRCCS, 00146 Rome, Italy
| | - Marco Tartaglia
- Molecular Genetics and Functional Genomics, Bambino Gesù Children's Hospital, IRCCS, 00146 Rome, Italy
| | - Lucas Gauthier
- Department of Molecular Genetics and Cytogenomics, Rare and Autoinflammatory Diseases Unit, University Hospital of Montpellier, 34295 Montpellier, France
| | - David Genevieve
- Montpellier University, Inserm U1183, Montpellier, France; Department of Clinical Genetics, University Hospital of Montpellier, 34295 Montpellier, France
| | - Mylène Tharreau
- Department of Molecular Genetics and Cytogenomics, Rare and Autoinflammatory Diseases Unit, University Hospital of Montpellier, 34295 Montpellier, France
| | - Noy Azoulay
- The Genetic Institute of Maccabi Health Services, Rehovot 7610000, Israel; Raphael Recanati Genetics Institute, Beilinson Hospital, Rabin Medical Center, Petach Tikva 49100, Israel
| | - Gal Zaks-Hoffer
- Raphael Recanati Genetics Institute, Beilinson Hospital, Rabin Medical Center, Petach Tikva 49100, Israel; Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 69978, Israel
| | - Nesia K Gilad
- Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 69978, Israel; Pediatric Genetics Unit, Schneider Children's Medical Center of Israel, Petah Tikvah 4920235, Israel
| | - Naama Orenstein
- Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 69978, Israel; Pediatric Genetics Unit, Schneider Children's Medical Center of Israel, Petah Tikvah 4920235, Israel
| | - Geneviève Bernard
- Division of Medical Genetics, Department of Specialized Medicine, Montreal Children's Hospital, McGill University Health Centre, QC H4A 3J1 Montreal, Canada; Departments of Neurology and Neurosurgery, Pediatrics and Human Genetics, McGill University, Montreal, Canada; Child Health and Human Development Program, Research Institute of the McGill University Health Centre, Montreal, Canada
| | - Isabelle Thiffault
- Genomic Medicine Center, Department of Pediatrics, Children's Mercy Kansas City, Kansas City, MO, USA; UKMC School of Medicine, University of Missouri Kansas City, Kansas City, MO, USA; Department of Pathology and Laboratory Medicine, Children's Mercy Kansas City, Kansas City, MO, USA
| | - Jonas Denecke
- Department of Pediatrics, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany
| | - Theresia Herget
- Institute of Human Genetics, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany
| | - Fanny Kortüm
- Institute of Human Genetics, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany
| | - Christian Kubisch
- Institute of Human Genetics, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany
| | - Robert Bähring
- Institute for Cellular and Integrative Physiology, Center for Experimental Medicine, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany.
| | - Stefan Kindler
- Institute of Human Genetics, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany.
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7
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Chen YT, Hong MR, Zhang XJ, Kostas J, Li Y, Kraus RL, Santarelli VP, Wang D, Gomez-Llorente Y, Brooun A, Strickland C, Soisson SM, Klein DJ, Ginnetti AT, Marino MJ, Stachel SJ, Ishchenko A. Identification, structural, and biophysical characterization of a positive modulator of human Kv3.1 channels. Proc Natl Acad Sci U S A 2023; 120:e2220029120. [PMID: 37812700 PMCID: PMC10589703 DOI: 10.1073/pnas.2220029120] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/23/2022] [Accepted: 08/16/2023] [Indexed: 10/11/2023] Open
Abstract
Voltage-gated potassium channels (Kv) are tetrameric membrane proteins that provide a highly selective pathway for potassium ions (K+) to diffuse across a hydrophobic cell membrane. These unique voltage-gated cation channels detect changes in membrane potential and, upon activation, help to return the depolarized cell to a resting state during the repolarization stage of each action potential. The Kv3 family of potassium channels is characterized by a high activation potential and rapid kinetics, which play a crucial role for the fast-spiking neuronal phenotype. Mutations in the Kv3.1 channel have been shown to have implications in various neurological diseases like epilepsy and Alzheimer's disease. Moreover, disruptions in neuronal circuitry involving Kv3.1 have been correlated with negative symptoms of schizophrenia. Here, we report the discovery of a novel positive modulator of Kv3.1, investigate its biophysical properties, and determine the cryo-EM structure of the compound in complex with Kv3.1. Structural analysis reveals the molecular determinants of positive modulation in Kv3.1 channels by this class of compounds and provides additional opportunities for rational drug design for the treatment of associated neurological disorders.
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Affiliation(s)
- Yun-Ting Chen
- Computational and Structural Chemistry, Merck & Co., Inc., Kenilworth, NJ07033
| | - Mee Ra Hong
- Computational and Structural Chemistry, Merck & Co., Inc., West Point, PA19486
| | - Xin-Jun Zhang
- Department of Neuroscience, Merck & Co., Inc., West Point, PA19486
| | - James Kostas
- Computational and Structural Chemistry, Merck & Co., Inc., West Point, PA19486
| | - Yuxing Li
- Department of Neuroscience, Merck & Co., Inc., West Point, PA19486
| | - Richard L. Kraus
- Department of Neuroscience, Merck & Co., Inc., West Point, PA19486
| | | | - Deping Wang
- Computational and Structural Chemistry, Merck & Co., Inc., West Point, PA19486
| | | | - Alexei Brooun
- Computational and Structural Chemistry, Merck & Co., Inc., West Point, PA19486
| | - Corey Strickland
- Computational and Structural Chemistry, Merck & Co., Inc., Kenilworth, NJ07033
| | - Stephen M. Soisson
- Computational and Structural Chemistry, Merck & Co., Inc., West Point, PA19486
| | - Daniel J. Klein
- Computational and Structural Chemistry, Merck & Co., Inc., West Point, PA19486
| | | | | | | | - Andrii Ishchenko
- Computational and Structural Chemistry, Merck & Co., Inc., West Point, PA19486
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8
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Chi G, Liang Q, Sridhar A, Cowgill JB, Sader K, Radjainia M, Qian P, Castro-Hartmann P, Venkaya S, Singh NK, McKinley G, Fernandez-Cid A, Mukhopadhyay SMM, Burgess-Brown NA, Delemotte L, Covarrubias M, Dürr KL. Cryo-EM structure of the human Kv3.1 channel reveals gating control by the cytoplasmic T1 domain. Nat Commun 2022; 13:4087. [PMID: 35840580 PMCID: PMC9287412 DOI: 10.1038/s41467-022-29594-w] [Citation(s) in RCA: 15] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2021] [Accepted: 03/24/2022] [Indexed: 11/26/2022] Open
Abstract
Kv3 channels have distinctive gating kinetics tailored for rapid repolarization in fast-spiking neurons. Malfunction of this process due to genetic variants in the KCNC1 gene causes severe epileptic disorders, yet the structural determinants for the unusual gating properties remain elusive. Here, we present cryo-electron microscopy structures of the human Kv3.1a channel, revealing a unique arrangement of the cytoplasmic tetramerization domain T1 which facilitates interactions with C-terminal axonal targeting motif and key components of the gating machinery. Additional interactions between S1/S2 linker and turret domain strengthen the interface between voltage sensor and pore domain. Supported by molecular dynamics simulations, electrophysiological and mutational analyses, we identify several residues in the S4/S5 linker which influence the gating kinetics and an electrostatic interaction between acidic residues in α6 of T1 and R449 in the pore-flanking S6T helices. These findings provide insights into gating control and disease mechanisms and may guide strategies for the design of pharmaceutical drugs targeting Kv3 channels.
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Affiliation(s)
- Gamma Chi
- Centre for Medicines Discovery, Nuffield Department of Medicine, University of Oxford, Roosevelt Drive, Oxford, OX3 7DQ, UK
- Structural Genomics Consortium, Nuffield Department of Medicine, University of Oxford, Roosevelt Drive, Oxford, OX3 7DQ, UK
| | - Qiansheng Liang
- Department of Neuroscience and Vickie and Jack Farber Institute for Neuroscience, Sidney Kimmel Medical College at Thomas Jefferson University, Philadelphia, PA, 19107, UK
| | - Akshay Sridhar
- Department of Applied Physics, Science for Life Laboratory, KTH, Solna, Sweden
| | - John B Cowgill
- Department of Applied Physics, Science for Life Laboratory, KTH, Solna, Sweden
| | - Kasim Sader
- Materials and Structural Analysis, Thermo Fisher Scientific, Achtseweg Noord 5, 5651 GG, Eindhoven, Netherlands
| | - Mazdak Radjainia
- Materials and Structural Analysis, Thermo Fisher Scientific, Achtseweg Noord 5, 5651 GG, Eindhoven, Netherlands
| | - Pu Qian
- Materials and Structural Analysis, Thermo Fisher Scientific, Achtseweg Noord 5, 5651 GG, Eindhoven, Netherlands
| | - Pablo Castro-Hartmann
- Materials and Structural Analysis, Thermo Fisher Scientific, Achtseweg Noord 5, 5651 GG, Eindhoven, Netherlands
| | - Shayla Venkaya
- Centre for Medicines Discovery, Nuffield Department of Medicine, University of Oxford, Roosevelt Drive, Oxford, OX3 7DQ, UK
- Structural Genomics Consortium, Nuffield Department of Medicine, University of Oxford, Roosevelt Drive, Oxford, OX3 7DQ, UK
- Exscientia Ltd., The Schrödinger Building, Heatley Road, The Oxford Science Park, Oxford, OX4 4GE, UK
| | - Nanki Kaur Singh
- Centre for Medicines Discovery, Nuffield Department of Medicine, University of Oxford, Roosevelt Drive, Oxford, OX3 7DQ, UK
- Structural Genomics Consortium, Nuffield Department of Medicine, University of Oxford, Roosevelt Drive, Oxford, OX3 7DQ, UK
| | - Gavin McKinley
- Centre for Medicines Discovery, Nuffield Department of Medicine, University of Oxford, Roosevelt Drive, Oxford, OX3 7DQ, UK
- Structural Genomics Consortium, Nuffield Department of Medicine, University of Oxford, Roosevelt Drive, Oxford, OX3 7DQ, UK
| | - Alejandra Fernandez-Cid
- Centre for Medicines Discovery, Nuffield Department of Medicine, University of Oxford, Roosevelt Drive, Oxford, OX3 7DQ, UK
- Structural Genomics Consortium, Nuffield Department of Medicine, University of Oxford, Roosevelt Drive, Oxford, OX3 7DQ, UK
- Exact Sciences Ltd., The Sherard Building, Edmund Halley Road, The Oxford Science Park, Oxford, OX4 4DQ, UK
| | - Shubhashish M M Mukhopadhyay
- Centre for Medicines Discovery, Nuffield Department of Medicine, University of Oxford, Roosevelt Drive, Oxford, OX3 7DQ, UK
- Structural Genomics Consortium, Nuffield Department of Medicine, University of Oxford, Roosevelt Drive, Oxford, OX3 7DQ, UK
- Exscientia Ltd., The Schrödinger Building, Heatley Road, The Oxford Science Park, Oxford, OX4 4GE, UK
| | - Nicola A Burgess-Brown
- Centre for Medicines Discovery, Nuffield Department of Medicine, University of Oxford, Roosevelt Drive, Oxford, OX3 7DQ, UK
- Structural Genomics Consortium, Nuffield Department of Medicine, University of Oxford, Roosevelt Drive, Oxford, OX3 7DQ, UK
- Exact Sciences Ltd., The Sherard Building, Edmund Halley Road, The Oxford Science Park, Oxford, OX4 4DQ, UK
| | - Lucie Delemotte
- Department of Applied Physics, Science for Life Laboratory, KTH, Solna, Sweden
| | - Manuel Covarrubias
- Department of Neuroscience and Vickie and Jack Farber Institute for Neuroscience, Sidney Kimmel Medical College at Thomas Jefferson University, Philadelphia, PA, 19107, UK
| | - Katharina L Dürr
- Centre for Medicines Discovery, Nuffield Department of Medicine, University of Oxford, Roosevelt Drive, Oxford, OX3 7DQ, UK.
- Structural Genomics Consortium, Nuffield Department of Medicine, University of Oxford, Roosevelt Drive, Oxford, OX3 7DQ, UK.
- OMass Therapeutics, Ltd., The Schrödinger Building, Heatley Road, The Oxford Science Park, Oxford, OX4 4GE, UK.
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9
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Xu Z, Khan S, Schnicker NJ, Baker S. Pentameric assembly of the Kv2.1 tetramerization domain. Acta Crystallogr D Struct Biol 2022; 78:792-802. [PMID: 35647925 PMCID: PMC9159280 DOI: 10.1107/s205979832200568x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2021] [Accepted: 05/25/2022] [Indexed: 11/25/2022] Open
Abstract
The Kv family of voltage-gated potassium channels regulate neuronal excitability. The biophysical characteristics of Kv channels can be matched to the needs of different neurons by forming homotetrameric or heterotetrameric channels within one of four subfamilies. The cytoplasmic tetramerization (T1) domain plays a major role in dictating the compatibility of different Kv subunits. The only Kv subfamily lacking a representative structure of the T1 domain is the Kv2 family. Here, X-ray crystallography was used to solve the structure of the human Kv2.1 T1 domain. The structure is similar to those of other T1 domains, but surprisingly formed a pentamer instead of a tetramer. In solution the Kv2.1 T1 domain also formed a pentamer, as determined by inline SEC-MALS-SAXS and negative-stain electron microscopy. The Kv2.1 T1-T1 interface involves electrostatic interactions, including a salt bridge formed by the negative charges in a previously described CDD motif, and inter-subunit coordination of zinc. It is shown that zinc binding is important for stability. In conclusion, the Kv2.1 T1 domain behaves differently from the other Kv T1 domains, which may reflect the versatility of Kv2.1, which can assemble with the regulatory KvS subunits and scaffold ER-plasma membrane contacts.
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Affiliation(s)
- Zhen Xu
- Protein and Crystallography Facility, University of Iowa, 51 Newton Road, Iowa City, IA 52242, USA
| | - Saif Khan
- Protein and Crystallography Facility, University of Iowa, 51 Newton Road, Iowa City, IA 52242, USA
| | - Nicholas J. Schnicker
- Protein and Crystallography Facility, University of Iowa, 51 Newton Road, Iowa City, IA 52242, USA
| | - Sheila Baker
- Department of Biochemistry and Molecular Biology, University of Iowa, 51 Newton Road, Iowa City, IA 52242, USA
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10
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Li J, Maghera J, Lamothe SM, Marco EJ, Kurata HT. Heteromeric Assembly of Truncated Neuronal Kv7 Channels: Implications for Neurologic Disease and Pharmacotherapy. Mol Pharmacol 2020; 98:192-202. [PMID: 32580997 DOI: 10.1124/mol.120.119644] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/04/2020] [Accepted: 06/11/2020] [Indexed: 02/14/2025] Open
Abstract
Neuronal voltage-gated potassium channels (Kv) are critical regulators of electrical activity in the central nervous system. Mutations in the KCNQ (Kv7) ion channel family are linked to epilepsy and neurodevelopmental disorders. These channels underlie the neuronal "M-current" and cluster in the axon initial segment to regulate the firing of action potentials. There is general consensus that KCNQ channel assembly and heteromerization are controlled by C-terminal helices. We identified a pediatric patient with neurodevelopmental disability, including autism traits, inattention and hyperactivity, and ataxia, who carries a de novo frameshift mutation in KCNQ3 (KCNQ3-FS534), leading to truncation of ∼300 amino acids in the C terminus. We investigated possible molecular mechanisms of channel dysfunction, including haplo-insufficiency or a dominant-negative effect caused by the assembly of truncated KCNQ3 and functional KCNQ2 subunits. We also used a recently recognized property of the KCNQ2-specific activator ICA-069673 to identify assembly of heteromeric channels. ICA-069673 exhibits a functional signature that depends on the subunit composition of KCNQ2/3 channels, allowing us to determine whether truncated KCNQ3 subunits can assemble with KCNQ2. Our findings demonstrate that although the KCNQ3-FS534 mutant does not generate functional channels on its own, large C-terminal truncations of KCNQ3 (including the KCNQ3-FS534 mutation) assemble efficiently with KCNQ2 but fail to promote or stabilize KCNQ2/KCNQ3 heteromeric channel expression. Therefore, the frequent assumption that pathologies linked to KCNQ3 truncations arise from haplo-insufficiency should be reconsidered in some cases. Subtype-specific channel activators like ICA-069673 are a reliable tool to identify heteromeric assembly of KCNQ2 and KCNQ3. SIGNIFICANCE STATEMENT: Mutations that truncate the C terminus of neuronal Kv7/KCNQ channels are linked to a spectrum of seizure disorders. One role of the multifunctional KCNQ C terminus is to mediate subtype-specific assembly of heteromeric KCNQ channels. This study describes the use of a subtype-specific Kv7 activator to assess assembly of heteromeric KCNQ2/KCNQ3 (Kv7.2/Kv7.3) channels and demonstrates that large disease-linked and experimentally generated C-terminal truncated KCNQ3 mutants retain the ability to assemble with KCNQ2.
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Affiliation(s)
- Jingru Li
- Department of Pharmacology, Alberta Diabetes Institute, University of Alberta, Edmonton, Alberta, Canada (J.L., J.M., S.M.L., H.T.K.) and Department of Neurodevelopmental Medicine, Cortica Healthcare, San Rafael, California (E.J.M.)
| | - Jasmine Maghera
- Department of Pharmacology, Alberta Diabetes Institute, University of Alberta, Edmonton, Alberta, Canada (J.L., J.M., S.M.L., H.T.K.) and Department of Neurodevelopmental Medicine, Cortica Healthcare, San Rafael, California (E.J.M.)
| | - Shawn M Lamothe
- Department of Pharmacology, Alberta Diabetes Institute, University of Alberta, Edmonton, Alberta, Canada (J.L., J.M., S.M.L., H.T.K.) and Department of Neurodevelopmental Medicine, Cortica Healthcare, San Rafael, California (E.J.M.)
| | - Elysa J Marco
- Department of Pharmacology, Alberta Diabetes Institute, University of Alberta, Edmonton, Alberta, Canada (J.L., J.M., S.M.L., H.T.K.) and Department of Neurodevelopmental Medicine, Cortica Healthcare, San Rafael, California (E.J.M.)
| | - Harley T Kurata
- Department of Pharmacology, Alberta Diabetes Institute, University of Alberta, Edmonton, Alberta, Canada (J.L., J.M., S.M.L., H.T.K.) and Department of Neurodevelopmental Medicine, Cortica Healthcare, San Rafael, California (E.J.M.)
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11
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The Structural Versatility of the BTB Domains of KCTD Proteins and Their Recognition of the GABA B Receptor. Biomolecules 2019; 9:biom9080323. [PMID: 31370201 PMCID: PMC6722564 DOI: 10.3390/biom9080323] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/21/2019] [Revised: 07/29/2019] [Accepted: 07/30/2019] [Indexed: 02/07/2023] Open
Abstract
Several recent investigations have demonstrated that members of the KCTD (Potassium Channel Tetramerization Domain) protein family are involved in fundamental processes. However, the paucity of structural data available on these proteins has frequently prevented the definition of their biochemical role(s). Fortunately, this scenario is rapidly changing as, in very recent years, several crystallographic structures have been reported. Although these investigations have provided very important insights into the function of KCTDs, they have also raised some puzzling issues. One is related to the observation that the BTB (broad-complex, tramtrack, and bric-à-brac) domain of these proteins presents a remarkable structural versatility, being able to adopt a variety of oligomeric states. To gain insights into this intriguing aspect, we performed extensive molecular dynamics simulations on several BTB domains of KCTD proteins in different oligomeric states (monomers, dimers, tetramers, and open/close pentamers). These studies indicate that KCTD-BTB domains are stable in the simulation timescales, even in their monomeric forms. Moreover, simulations also show that the dynamic behavior of open pentameric states is strictly related to their functional roles and that different KCTDs may form stable hetero-oligomers. Molecular dynamics (MD) simulations also provided a dynamic view of the complex formed by KCTD16 and the GABAB2 receptor, whose structure has been recently reported. Finally, simulations carried out on the isolated fragment of the GABAB2 receptor that binds KCTD16 indicate that it is able to assume the local conformation required for the binding to KCTD.
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12
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Structural basis for auxiliary subunit KCTD16 regulation of the GABA B receptor. Proc Natl Acad Sci U S A 2019; 116:8370-8379. [PMID: 30971491 DOI: 10.1073/pnas.1903024116] [Citation(s) in RCA: 37] [Impact Index Per Article: 6.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022] Open
Abstract
Metabotropic GABAB receptors mediate a significant fraction of inhibitory neurotransmission in the brain. Native GABAB receptor complexes contain the principal subunits GABAB1 and GABAB2, which form an obligate heterodimer, and auxiliary subunits, known as potassium channel tetramerization domain-containing proteins (KCTDs). KCTDs interact with GABAB receptors and modify the kinetics of GABAB receptor signaling. Little is known about the molecular mechanism governing the direct association and functional coupling of GABAB receptors with these auxiliary proteins. Here, we describe the high-resolution structure of the KCTD16 oligomerization domain in complex with part of the GABAB2 receptor. A single GABAB2 C-terminal peptide is bound to the interior of an open pentamer formed by the oligomerization domain of five KCTD16 subunits. Mutation of specific amino acids identified in the structure of the GABAB2-KCTD16 interface disrupted both the biochemical association and functional modulation of GABAB receptors and G protein-activated inwardly rectifying K+ channel (GIRK) channels. These interfacial residues are conserved among KCTDs, suggesting a common mode of KCTD interaction with GABAB receptors. Defining the binding interface of GABAB receptor and KCTD reveals a potential regulatory site for modulating GABAB-receptor function in the brain.
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13
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Kaczmarek LK, Zhang Y. Kv3 Channels: Enablers of Rapid Firing, Neurotransmitter Release, and Neuronal Endurance. Physiol Rev 2017; 97:1431-1468. [PMID: 28904001 PMCID: PMC6151494 DOI: 10.1152/physrev.00002.2017] [Citation(s) in RCA: 127] [Impact Index Per Article: 15.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/24/2017] [Revised: 04/24/2017] [Accepted: 05/05/2017] [Indexed: 12/11/2022] Open
Abstract
The intrinsic electrical characteristics of different types of neurons are shaped by the K+ channels they express. From among the more than 70 different K+ channel genes expressed in neurons, Kv3 family voltage-dependent K+ channels are uniquely associated with the ability of certain neurons to fire action potentials and to release neurotransmitter at high rates of up to 1,000 Hz. In general, the four Kv3 channels Kv3.1-Kv3.4 share the property of activating and deactivating rapidly at potentials more positive than other channels. Each Kv3 channel gene can generate multiple protein isoforms, which contribute to the high-frequency firing of neurons such as auditory brain stem neurons, fast-spiking GABAergic interneurons, and Purkinje cells of the cerebellum, and to regulation of neurotransmitter release at the terminals of many neurons. The different Kv3 channels have unique expression patterns and biophysical properties and are regulated in different ways by protein kinases. In this review, we cover the function, localization, and modulation of Kv3 channels and describe how levels and properties of the channels are altered by changes in ongoing neuronal activity. We also cover how the protein-protein interaction of these channels with other proteins affects neuronal functions, and how mutations or abnormal regulation of Kv3 channels are associated with neurological disorders such as ataxias, epilepsies, schizophrenia, and Alzheimer's disease.
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Affiliation(s)
- Leonard K Kaczmarek
- Departments of Pharmacology and of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut
| | - Yalan Zhang
- Departments of Pharmacology and of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut
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14
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Fuchs JR, Darlington SW, Green JT, Morielli AD. Cerebellar learning modulates surface expression of a voltage-gated ion channel in cerebellar cortex. Neurobiol Learn Mem 2017; 142:252-262. [PMID: 28512010 DOI: 10.1016/j.nlm.2017.05.009] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/10/2017] [Revised: 05/05/2017] [Accepted: 05/12/2017] [Indexed: 11/30/2022]
Abstract
Numerous experiments using ex vivo electrophysiology suggest that mammalian learning and memory involves regulation of voltage-gated ion channels in terms of changes in function. Yet, little is known about learning-related regulation of voltage-gated ion channels in terms of changes in expression. In two experiments, we examined changes in cell surface expression of the voltage-gated potassium channel alpha-subunit Kv1.2 in a discrete region of cerebellar cortex after eyeblink conditioning (EBC), a well-studied form of cerebellar-dependent learning. Kv1.2 in cerebellar cortex is expressed almost entirely in basket cells, primarily in the axon terminal pinceaux (PCX) region, and Purkinje cells, primarily in dendrites. Cell surface expression of Kv1.2 was measured using both multiphoton microscopy, which allowed measurement confined to the PCX region, and biotinylation/western blot, which measured total cell surface expression. In the first experiment, rats underwent three sessions of EBC, explicitly unpaired stimulus exposure, or context-only exposure and the results revealed a decrease in Kv1.2 cell surface expression in the unpaired group as measured with microscopy but no change as measured with western blot. In the second experiment, the same three training groups underwent only one half of a session of training, and the results revealed an increase in Kv1.2 cell surface expression in the unpaired group as measured with western blot but no change as measured with microscopy. In addition, rats in the EBC group that did not express conditioned responses (CRs) exhibited the same increase in Kv1.2 cell surface expression as the unpaired group. The overall pattern of results suggests that cell surface expression of Kv1.2 is changed with exposure to EBC stimuli in the absence, or prior to the emergence, of CRs.
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Affiliation(s)
- Jason R Fuchs
- Department of Psychological Science, University of Vermont, Burlington, VT 05405, United States
| | - Shelby W Darlington
- Department of Psychological Science, University of Vermont, Burlington, VT 05405, United States
| | - John T Green
- Department of Psychological Science, University of Vermont, Burlington, VT 05405, United States
| | - Anthony D Morielli
- Department of Pharmacology, University of Vermont, Burlington, VT 05405, United States.
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15
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Kochańczyk T, Nowakowski M, Wojewska D, Kocyła A, Ejchart A, Koźmiński W, Krężel A. Metal-coupled folding as the driving force for the extreme stability of Rad50 zinc hook dimer assembly. Sci Rep 2016; 6:36346. [PMID: 27808280 PMCID: PMC5093744 DOI: 10.1038/srep36346] [Citation(s) in RCA: 33] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2016] [Accepted: 10/14/2016] [Indexed: 01/26/2023] Open
Abstract
The binding of metal ions at the interface of protein complexes presents a unique and poorly understood mechanism of molecular assembly. A remarkable example is the Rad50 zinc hook domain, which is highly conserved and facilitates the Zn2+-mediated homodimerization of Rad50 proteins. Here, we present a detailed analysis of the structural and thermodynamic effects governing the formation and stability (logK12 = 20.74) of this evolutionarily conserved protein assembly. We have dissected the determinants of the stability contributed by the small β-hairpin of the domain surrounding the zinc binding motif and the coiled-coiled regions using peptides of various lengths from 4 to 45 amino acid residues, alanine substitutions and peptide bond-to-ester perturbations. In the studied series of peptides, an >650 000-fold increase of the formation constant of the dimeric complex arises from favorable enthalpy because of the increased acidity of the cysteine thiols in metal-free form and the structural properties of the dimer. The dependence of the enthalpy on the domain fragment length is partially compensated by the entropic penalty of domain folding, indicating enthalpy-entropy compensation. This study facilitates understanding of the metal-mediated protein-protein interactions in which the metal ion is critical for the tight association of protein subunits.
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Affiliation(s)
- Tomasz Kochańczyk
- Department of Chemical Biology, Faculty of Biotechnology, University of Wrocław, Joliot-Curie 14a, 50-383 Wrocław, Poland
| | - Michał Nowakowski
- Biological and Chemical Research Center, Faculty of Chemistry, University of Warsaw, Żwirki i Wigury 101, 02-089 Warsaw, Poland
| | - Dominika Wojewska
- Department of Chemical Biology, Faculty of Biotechnology, University of Wrocław, Joliot-Curie 14a, 50-383 Wrocław, Poland
| | - Anna Kocyła
- Department of Chemical Biology, Faculty of Biotechnology, University of Wrocław, Joliot-Curie 14a, 50-383 Wrocław, Poland
| | - Andrzej Ejchart
- Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02-106, Warsaw, Poland
| | - Wiktor Koźmiński
- Biological and Chemical Research Center, Faculty of Chemistry, University of Warsaw, Żwirki i Wigury 101, 02-089 Warsaw, Poland
| | - Artur Krężel
- Department of Chemical Biology, Faculty of Biotechnology, University of Wrocław, Joliot-Curie 14a, 50-383 Wrocław, Poland
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16
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Moen MN, Fjær R, Hamdani EH, Laerdahl JK, Menchini RJ, Vigeland MD, Sheng Y, Undlien DE, Hassel B, Salih MA, El Khashab HY, Selmer KK, Chaudhry FA. Pathogenic variants in KCTD7 perturb neuronal K+ fluxes and glutamine transport. Brain 2016; 139:3109-3120. [PMID: 27742667 DOI: 10.1093/brain/aww244] [Citation(s) in RCA: 28] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/23/2015] [Revised: 06/11/2016] [Accepted: 08/17/2016] [Indexed: 12/11/2022] Open
Abstract
Progressive myoclonus epilepsy is a heterogeneous group of disorders characterized by myoclonic and tonic-clonic seizures, ataxia and cognitive decline. We here present two affected brothers. At 9 months of age the elder brother developed ataxia and myoclonic jerks. In his second year he lost the ability to walk and talk, and he developed drug-resistant progressive myoclonus epilepsy. The cerebrospinal fluid level of glutamate was decreased while glutamine was increased. His younger brother manifested similar symptoms from 6 months of age. By exome sequencing of the proband we identified a novel homozygous frameshift variant in the potassium channel tetramerization domain 7 (KCTD7) gene (NM_153033.1:c.696delT: p.F232fs), which results in a truncated protein. The identified F232fs variant is inherited in an autosomal recessive manner, and the healthy consanguineous parents carry the variant in a heterozygous state. Bioinformatic analyses and structure modelling showed that KCTD7 is a highly conserved protein, structurally similar to KCTD5 and several voltage-gated potassium channels, and that it may form homo- or heteromultimers. By heterologous expression in Xenopus laevis oocytes, we demonstrate that wild-type KCTD7 hyperpolarizes cells in a K+ dependent manner and regulates activity of the neuronal glutamine transporter SAT2 (Slc38a2), while the F232fs variant impairs K+ fluxes and obliterates SAT2-dependent glutamine transport. Characterization of four additional disease-causing variants (R94W, R184C, N273I, Y276C) bolster these results and reveal the molecular mechanisms involved in the pathophysiology of KCTD7-related progressive myoclonus epilepsy. Thus, our data demonstrate that KCTD7 has an impact on K+ fluxes, neurotransmitter synthesis and neuronal function, and that malfunction of the encoded protein may lead to progressive myoclonus epilepsy.
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Affiliation(s)
- Marivi Nabong Moen
- 1 The Institute of Basic Medical Sciences, Department of Molecular Medicine, University of Oslo, Oslo, Norway
| | - Roar Fjær
- 2 Department of Medical Genetics, Oslo University Hospital and University of Oslo, Norway
| | - El Hassan Hamdani
- 1 The Institute of Basic Medical Sciences, Department of Molecular Medicine, University of Oslo, Oslo, Norway.,3 Department of Medical Biochemistry, Oslo University Hospital, Oslo, Norway
| | - Jon K Laerdahl
- 4 Department of Microbiology, Oslo University Hospital, Oslo, Norway.,5 Bioinformatics Core Facility, Department of Informatics, University of Oslo, Oslo, Norway
| | - Robin Johansen Menchini
- 1 The Institute of Basic Medical Sciences, Department of Molecular Medicine, University of Oslo, Oslo, Norway
| | - Magnus Dehli Vigeland
- 2 Department of Medical Genetics, Oslo University Hospital and University of Oslo, Norway
| | - Ying Sheng
- 2 Department of Medical Genetics, Oslo University Hospital and University of Oslo, Norway
| | - Dag Erik Undlien
- 2 Department of Medical Genetics, Oslo University Hospital and University of Oslo, Norway
| | - Bjørnar Hassel
- 6 Department of Complex Neurology and Neurohabilitation, Oslo University Hospital, Oslo, Norway
| | - Mustafa A Salih
- 7 Division of Paediatric Neurology, College of Medicine, King Saud University, Riyadh, Saudi Arabia
| | - Heba Y El Khashab
- 7 Division of Paediatric Neurology, College of Medicine, King Saud University, Riyadh, Saudi Arabia.,8 Department of Paediatrics, Ain Shams University, Cairo, Egypt
| | - Kaja Kristine Selmer
- 2 Department of Medical Genetics, Oslo University Hospital and University of Oslo, Norway.,9 National Centre for Rare Epilepsy-related Disorders, Oslo University Hospital, Oslo, Norway
| | - Farrukh Abbas Chaudhry
- 1 The Institute of Basic Medical Sciences, Department of Molecular Medicine, University of Oslo, Oslo, Norway .,3 Department of Medical Biochemistry, Oslo University Hospital, Oslo, Norway
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17
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Peralta FA, Huidobro-Toro JP. Zinc as Allosteric Ion Channel Modulator: Ionotropic Receptors as Metalloproteins. Int J Mol Sci 2016; 17:E1059. [PMID: 27384555 PMCID: PMC4964435 DOI: 10.3390/ijms17071059] [Citation(s) in RCA: 30] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/18/2016] [Revised: 06/20/2016] [Accepted: 06/22/2016] [Indexed: 12/20/2022] Open
Abstract
Zinc is an essential metal to life. This transition metal is a structural component of many proteins and is actively involved in the catalytic activity of cell enzymes. In either case, these zinc-containing proteins are metalloproteins. However, the amino acid residues that serve as ligands for metal coordination are not necessarily the same in structural proteins compared to enzymes. While crystals of structural proteins that bind zinc reveal a higher preference for cysteine sulfhydryls rather than histidine imidazole rings, catalytic enzymes reveal the opposite, i.e., a greater preference for the histidines over cysteines for catalysis, plus the influence of carboxylic acids. Based on this paradigm, we reviewed the putative ligands of zinc in ionotropic receptors, where zinc has been described as an allosteric modulator of channel receptors. Although these receptors do not strictly qualify as metalloproteins since they do not normally bind zinc in structural domains, they do transitorily bind zinc at allosteric sites, modifying transiently the receptor channel's ion permeability. The present contribution summarizes current information showing that zinc allosteric modulation of receptor channels occurs by the preferential metal coordination to imidazole rings as well as to the sulfhydryl groups of cysteine in addition to the carboxyl group of acid residues, as with enzymes and catalysis. It is remarkable that most channels, either voltage-sensitive or transmitter-gated receptor channels, are susceptible to zinc modulation either as positive or negative regulators.
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Affiliation(s)
- Francisco Andrés Peralta
- Laboratorio de Farmacología de Nucleótidos, Laboratorio de Farmacología, Departamento de Biología, Facultad de Química y Biología, y Centro para el Desarrollo de Nanociencias y Nanotecnología (CEDENNA), Universidad de Santiago de Chile, Alameda Libertador B. O'Higgins, 3363 Santiago, Chile.
| | - Juan Pablo Huidobro-Toro
- Laboratorio de Farmacología de Nucleótidos, Laboratorio de Farmacología, Departamento de Biología, Facultad de Química y Biología, y Centro para el Desarrollo de Nanociencias y Nanotecnología (CEDENNA), Universidad de Santiago de Chile, Alameda Libertador B. O'Higgins, 3363 Santiago, Chile.
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18
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Positive Allosteric Modulation of Kv Channels by Sevoflurane: Insights into the Structural Basis of Inhaled Anesthetic Action. PLoS One 2015; 10:e0143363. [PMID: 26599217 PMCID: PMC4657974 DOI: 10.1371/journal.pone.0143363] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/26/2015] [Accepted: 11/04/2015] [Indexed: 11/19/2022] Open
Abstract
Inhalational general anesthesia results from the poorly understood interactions of haloethers with multiple protein targets, which prominently includes ion channels in the nervous system. Previously, we reported that the commonly used inhaled anesthetic sevoflurane potentiates the activity of voltage-gated K+ (Kv) channels, specifically, several mammalian Kv1 channels and the Drosophila K-Shaw2 channel. Also, previous work suggested that the S4-S5 linker of K-Shaw2 plays a role in the inhibition of this Kv channel by n-alcohols and inhaled anesthetics. Here, we hypothesized that the S4-S5 linker is also a determinant of the potentiation of Kv1.2 and K-Shaw2 by sevoflurane. Following functional expression of these Kv channels in Xenopus oocytes, we found that converse mutations in Kv1.2 (G329T) and K-Shaw2 (T330G) dramatically enhance and inhibit the potentiation of the corresponding conductances by sevoflurane, respectively. Additionally, Kv1.2-G329T impairs voltage-dependent gating, which suggests that Kv1.2 modulation by sevoflurane is tied to gating in a state-dependent manner. Toward creating a minimal Kv1.2 structural model displaying the putative sevoflurane binding sites, we also found that the positive modulations of Kv1.2 and Kv1.2-G329T by sevoflurane and other general anesthetics are T1-independent. In contrast, the positive sevoflurane modulation of K-Shaw2 is T1-dependent. In silico docking and molecular dynamics-based free-energy calculations suggest that sevoflurane occupies distinct sites near the S4-S5 linker, the pore domain and around the external selectivity filter. We conclude that the positive allosteric modulation of the Kv channels by sevoflurane involves separable processes and multiple sites within regions intimately involved in channel gating.
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19
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Barone D, Balasco N, Vitagliano L. KCTD5 is endowed with large, functionally relevant, interdomain motions. J Biomol Struct Dyn 2015; 34:1725-35. [PMID: 26336981 DOI: 10.1080/07391102.2015.1090343] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/23/2022]
Abstract
The KCTD family is an emerging class of proteins that are involved in important biological processes whose biochemical and structural properties are rather poorly characterized or even completely undefined. We here used KCTD5, the only member of the family with a known three-dimensional structure, to gain insights into the intrinsic structural stability of the C-terminal domain (CTD) and into the mutual dynamic interplay between the two domains of the protein. Molecular dynamics (MD) simulations indicate that in the simulation timescale (120 ns), the pentameric assembly of the CTD is endowed with a significant intrinsic stability. Moreover, MD analyses also led to the identification of exposed β-strand residues. Being these regions intrinsically sticky, they could be involved in the substrate recognition. More importantly, simulations conducted on the full-length protein provide interesting information of the relative motions between the BTB domain and the CTD of the protein. Indeed, the dissection of the overall motion of the protein is indicative of a large interdomain twisting associated with limited bending movements. Notably, MD data indicate that the entire interdomain motion is pivoted by a single residue (Ser150) of the hinge region that connects the domains. The functional relevance of these motions was evaluated in the context of the functional macromolecular machinery in which KCTD5 is involved. This analysis indicates that the interdomain twisting motion here characterized may be important for the correct positioning of the substrate to be ubiquitinated with respect to the other factors of the ubiquitination machinery.
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Affiliation(s)
- Daniela Barone
- a Institute of Biostructures and Bioimaging, C.N.R. , Via Mezzocannone 16, Naples I-80134 , Italy.,b Dipartimento di Scienze e Tecnologie Ambientali Biologiche e Farmaceutiche , Seconda Università di Napoli , Caserta 81100 , Italy
| | - Nicole Balasco
- a Institute of Biostructures and Bioimaging, C.N.R. , Via Mezzocannone 16, Naples I-80134 , Italy.,b Dipartimento di Scienze e Tecnologie Ambientali Biologiche e Farmaceutiche , Seconda Università di Napoli , Caserta 81100 , Italy
| | - Luigi Vitagliano
- a Institute of Biostructures and Bioimaging, C.N.R. , Via Mezzocannone 16, Naples I-80134 , Italy
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20
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The tetramerization domain potentiates Kv4 channel function by suppressing closed-state inactivation. Biophys J 2015; 107:1090-1104. [PMID: 25185545 DOI: 10.1016/j.bpj.2014.07.038] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/12/2014] [Revised: 05/25/2014] [Accepted: 07/01/2014] [Indexed: 01/26/2023] Open
Abstract
A-type Kv4 potassium channels undergo a conformational change toward a nonconductive state at negative membrane potentials, a dynamic process known as pre-open closed states or closed-state inactivation (CSI). CSI causes inhibition of channel activity without the prerequisite of channel opening, thus providing a dynamic regulation of neuronal excitability, dendritic signal integration, and synaptic plasticity at resting. However, the structural determinants underlying Kv4 CSI remain largely unknown. We recently showed that the auxiliary KChIP4a subunit contains an N-terminal Kv4 inhibitory domain (KID) that directly interacts with Kv4.3 channels to enhance CSI. In this study, we utilized the KChIP4a KID to probe key structural elements underlying Kv4 CSI. Using fluorescence resonance energy transfer two-hybrid mapping and bimolecular fluorescence complementation-based screening combined with electrophysiology, we identified the intracellular tetramerization (T1) domain that functions to suppress CSI and serves as a receptor for the binding of KID. Disrupting the Kv4.3 T1-T1 interaction interface by mutating C110A within the C3H1 motif of T1 domain facilitated CSI and ablated the KID-mediated enhancement of CSI. Furthermore, replacing the Kv4.3 T1 domain with the T1 domain from Kv1.4 (without the C3H1 motif) or Kv2.1 (with the C3H1 motif) resulted in channels functioning with enhanced or suppressed CSI, respectively. Taken together, our findings reveal a novel (to our knowledge) role of the T1 domain in suppressing Kv4 CSI, and that KChIP4a KID directly interacts with the T1 domain to facilitate Kv4.3 CSI, thus leading to inhibition of channel function.
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21
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Abstract
In excitable cells, ion channels are frequently challenged by repetitive stimuli, and their responses shape cellular behavior by regulating the duration and termination of bursts of action potentials. We have investigated the behavior of Shaker family voltage-gated potassium (Kv) channels subjected to repetitive stimuli, with a particular focus on Kv1.2. Genetic deletion of this subunit results in complete mortality within 2 weeks of birth in mice, highlighting a critical physiological role for Kv1.2. Kv1.2 channels exhibit a unique property described previously as "prepulse potentiation," in which activation by a depolarizing step facilitates activation in a subsequent pulse. In this study, we demonstrate that this property enables Kv1.2 channels to exhibit use-dependent activation during trains of very brief depolarizations. Also, Kv subunits usually assemble into heteromeric channels in the central nervous system, generating diversity of function and sensitivity to signaling mechanisms. We demonstrate that other Kv1 channel types do not exhibit use-dependent activation, but this property is conferred in heteromeric channel complexes containing even a single Kv1.2 subunit. This regulatory mechanism is observed in mammalian cell lines as well as primary cultures of hippocampal neurons. Our findings illustrate that use-dependent activation is a unique property of Kv1.2 that persists in heteromeric channel complexes and may influence function of hippocampal neurons.
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22
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de Paola I, Pirone L, Palmieri M, Balasco N, Esposito L, Russo L, Mazzà D, Di Marcotullio L, Di Gaetano S, Malgieri G, Vitagliano L, Pedone E, Zaccaro L. Cullin3-BTB interface: a novel target for stapled peptides. PLoS One 2015; 10:e0121149. [PMID: 25848797 PMCID: PMC4388676 DOI: 10.1371/journal.pone.0121149] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/18/2014] [Accepted: 02/06/2015] [Indexed: 12/21/2022] Open
Abstract
Cullin3 (Cul3), a key factor of protein ubiquitination, is able to interact with dozens of different proteins containing a BTB (Bric-a-brac, Tramtrack and Broad Complex) domain. We here targeted the Cul3–BTB interface by using the intriguing approach of stabilizing the α-helical conformation of Cul3-based peptides through the “stapling” with a hydrocarbon cross-linker. In particular, by combining theoretical and experimental techniques, we designed and characterized stapled Cul3-based peptides embedding the helix 2 of the protein (residues 49–68). Intriguingly, CD and NMR experiments demonstrate that these stapled peptides were able to adopt the helical structure that the fragment assumes in the parent protein. We also show that some of these peptides were able to bind to the BTB of the tetrameric KCTD11, a substrate adaptor involved in HDAC1 degradation, with high affinity (~ 300–600 nM). Cul3-derived staple peptides are also able to bind the BTB of the pentameric KCTD5. Interestingly, the affinity of these peptides is of the same order of magnitude of that reported for the interaction of full-length Cul3 with some BTB containing proteins. Moreover, present data indicate that stapling endows these peptides with an increased serum stability. Altogether, these findings indicate that the designed stapled peptides can efficiently mimic protein-protein interactions and are potentially able to modulate fundamental biological processes involving Cul3.
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Affiliation(s)
- Ivan de Paola
- Institute of Biostructures and Bioimaging, C.N.R., Napoli, Italy
| | | | | | - Nicole Balasco
- Institute of Biostructures and Bioimaging, C.N.R., Napoli, Italy
- Second University of Napoli, Caserta, Italy
| | - Luciana Esposito
- Institute of Biostructures and Bioimaging, C.N.R., Napoli, Italy
- Interuniversity Centre for Research on Bioactive Peptides (CIRPEB), Napoli, Italy
| | | | - Daniela Mazzà
- Department of Molecular Medicine, La Sapienza University, Roma, Italy
| | | | - Sonia Di Gaetano
- Institute of Biostructures and Bioimaging, C.N.R., Napoli, Italy
- Interuniversity Centre for Research on Bioactive Peptides (CIRPEB), Napoli, Italy
| | | | - Luigi Vitagliano
- Institute of Biostructures and Bioimaging, C.N.R., Napoli, Italy
- Interuniversity Centre for Research on Bioactive Peptides (CIRPEB), Napoli, Italy
| | - Emilia Pedone
- Institute of Biostructures and Bioimaging, C.N.R., Napoli, Italy
- Interuniversity Centre for Research on Bioactive Peptides (CIRPEB), Napoli, Italy
- * E-mail: (EP); (LZ)
| | - Laura Zaccaro
- Institute of Biostructures and Bioimaging, C.N.R., Napoli, Italy
- Interuniversity Centre for Research on Bioactive Peptides (CIRPEB), Napoli, Italy
- * E-mail: (EP); (LZ)
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23
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Yang SN, Shi Y, Yang G, Li Y, Yu J, Berggren PO. Ionic mechanisms in pancreatic β cell signaling. Cell Mol Life Sci 2014; 71:4149-77. [PMID: 25052376 PMCID: PMC11113777 DOI: 10.1007/s00018-014-1680-6] [Citation(s) in RCA: 65] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/22/2014] [Revised: 07/03/2014] [Accepted: 07/10/2014] [Indexed: 01/07/2023]
Abstract
The function and survival of pancreatic β cells critically rely on complex electrical signaling systems composed of a series of ionic events, namely fluxes of K(+), Na(+), Ca(2+) and Cl(-) across the β cell membranes. These electrical signaling systems not only sense events occurring in the extracellular space and intracellular milieu of pancreatic islet cells, but also control different β cell activities, most notably glucose-stimulated insulin secretion. Three major ion fluxes including K(+) efflux through ATP-sensitive K(+) (KATP) channels, the voltage-gated Ca(2+) (CaV) channel-mediated Ca(2+) influx and K(+) efflux through voltage-gated K(+) (KV) channels operate in the β cell. These ion fluxes set the resting membrane potential and the shape, rate and pattern of firing of action potentials under different metabolic conditions. The KATP channel-mediated K(+) efflux determines the resting membrane potential and keeps the excitability of the β cell at low levels. Ca(2+) influx through CaV1 channels, a major type of β cell CaV channels, causes the upstroke or depolarization phase of the action potential and regulates a wide range of β cell functions including the most elementary β cell function, insulin secretion. K(+) efflux mediated by KV2.1 delayed rectifier K(+) channels, a predominant form of β cell KV channels, brings about the downstroke or repolarization phase of the action potential, which acts as a brake for insulin secretion owing to shutting down the CaV channel-mediated Ca(2+) entry. These three ion channel-mediated ion fluxes are the most important ionic events in β cell signaling. This review concisely discusses various ionic mechanisms in β cell signaling and highlights KATP channel-, CaV1 channel- and KV2.1 channel-mediated ion fluxes.
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Affiliation(s)
- Shao-Nian Yang
- The Rolf Luft Research Center for Diabetes and Endocrinology, Karolinska Institutet, SE-171 76, Stockholm, Sweden,
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24
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Bocksteins E, Mayeur E, Van Tilborg A, Regnier G, Timmermans JP, Snyders DJ. The subfamily-specific interaction between Kv2.1 and Kv6.4 subunits is determined by interactions between the N- and C-termini. PLoS One 2014; 9:e98960. [PMID: 24901643 PMCID: PMC4047056 DOI: 10.1371/journal.pone.0098960] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/14/2014] [Accepted: 05/08/2014] [Indexed: 12/14/2022] Open
Abstract
The "silent" voltage-gated potassium (KvS) channel subunit Kv6.4 does not form electrically functional homotetramers at the plasma membrane but assembles with Kv2.1 subunits, generating functional Kv2.1/Kv6.4 heterotetramers. The N-terminal T1 domain determines the subfamily-specific assembly of Kv1-4 subunits by preventing interactions between subunits that belong to different subfamilies. For Kv6.4, yeast-two-hybrid experiments showed an interaction of the Kv6.4 N-terminus with the Kv2.1 N-terminus, but unexpectedly also with the Kv3.1 N-terminus. We confirmed this interaction by Fluorescence Resonance Energy Transfer (FRET) and co-immunoprecipitation (co-IP) using N-terminal Kv3.1 and Kv6.4 fragments. However, full-length Kv3.1 and Kv6.4 subunits do not form heterotetramers at the plasma membrane. Therefore, additional interactions between the Kv6.4 and Kv2.1 subunits should be important in the Kv2.1/Kv6.4 subfamily-specificity. Using FRET and co-IP approaches with N- and C-terminal fragments we observed that the Kv6.4 C-terminus physically interacts with the Kv2.1 N-terminus but not with the Kv3.1 N-terminus. The N-terminal amino acid sequence CDD which is conserved between Kv2 and KvS subunits appeared to be a key determinant since charge reversals with arginine substitutions abolished the interaction between the N-terminus of Kv2.1 and the C-terminus of both Kv2.1 and Kv6.4. In addition, the Kv6.4(CKv3.1) chimera in which the C-terminus of Kv6.4 was replaced by the corresponding domain of Kv3.1, disrupted the assembly with Kv2.1. These results indicate that the subfamily-specific Kv2.1/Kv6.4 heterotetramerization is determined by interactions between Kv2.1 and Kv6.4 that involve both the N- and C-termini in which the conserved N-terminal CDD sequence plays a key role.
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Affiliation(s)
- Elke Bocksteins
- Laboratory for Molecular Biophysics, Physiology and Pharmacology, Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgium
| | - Evy Mayeur
- Laboratory for Molecular Biophysics, Physiology and Pharmacology, Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgium
| | - Abbi Van Tilborg
- Laboratory for Molecular Biophysics, Physiology and Pharmacology, Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgium
| | - Glenn Regnier
- Laboratory for Molecular Biophysics, Physiology and Pharmacology, Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgium
| | - Jean-Pierre Timmermans
- Laboratory of Cell Biology and Histology, Department of Veterinary Sciences, University of Antwerp, Antwerp, Belgium
| | - Dirk J. Snyders
- Laboratory for Molecular Biophysics, Physiology and Pharmacology, Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgium
- * E-mail:
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25
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Abstract
Here we present the structure of the T1 domain derived from the voltage-dependent potassium channel K(v)1.3 of Homo sapiens sapiens at 1.2 Å resolution crystallized under near-physiological conditions. The crystals were grown without precipitant in 150 mM KP(i), pH 6.25. The crystals show I4 symmetry typical of the natural occurring tetrameric assembly of the single subunits. The obtained structural model is based on the highest resolution currently achieved for tetramerization domains of voltage-gated potassium channels. We identified an identical fold of the monomer but inside the tetramer the single monomers show a significant rotation which leads to a different orientation of the tetramer compared to other known structures. Such a rotational movement inside the tetrameric assembly might influence the gating properties of the channel. In addition we see two distinct side chain configurations for amino acids located in the top layer proximal to the membrane (Tyr109, Arg116, Ser129, Glu140, Met142, Arg146), and amino acids in the bottom layer of the T1-domain distal from the membrane (Val55, Ile56, Leu77, Arg86). The relative populations of these two states are ranging from 50:50 for Val55, Tyr109, Arg116, Ser129, Glu140, 60:40 for Met142, 65:35 for Arg86, 70:30 for Arg146, and 80:20 for Ile56 and Leu77. The data suggest that in solution these amino acids are involved in an equilibrium of conformational states that may be coupled to the functional states of the whole potassium channel.
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26
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Hu X, Gan S, Xie G, Li L, Chen C, Ding X, Han M, Xiang S, Zhang J. KCTD10 is critical for heart and blood vessel development of zebrafish. Acta Biochim Biophys Sin (Shanghai) 2014; 46:377-86. [PMID: 24705121 DOI: 10.1093/abbs/gmu017] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/26/2023] Open
Abstract
KCTD10 is a member of the PDIP1 family, which is highly conserved during evolution, sharing a lot of similarities among human, mouse, and zebrafish. Recently, zebrafish KCTD13 has been identified to play an important role in the early development of brain and autism. However, the specific function of KCTD10 remains to be elucidated. In this study, experiments were carried out to determine the expression pattern of zebrafish KCTD10 mRNA during embryonic development. It was found that KCTD10 is a maternal gene and KCTD10 is of great importance in the shaping of heart and blood vessels. Our data provide direct clues that knockdown of KCTD10 resulted in severe pericardial edema and loss of heart formation indicated by morphological observation and crucial heart markers like amhc, vmhc, and cmlc2. The heart defect caused by KCTD10 is linked to RhoA and PCNA. Flk-1 staining revealed that intersomitic vessels were lost in the trunk, although angioblasts could migrate to the midline. These findings could be helpful to better understand the determinants responsible for the heart and blood vessel defects.
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Affiliation(s)
- Xiang Hu
- Key Laboratory of Protein Chemistry and Development Biology of State Education Ministry of China, College of Life Sciences, Hunan Normal University, Changsha 410081, China
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27
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Heinemann SH, Hoshi T, Westerhausen M, Schiller A. Carbon monoxide--physiology, detection and controlled release. Chem Commun (Camb) 2014; 50:3644-60. [PMID: 24556640 PMCID: PMC4072318 DOI: 10.1039/c3cc49196j] [Citation(s) in RCA: 302] [Impact Index Per Article: 27.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Carbon monoxide (CO) is increasingly recognized as a cell-signalling molecule akin to nitric oxide (NO). CO has attracted particular attention as a potential therapeutic agent because of its reported anti-hypertensive, anti-inflammatory and cell-protective effects. We discuss recent progress in identifying new effector systems and elucidating the mechanisms of action of CO on, e.g., ion channels, as well as the design of novel methods to monitor CO in cellular environments. We also report on recent developments in the area of CO-releasing molecules (CORMs) and materials for controlled CO application. Novel triggers for CO release, metal carbonyls and degradation mechanisms of CORMs are highlighted. In addition, potential formulations of CORMs for targeted CO release are discussed.
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Affiliation(s)
- Stefan H. Heinemann
- Center for Molecular Biomedicine (CMB), Department of Biophysics, Friedrich Schiller University Jena & Jena University Hospital, Hans-Knöll-Straße 2, D-07745 Jena, Germany
| | - Toshinori Hoshi
- Department of Physiology, University of Pennsylvania, 415 Curie Boulevard, 605 CRB, Philadelphia, PA 19104-6085, USA
| | - Matthias Westerhausen
- Institute for Inorganic and Analytical Chemistry, Friedrich Schiller University Jena, Humboldtstr. 8, D-07743 Jena, Germany
| | - Alexander Schiller
- Institute for Inorganic and Analytical Chemistry, Friedrich Schiller University Jena, Humboldtstr. 8, D-07743 Jena, Germany
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28
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Esperante SA, Noval MG, Altieri TA, de Oliveira GAP, Silva JL, de Prat-Gay G. Fine modulation of the respiratory syncytial virus M2-1 protein quaternary structure by reversible zinc removal from its Cys(3)-His(1) motif. Biochemistry 2013; 52:6779-89. [PMID: 23984912 DOI: 10.1021/bi401029q] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
Human respiratory syncytial virus (hRSV) is a worldwide distributed pathogen that causes respiratory disease mostly in infants and the elderly. The M2-1 protein of hRSV functions as a transcription antiterminator and partakes in virus particle budding. It is present only in Pneumovirinae, namely, Pneumovirus (RSV) and Metapneumovirus, making it an interesting target for specific antivirals. hRSV M2-1 is a tight tetramer bearing a Cys3-His1 zinc-binding motif, present in Ebola VP30 protein and some eukaryotic proteins, whose integrity was shown to be essential for protein function but without a biochemical mechanistic basis. We showed that removal of the zinc atom causes dissociation to a monomeric apo-M2-1 species. Surprisingly, the secondary structure and stability of the apo-monomer is indistinguishable from that of the M2-1 tetramer. Dissociation reported by a highly sensitive tryptophan residue is much increased at pH 5.0 compared to pH 7.0, suggesting a histidine protonation cooperating in zinc removal. The monomeric apo form binds RNA at least as well as the tetramer, and this interaction is outcompeted by the phosphoprotein P, the RNA polymerase cofactor. The role of zinc goes beyond stabilization of local structure, finely tuning dissociation to a fully folded and binding competent monomer. Removal of zinc is equivalent to the disruption of the motif by mutation, only that the former is potentially reversible in the cellular context. Thus, this process could be triggered by a natural chelator such as glutathione or thioneins, where reversibility strongly suggests a modulatory role in the participation of M2-1 in the assembly of the polymerase complex or in virion budding.
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Affiliation(s)
- Sebastián A Esperante
- Protein Structure-Function and Engineering Laboratory, Fundación Instituto Leloir and IIBA-Conicet , Patricias Argentinas 435, (1405) Buenos Aires, Argentina
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29
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Skoblov M, Marakhonov A, Marakasova E, Guskova A, Chandhoke V, Birerdinc A, Baranova A. Protein partners of KCTD proteins provide insights about their functional roles in cell differentiation and vertebrate development. Bioessays 2013; 35:586-96. [PMID: 23592240 DOI: 10.1002/bies.201300002] [Citation(s) in RCA: 70] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
The KCTD family includes tetramerization (T1) domain containing proteins with diverse biological effects. We identified a novel member of the KCTD family, BTBD10. A comprehensive analysis of protein-protein interactions (PPIs) allowed us to put forth a number of testable hypotheses concerning the biological functions for individual KCTD proteins. In particular, we predict that KCTD20 participates in the AKT-mTOR-p70 S6k signaling cascade, KCTD5 plays a role in cytokinesis in a NEK6 and ch-TOG-dependent manner, KCTD10 regulates the RhoA/RhoB pathway. Developmental regulator KCTD15 represses AP-2α and contributes to energy homeostasis by suppressing early adipogenesis. TNFAIP1-like KCTD proteins may participate in post-replication DNA repair through PCNA ubiquitination. KCTD12 may suppress the proliferation of gastrointestinal cells through interference with GABAb signaling. KCTD9 deserves experimental attention as the only eukaryotic protein with a DNA-like pentapeptide repeat domain. The value of manual curation of PPIs and analysis of existing high-throughput data should not be underestimated.
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Affiliation(s)
- Mikhail Skoblov
- Research Center for Medical Genetics RAMS, Moscow, Russian Federation, Russia
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30
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Barry J, Gu C. Coupling mechanical forces to electrical signaling: molecular motors and the intracellular transport of ion channels. Neuroscientist 2013; 19:145-59. [PMID: 22910031 PMCID: PMC3625366 DOI: 10.1177/1073858412456088] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
Proper localization of various ion channels is fundamental to neuronal functions, including postsynaptic potential plasticity, dendritic integration, action potential initiation and propagation, and neurotransmitter release. Microtubule-based forward transport mediated by kinesin motors plays a key role in placing ion channel proteins to correct subcellular compartments. PDZ- and coiled-coil-domain proteins function as adaptor proteins linking ionotropic glutamate and GABA receptors to various kinesin motors, respectively. Recent studies show that several voltage-gated ion channel/transporter proteins directly bind to kinesins during forward transport. Three major regulatory mechanisms underlying intracellular transport of ion channels are also revealed. These studies contribute to understanding how mechanical forces are coupled to electrical signaling and illuminating pathogenic mechanisms in neurodegenerative diseases.
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Affiliation(s)
- Joshua Barry
- The Molecular, Cellular and Developmental Biology Graduate Program, The Ohio State University, Columbus, OH, USA
| | - Chen Gu
- The Molecular, Cellular and Developmental Biology Graduate Program, The Ohio State University, Columbus, OH, USA
- Department of Neuroscience, The Ohio State University, Columbus, OH, USA
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31
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Barry J, Xu M, Gu Y, Dangel AW, Jukkola P, Shrestha C, Gu C. Activation of conventional kinesin motors in clusters by Shaw voltage-gated K+ channels. J Cell Sci 2013; 126:2027-41. [PMID: 23487040 DOI: 10.1242/jcs.122234] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/03/2023] Open
Abstract
The conventional kinesin motor transports many different cargos to specific locations in neurons. How cargos regulate motor function remains unclear. Here we focus on KIF5, the heavy chain of conventional kinesin, and report that the Kv3 (Shaw) voltage-gated K(+) channel, the only known tetrameric KIF5-binding protein, clusters and activates KIF5 motors during axonal transport. Endogenous KIF5 often forms clusters along axons, suggesting a potential role of KIF5-binding proteins. Our biochemical assays reveal that the high-affinity multimeric binding between the Kv3.1 T1 domain and KIF5B requires three basic residues in the KIF5B tail. Kv3.1 T1 competes with the motor domain and microtubules, but not with kinesin light chain 1 (KLC1), for binding to the KIF5B tail. Live-cell imaging assays show that four KIF5-binding proteins, Kv3.1, KLC1 and two synaptic proteins SNAP25 and VAMP2, differ in how they regulate KIF5B distribution. Only Kv3.1 markedly increases the frequency and number of KIF5B-YFP anterograde puncta. Deletion of Kv3.1 channels reduces KIF5 clusters in mouse cerebellar neurons. Therefore, clustering and activation of KIF5 motors by Kv3 regulate the motor number in carrier vesicles containing the channel proteins, contributing not only to the specificity of Kv3 channel transport, but also to the cargo-mediated regulation of motor function.
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Affiliation(s)
- Joshua Barry
- Molecular, Cellular and Developmental Biology Graduate Program, The Ohio State University, Columbus, OH 43210, USA
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Gu Y, Barry J, Gu C. Kv3 channel assembly, trafficking and activity are regulated by zinc through different binding sites. J Physiol 2013; 591:2491-507. [PMID: 23420657 DOI: 10.1113/jphysiol.2013.251983] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/28/2023] Open
Abstract
Zinc, a divalent heavy metal ion and an essential mineral for life, regulates synaptic transmission and neuronal excitability via ion channels. However, its binding sites and regulatory mechanisms are poorly understood. Here, we report that Kv3 channel assembly, localization and activity are regulated by zinc through different binding sites. Local perfusion of zinc reversibly reduced spiking frequency of cultured neurons most likely by suppressing Kv3 channels. Indeed, zinc inhibited Kv3.1 channel activity and slowed activation kinetics, independent of its site in the N-terminal T1 domain. Biochemical assays surprisingly identified a novel zinc-binding site in the Kv3.1 C-terminus, critical for channel activity and axonal targeting, but not for the zinc inhibition. Finally, mutagenesis revealed an important role of the junction between the first transmembrane (TM) segment and the first extracellular loop in sensing zinc. Its mutant enabled fast spiking with relative resistance to the zinc inhibition. Therefore, our studies provide novel mechanistic insights into the multifaceted regulation of Kv3 channel activity and localization by divalent heavy metal ions.
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Affiliation(s)
- Yuanzheng Gu
- 182 Rightmire Hall, 1060 Carmack Road, The Ohio State University, Columbus, OH 43210, USA.
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Smith KE, Wilkie SE, Tebbs-Warner JT, Jarvis BJ, Gallasch L, Stocker M, Hunt DM. Functional analysis of missense mutations in Kv8.2 causing cone dystrophy with supernormal rod electroretinogram. J Biol Chem 2012; 287:43972-83. [PMID: 23115240 DOI: 10.1074/jbc.m112.388033] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
Abstract
Mutations in KCNV2 have been proposed as the molecular basis for cone dystrophy with supernormal rod electroretinogram. KCNV2 codes for the modulatory voltage-gated potassium channel α-subunit, Kv8.2, which is incapable of forming functional channels on its own. Functional heteromeric channels are however formed with Kv2.1 in heterologous expression systems, with both α-subunit genes expressed in rod and cone photoreceptors. Of the 30 mutations identified in the KCNV2 gene, we have selected three missense mutations localized in the potassium channel pore and two missense mutations localized in the tetramerization domain for analysis. We characterized the differences between homomeric Kv2.1 and heteromeric Kv2.1/Kv8.2 channels and investigated the influence of the selected mutations on the function of heteromeric channels. We found that two pore mutations (W467G and G478R) led to the formation of nonconducting heteromeric Kv2.1/Kv8.2 channels, whereas the mutations localized in the tetramerization domain prevented heteromer generation and resulted in the formation of homomeric Kv2.1 channels only. Consequently, our study suggests the existence of two distinct molecular mechanisms involved in the disease pathology.
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Affiliation(s)
- Katie E Smith
- University College London Institute of Ophthalmology, London EC1V 9EL, United Kingdom
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Seddik R, Jungblut SP, Silander OK, Rajalu M, Fritzius T, Besseyrias V, Jacquier V, Fakler B, Gassmann M, Bettler B. Opposite effects of KCTD subunit domains on GABA(B) receptor-mediated desensitization. J Biol Chem 2012; 287:39869-77. [PMID: 23035119 DOI: 10.1074/jbc.m112.412767] [Citation(s) in RCA: 44] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
GABA(B) receptors assemble from principle and auxiliary subunits. The principle subunits GABA(B1) and GABA(B2) form functional heteromeric GABA(B(1,2)) receptors that associate with homotetramers of auxiliary KCTD8, -12, -12b, or -16 (named after their K(+) channel tetramerization domain) subunits. These auxiliary subunits constitute receptor subtypes with distinct functional properties. KCTD12 and -12b generate desensitizing receptor responses while KCTD8 and -16 generate largely non-desensitizing receptor responses. The structural elements of the KCTDs underlying these differences in desensitization are unknown. KCTDs are modular proteins comprising a T1 tetramerization domain, which binds to GABA(B2), and a H1 homology domain. KCTD8 and -16 contain an additional C-terminal H2 homology domain that is not sequence-related to the H1 domains. No functions are known for the H1 and H2 domains. Here we addressed which domains and sequence motifs in KCTD proteins regulate desensitization of the receptor response. We found that the H1 domains in KCTD12 and -12b mediate desensitization through a particular sequence motif, T/NFLEQ, which is not present in the H1 domains of KCTD8 and -16. In addition, the H2 domains in KCTD8 and -16 inhibit desensitization when expressed C-terminal to the H1 domains but not when expressed as a separate protein in trans. Intriguingly, the inhibitory effect of the H2 domain is sequence-independent, suggesting that the H2 domain sterically hinders desensitization by the H1 domain. Evolutionary analysis supports that KCTD12 and -12b evolved desensitizing properties by liberating their H1 domains from antagonistic H2 domains and acquisition of the T/NFLEQ motif.
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Affiliation(s)
- Riad Seddik
- Department of Biomedicine, University of Basel, 4056 Basel, Switzerland
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Blunck R, Batulan Z. Mechanism of electromechanical coupling in voltage-gated potassium channels. Front Pharmacol 2012; 3:166. [PMID: 22988442 PMCID: PMC3439648 DOI: 10.3389/fphar.2012.00166] [Citation(s) in RCA: 70] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/30/2012] [Accepted: 08/24/2012] [Indexed: 01/10/2023] Open
Abstract
Voltage-gated ion channels play a central role in the generation of action potentials in the nervous system. They are selective for one type of ion - sodium, calcium, or potassium. Voltage-gated ion channels are composed of a central pore that allows ions to pass through the membrane and four peripheral voltage sensing domains that respond to changes in the membrane potential. Upon depolarization, voltage sensors in voltage-gated potassium channels (Kv) undergo conformational changes driven by positive charges in the S4 segment and aided by pairwise electrostatic interactions with the surrounding voltage sensor. Structure-function relations of Kv channels have been investigated in detail, and the resulting models on the movement of the voltage sensors now converge to a consensus; the S4 segment undergoes a combined movement of rotation, tilt, and vertical displacement in order to bring 3-4e(+) each through the electric field focused in this region. Nevertheless, the mechanism by which the voltage sensor movement leads to pore opening, the electromechanical coupling, is still not fully understood. Thus, recently, electromechanical coupling in different Kv channels has been investigated with a multitude of techniques including electrophysiology, 3D crystal structures, fluorescence spectroscopy, and molecular dynamics simulations. Evidently, the S4-S5 linker, the covalent link between the voltage sensor and pore, plays a crucial role. The linker transfers the energy from the voltage sensor movement to the pore domain via an interaction with the S6 C-termini, which are pulled open during gating. In addition, other contact regions have been proposed. This review aims to provide (i) an in-depth comparison of the molecular mechanisms of electromechanical coupling in different Kv channels; (ii) insight as to how the voltage sensor and pore domain influence one another; and (iii) theoretical predictions on the movement of the cytosolic face of the Kv channels during gating.
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Affiliation(s)
- Rikard Blunck
- Groupe d’étude des protéines membranairesMontreal, QC, Canada
- Department of Physiology, Université de MontréalMontreal, QC, Canada
- Department of Physics, Université de MontréalMontreal, QC, Canada
| | - Zarah Batulan
- Groupe d’étude des protéines membranairesMontreal, QC, Canada
- Department of Physiology, Université de MontréalMontreal, QC, Canada
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Barros F, Domínguez P, de la Peña P. Cytoplasmic domains and voltage-dependent potassium channel gating. Front Pharmacol 2012; 3:49. [PMID: 22470342 PMCID: PMC3311039 DOI: 10.3389/fphar.2012.00049] [Citation(s) in RCA: 40] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/24/2011] [Accepted: 03/05/2012] [Indexed: 12/20/2022] Open
Abstract
The basic architecture of the voltage-dependent K+ channels (Kv channels) corresponds to a transmembrane protein core in which the permeation pore, the voltage-sensing components and the gating machinery (cytoplasmic facing gate and sensor–gate coupler) reside. Usually, large protein tails are attached to this core, hanging toward the inside of the cell. These cytoplasmic regions are essential for normal channel function and, due to their accessibility to the cytoplasmic environment, constitute obvious targets for cell-physiological control of channel behavior. Here we review the present knowledge about the molecular organization of these intracellular channel regions and their role in both setting and controlling Kv voltage-dependent gating properties. This includes the influence that they exert on Kv rapid/N-type inactivation and on activation/deactivation gating of Shaker-like and eag-type Kv channels. Some illustrative examples about the relevance of these cytoplasmic domains determining the possibilities for modulation of Kv channel gating by cellular components are also considered.
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Affiliation(s)
- Francisco Barros
- Departamento de Bioquímica y Biología Molecular, Universidad de Oviedo Oviedo, Asturias, Spain
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Lo T, Tsai CF, Shih YRV, Wang YT, Lu SC, Sung TY, Hsu WL, Chen YJ, Lee OK. Phosphoproteomic Analysis of Human Mesenchymal Stromal Cells during Osteogenic Differentiation. J Proteome Res 2011; 11:586-98. [DOI: 10.1021/pr200868p] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Affiliation(s)
- Ting Lo
- Department of Medical Research and Education and ‡Department of Orthopaedics and Traumatology, Taipei Veterans General Hospital, Taipei, Taiwan
- Institute of Clinical Medicine and ∥Stem Cell Research Center, National Yang-Ming University, Taipei, Taiwan
- Institute of Chemistry and Genomics Research Center, ¶Chemical Biology and Molecular Biophysics Program, Taiwan International Graduate Program, Institute of Chemistry, and #Institute of Information Science, Academia Sinica, Taipei, Taiwan
- Department of Chemistry and ○Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan
| | - Chia-Feng Tsai
- Department of Medical Research and Education and ‡Department of Orthopaedics and Traumatology, Taipei Veterans General Hospital, Taipei, Taiwan
- Institute of Clinical Medicine and ∥Stem Cell Research Center, National Yang-Ming University, Taipei, Taiwan
- Institute of Chemistry and Genomics Research Center, ¶Chemical Biology and Molecular Biophysics Program, Taiwan International Graduate Program, Institute of Chemistry, and #Institute of Information Science, Academia Sinica, Taipei, Taiwan
- Department of Chemistry and ○Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan
| | - Yu-Ru V. Shih
- Department of Medical Research and Education and ‡Department of Orthopaedics and Traumatology, Taipei Veterans General Hospital, Taipei, Taiwan
- Institute of Clinical Medicine and ∥Stem Cell Research Center, National Yang-Ming University, Taipei, Taiwan
- Institute of Chemistry and Genomics Research Center, ¶Chemical Biology and Molecular Biophysics Program, Taiwan International Graduate Program, Institute of Chemistry, and #Institute of Information Science, Academia Sinica, Taipei, Taiwan
- Department of Chemistry and ○Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan
| | - Yi-Ting Wang
- Department of Medical Research and Education and ‡Department of Orthopaedics and Traumatology, Taipei Veterans General Hospital, Taipei, Taiwan
- Institute of Clinical Medicine and ∥Stem Cell Research Center, National Yang-Ming University, Taipei, Taiwan
- Institute of Chemistry and Genomics Research Center, ¶Chemical Biology and Molecular Biophysics Program, Taiwan International Graduate Program, Institute of Chemistry, and #Institute of Information Science, Academia Sinica, Taipei, Taiwan
- Department of Chemistry and ○Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan
| | - Sheng-Chieh Lu
- Department of Medical Research and Education and ‡Department of Orthopaedics and Traumatology, Taipei Veterans General Hospital, Taipei, Taiwan
- Institute of Clinical Medicine and ∥Stem Cell Research Center, National Yang-Ming University, Taipei, Taiwan
- Institute of Chemistry and Genomics Research Center, ¶Chemical Biology and Molecular Biophysics Program, Taiwan International Graduate Program, Institute of Chemistry, and #Institute of Information Science, Academia Sinica, Taipei, Taiwan
- Department of Chemistry and ○Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan
| | - Ting-Yi Sung
- Department of Medical Research and Education and ‡Department of Orthopaedics and Traumatology, Taipei Veterans General Hospital, Taipei, Taiwan
- Institute of Clinical Medicine and ∥Stem Cell Research Center, National Yang-Ming University, Taipei, Taiwan
- Institute of Chemistry and Genomics Research Center, ¶Chemical Biology and Molecular Biophysics Program, Taiwan International Graduate Program, Institute of Chemistry, and #Institute of Information Science, Academia Sinica, Taipei, Taiwan
- Department of Chemistry and ○Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan
| | - Wen-Lian Hsu
- Department of Medical Research and Education and ‡Department of Orthopaedics and Traumatology, Taipei Veterans General Hospital, Taipei, Taiwan
- Institute of Clinical Medicine and ∥Stem Cell Research Center, National Yang-Ming University, Taipei, Taiwan
- Institute of Chemistry and Genomics Research Center, ¶Chemical Biology and Molecular Biophysics Program, Taiwan International Graduate Program, Institute of Chemistry, and #Institute of Information Science, Academia Sinica, Taipei, Taiwan
- Department of Chemistry and ○Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan
| | - Yu-Ju Chen
- Department of Medical Research and Education and ‡Department of Orthopaedics and Traumatology, Taipei Veterans General Hospital, Taipei, Taiwan
- Institute of Clinical Medicine and ∥Stem Cell Research Center, National Yang-Ming University, Taipei, Taiwan
- Institute of Chemistry and Genomics Research Center, ¶Chemical Biology and Molecular Biophysics Program, Taiwan International Graduate Program, Institute of Chemistry, and #Institute of Information Science, Academia Sinica, Taipei, Taiwan
- Department of Chemistry and ○Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan
| | - Oscar K. Lee
- Department of Medical Research and Education and ‡Department of Orthopaedics and Traumatology, Taipei Veterans General Hospital, Taipei, Taiwan
- Institute of Clinical Medicine and ∥Stem Cell Research Center, National Yang-Ming University, Taipei, Taiwan
- Institute of Chemistry and Genomics Research Center, ¶Chemical Biology and Molecular Biophysics Program, Taiwan International Graduate Program, Institute of Chemistry, and #Institute of Information Science, Academia Sinica, Taipei, Taiwan
- Department of Chemistry and ○Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan
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Xu M, Gu Y, Barry J, Gu C. Kinesin I transports tetramerized Kv3 channels through the axon initial segment via direct binding. J Neurosci 2010; 30:15987-6001. [PMID: 21106837 PMCID: PMC2996050 DOI: 10.1523/jneurosci.3565-10.2010] [Citation(s) in RCA: 48] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/09/2010] [Revised: 09/28/2010] [Accepted: 10/01/2010] [Indexed: 01/13/2023] Open
Abstract
Precise targeting of various voltage-gated ion channels to proper membrane domains is crucial for their distinct roles in neuronal excitability and synaptic transmission. How each channel protein is transported within the cytoplasm is poorly understood. Here, we report that KIF5/kinesin I transports Kv3.1 voltage-gated K(+) (Kv) channels through the axon initial segment (AIS) via direct binding. First, we have identified a novel interaction between Kv3.1 and KIF5, confirmed by immunoprecipitation from mouse brain lysates and by pull-down assays with exogenously expressed proteins. The interaction is mediated by a direct binding between the Kv3.1 N-terminal T1 domain and a conserved region in KIF5 tail domains, in which proper T1 tetramerization is crucial. Overexpression of this region of KIF5B markedly reduces axonal levels of Kv3.1bHA. In mature hippocampal neurons, endogenous Kv3.1b and KIF5 colocalize. Suppressing the endogenous KIF5B level by RNA interference significantly reduces the Kv3.1b axonal level. Furthermore, mutating the Zn(2+)-binding site within T1 markedly decreases channel axonal targeting and forward trafficking, likely through disrupting T1 tetramerization and hence eliminating the binding to KIF5 tail. The mutation also alters channel activity. Interestingly, coexpression of the YFP (yellow fluorescent protein)-tagged KIF5B assists dendritic Kv3.1a and even mutants with a faulty axonal targeting motif to penetrate the AIS. Finally, fluorescently tagged Kv3.1 channels colocalize and comove with KIF5B along axons revealed by two-color time-lapse imaging. Our findings suggest that the binding to KIF5 ensures properly assembled and functioning Kv3.1 channels to be transported into axons.
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Affiliation(s)
- Mingxuan Xu
- Department of Neuroscience and Center for Molecular Neurobiology
| | - Yuanzheng Gu
- Department of Neuroscience and Center for Molecular Neurobiology
| | - Joshua Barry
- Molecular, Cellular, and Developmental Biology Graduate Program, The Ohio State University, Columbus, Ohio 43210
| | - Chen Gu
- Department of Neuroscience and Center for Molecular Neurobiology
- Molecular, Cellular, and Developmental Biology Graduate Program, The Ohio State University, Columbus, Ohio 43210
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39
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Liang P, Chen H, Cui Y, Lei L, Wang K. Functional rescue of Kv4.3 channel tetramerization mutants by KChIP4a. Biophys J 2010; 98:2867-76. [PMID: 20550899 DOI: 10.1016/j.bpj.2010.03.044] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/24/2009] [Revised: 03/12/2010] [Accepted: 03/15/2010] [Indexed: 11/16/2022] Open
Abstract
KChIP4a shows a high homology with other members of the family of Kv channel-interacting proteins (KChIPs) in the conserved C-terminal core region, but exhibits a unique modulation of Kv4 channel gating and surface expression. Unlike KChIP1, the KChIP4 splice variant KChIP4a has been shown to inhibit surface expression and function as a suppressor of channel inactivation of Kv4. In this study, we sought to determine whether the multitasking KChIP4a modulates Kv4 function in a clamping fashion similar to that shown by KChIP1. Injection of Kv4.3 T1 zinc mutants into Xenopus oocytes resulted in the nonfunctional expression of Kv4.3 channels. Coexpression of Kv4.3 zinc mutants with WT KChIP4a gave rise to the functional expression of Kv4.3 current. Oocyte surface labeling results confirm the correlation between functional rescue and enhanced surface expression of zinc mutant proteins. Chimeric mutations that replace the Kv4.3 N-terminus with N-terminal KChIP4a or N-terminal deletion of KChIP4a further demonstrate that the functional rescue of Kv4.3 channel tetramerization mutants depends on the KChIP4a core region, but not its N-terminus. Structure-guided mutation of two critical residues of core KChIP4a attenuated functional rescue and tetrameric assembly. Moreover, size exclusion chromatography combined with fast protein liquid chromatography showed that KChIP4a can drive zinc mutant monomers to assemble as tetramers. Taken together, our results show that KChIP4a can rescue the function of tetramerization-defective Kv4 monomers. Therefore, we propose that core KChIP4a functions to promote tetrameric assembly and enhance surface expression of Kv4 channels by a clamping action, whereas its N-terminus inhibits surface expression of Kv4 by a mechanism that remains elusive.
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Affiliation(s)
- Ping Liang
- Department of Neurobiology, Neuroscience Research Institute, Peking University Health Science Center, Beijing, China
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40
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Kihira Y, Hermanstyne TO, Misonou H. Formation of heteromeric Kv2 channels in mammalian brain neurons. J Biol Chem 2010; 285:15048-15055. [PMID: 20202934 DOI: 10.1074/jbc.m109.074260] [Citation(s) in RCA: 53] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
The formation of heteromeric tetramers is a common feature of voltage-gated potassium (Kv) channels. This results in the generation of a variety of tetrameric Kv channels that exhibit distinct biophysical and biochemical characteristics. Kv2 delayed rectifier channels are, however, unique exceptions. It has been previously shown that mammalian Kv2.1 and Kv2.2 are localized in distinct domains of neuronal membranes and are not capable of forming heteromeric channels with each other (Hwang, P. M., Glatt, C. E., Bredt, D. S., Yellen, G., and Snyder, S. H. (1992) Neuron 8, 473-481). In this study, we report a novel form of rat Kv2.2, Kv2.2(long), which has not been previously recognized. Our data indicate that Kv2.2(long) is the predominant form of Kv2.2 expressed in cortical pyramidal neurons. In contrast to the previous findings, we also found that rat Kv2.1 and Kv2.2(long) are colocalized in the somata and proximal dendrites of cortical pyramidal neurons and are capable of forming functional heteromeric delayed rectifier channels. Our results suggest that the delayed rectifier currents, which regulate action potential firing, are encoded by heteromeric Kv2 channels in cortical neurons.
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Affiliation(s)
- Yoshitaka Kihira
- Department of Neural and Pain Sciences, Dental School, University of Maryland, Baltimore, Maryland 21201
| | - Tracey O Hermanstyne
- Department of Neural and Pain Sciences, Dental School, University of Maryland, Baltimore, Maryland 21201; Program in Neuroscience, University of Maryland, Baltimore, Maryland 21201
| | - Hiroaki Misonou
- Department of Neural and Pain Sciences, Dental School, University of Maryland, Baltimore, Maryland 21201; Program in Neuroscience, University of Maryland, Baltimore, Maryland 21201.
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Mutations in Grxcr1 are the basis for inner ear dysfunction in the pirouette mouse. Am J Hum Genet 2010; 86:148-60. [PMID: 20137774 DOI: 10.1016/j.ajhg.2010.01.016] [Citation(s) in RCA: 38] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/17/2009] [Revised: 01/18/2010] [Accepted: 01/19/2010] [Indexed: 01/03/2023] Open
Abstract
Recessive mutations at the mouse pirouette (pi) locus result in hearing loss and vestibular dysfunction due to neuroepithelial defects in the inner ear. Using a positional cloning strategy, we have identified mutations in the gene Grxcr1 (glutaredoxin cysteine-rich 1) in five independent allelic strains of pirouette mice. We also provide sequence data of GRXCR1 from humans with profound hearing loss suggesting that pirouette is a model for studying the mechanism of nonsyndromic deafness DFNB25. Grxcr1 encodes a 290 amino acid protein that contains a region of similarity to glutaredoxin proteins and a cysteine-rich region at its C terminus. Grxcr1 is expressed in sensory epithelia of the inner ear, and its encoded protein is localized along the length of stereocilia, the actin-filament-rich mechanosensory structures at the apical surface of auditory and vestibular hair cells. The precise architecture of hair cell stereocilia is essential for normal hearing. Loss of function of Grxcr1 in homozygous pirouette mice results in abnormally thin and slightly shortened stereocilia. When overexpressed in transfected cells, GRXCR1 localizes along the length of actin-filament-rich structures at the dorsal-apical surface and induces structures with greater actin filament content and/or increased lengths in a subset of cells. Our results suggest that deafness in pirouette mutants is associated with loss of GRXCR1 function in modulating actin cytoskeletal architecture in the developing stereocilia of sensory hair cells.
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Hou S, Vigeland LE, Zhang G, Xu R, Li M, Heinemann SH, Hoshi T. Zn2+ activates large conductance Ca2+-activated K+ channel via an intracellular domain. J Biol Chem 2009; 285:6434-42. [PMID: 20037152 DOI: 10.1074/jbc.m109.069211] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023] Open
Abstract
Zinc is an essential trace element and plays crucial roles in normal development, often as an integral structural component of transcription factors and enzymes. Recent evidence suggests that intracellular Zn(2+) functions as a signaling molecule, mediating a variety of important physiological phenomena. However, the immediate effectors of intracellular Zn(2+) signaling are not well known. We show here that intracellular Zn(2+) potently and reversibly activates large-conductance voltage- and Ca(2+)-activated Slo1 K(+) (BK) channels. The full effect of Zn(2+) requires His(365) in the RCK1 (regulator of conductance for K(+)) domain of the channel. Furthermore, mutation of two nearby acidic residues, Asp(367) and Glu(399), also reduced activation of the channel by Zn(2+), suggesting a possible structural arrangement for Zn(2+) binding by the aforementioned residues. Extracellular Zn(2+) activated Slo1 BK channels when coexpressed with Zn(2+)-permeable TRPM7 (transient receptor potential melastatin 7) channels. The results thus demonstrate that Slo1 BK channels represent a positive and direct effector of Zn(2+) signaling and may participate in sculpting cellular response to an increase in intracellular Zn(2+) concentration.
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Affiliation(s)
- Shangwei Hou
- Department of Physiology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
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Burg ED, Platoshyn O, Tsigelny IF, Lozano-Ruiz B, Rana BK, Yuan JXJ. Tetramerization domain mutations in KCNA5 affect channel kinetics and cause abnormal trafficking patterns. Am J Physiol Cell Physiol 2009; 298:C496-509. [PMID: 20018952 DOI: 10.1152/ajpcell.00464.2009] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
The activity of voltage-gated K(+) (K(V)) channels plays an important role in regulating pulmonary artery smooth muscle cell (PASMC) contraction, proliferation, and apoptosis. The highly conserved NH(2)-terminal tetramerization domain (T1) of K(V) channels is important for proper channel assembly, association with regulatory K(V) beta-subunits, and localization of the channel to the plasma membrane. We recently reported two nonsynonymous mutations (G182R and E211D) in the KCNA5 gene of patients with idiopathic pulmonary arterial hypertension, which localize to the T1 domain of KCNA5. To study the electrophysiological properties and expression patterns of the mutants compared with the wild-type (WT) channel in vitro, we transfected HEK-293 cells with WT KCNA5, G182R, E211D, or the double mutant G182R/E211D channel. The mutants form functional channels; however, whole cell current kinetic differences between WT and mutant channels exist. Steady-state inactivation curves of the G182R and G182R/E211D channels reveal accelerated inactivation; the mutant channels inactivated at more hyperpolarized potentials compared with the WT channel. Channel protein expression was also decreased by the mutations. Compared with the WT channel, which was present in its mature glycosylated form, the mutant channels are present in greater proportion in their immature form in HEK-293 cells. Furthermore, G182R protein level is greatly reduced in COS-1 cells compared with WT. Immunostaining data support the hypothesis that, while WT protein localizes to the plasma membrane, mutant protein is mainly retained in intracellular packets. Overall, these data support a role for the T1 domain in channel kinetics as well as in KCNA5 channel subcellular localization.
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Affiliation(s)
- Elyssa D Burg
- Dept. of Medicine, Univ. of California, San Diego, 9500 Gilman Dr., MC 0725, La Jolla, CA 92093-0725, USA
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Bocksteins E, Labro AJ, Mayeur E, Bruyns T, Timmermans JP, Adriaensen D, Snyders DJ. Conserved negative charges in the N-terminal tetramerization domain mediate efficient assembly of Kv2.1 and Kv2.1/Kv6.4 channels. J Biol Chem 2009; 284:31625-34. [PMID: 19717558 DOI: 10.1074/jbc.m109.039479] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Voltage-gated potassium (Kv) channels are transmembrane tetramers of individual alpha-subunits. Eight different Shaker-related Kv subfamilies have been identified in which the tetramerization domain T1, located on the intracellular N terminus, facilitates and controls the assembly of both homo- and heterotetrameric channels. Only the Kv2 alpha-subunits are able to form heterotetramers with members of the silent Kv subfamilies (Kv5, Kv6, Kv8, and Kv9). The T1 domain contains two subdomains, A and B box, which presumably determine subfamily specificity by preventing incompatible subunits to assemble. In contrast, little is known about the involvement of the A/B linker sequence. Both Kv2 and silent Kv subfamilies contain a fully conserved and negatively charged sequence (CDD) in this linker that is lacking in the other subfamilies. Neutralizing these aspartates in Kv2.1 by mutating them to alanines did not affect the gating properties, but reduced the current density moderately. However, charge reversal arginine substitutions strongly reduced the current density of these homotetrameric mutant Kv2.1 channels and immunocytochemistry confirmed the reduced expression at the plasma membrane. Förster resonance energy transfer measurements using confocal microscopy showed that the latter was not due to impaired trafficking, but to a failure to assemble the tetramer. This was further confirmed with co-immunoprecipitation experiments. The corresponding arginine substitution in Kv6.4 prevented its heterotetrameric interaction with Kv2.1. These results indicate that these aspartates (especially the first one) in the A/B box linker of the T1 domain are required for efficient assembly of both homotetrameric Kv2.1 and heterotetrameric Kv2.1/silent Kv6.4 channels.
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Affiliation(s)
- Elke Bocksteins
- Department of Biomedical Sciences, Laboratory for Molecular Biophysics, Physiology and Pharmacology, University of Antwerp, CDE, Universiteitsplein 1, 2610 Antwerp, Belgium
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Davis AM, Berg JM. Homodimerization and heterodimerization of minimal zinc(II)-binding-domain peptides of T-cell proteins CD4, CD8alpha, and Lck. J Am Chem Soc 2009; 131:11492-7. [PMID: 19624124 PMCID: PMC2769085 DOI: 10.1021/ja9028928] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
Metal-mediated protein oligomerization is an emerging mode of protein-protein interaction. The C-terminal cytosolic domains of T-cell coreceptors CD4 and CD8alpha form zinc-bridged heterodimers with the N-terminal region of the kinase Lck, with each protein contributing two cysteinate ligands to the complex. Using size exclusion chromatography, (1)H NMR, and UV/visible absorption spectroscopy with cobalt(II) as a spectroscopic probe, we demonstrate that small peptides derived from these regions form metal-bridged heterodimers but also homodimers, in contrast to previous reports. The Lck-CD4 and Lck-CD8alpha cobalt(II)-bridged heterodimer complexes are more stable than the corresponding (Lck)(2)cobalt(II) complex by factors of 11 +/- 4 and 22 +/- 9, respectively. These studies were aided by the discovery that cobalt(II) complexes with a cobalt(II)(-Cys-X-X-Cys-)(-Cys-X-Cys-) chromophore show unusual optical spectra with one component of the visible d-d ((4)A(2)-to-(4)T(1)(P)) transition red-shifted and well separated from the other components. These results provide insights into the basis of specificity of metal-bridged complex formation and on the potential biological significance of metal-bridged homodimers in T-cells.
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Affiliation(s)
| | - Jeremy M. Berg
- Contribution from the Laboratory of Molecular Biology, National Institute of Diabetes, Digestive & Kidney Disorders, National Institutes of Health, Bethesda, Maryland 20892
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Winklmeier A, Weyand M, Schreier C, Kalbitzer HR, Kremer W. Crystallization and preliminary X-ray diffraction studies of the tetramerization domain derived from the human potassium channel Kv1.3. Acta Crystallogr Sect F Struct Biol Cryst Commun 2009; 65:688-91. [PMID: 19574640 DOI: 10.1107/s1744309109019514] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/24/2009] [Accepted: 05/22/2009] [Indexed: 11/10/2022]
Abstract
The tetramerization domain (T1 domain) derived from the voltage-dependent potassium channel Kv1.3 of Homo sapiens was recombinantly expressed in Escherichia coli and purified. The crystals were first grown in an NMR tube in 150 mM potassium phosphate pH 6.5 in the absence of additional precipitants. The crystals showed I4 symmetry characteristic of the naturally occurring tetrameric assembly of the single subunits. A complete native data set was collected to 1.2 A resolution at 100 K using synchrotron radiation.
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Affiliation(s)
- Andreas Winklmeier
- Department of Biophysics and Physical Biochemistry, University of Regensburg, D-93040 Regensburg, Germany
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Dementieva IS, Tereshko V, McCrossan ZA, Solomaha E, Araki D, Xu C, Grigorieff N, Goldstein SAN. Pentameric assembly of potassium channel tetramerization domain-containing protein 5. J Mol Biol 2009; 387:175-91. [PMID: 19361449 PMCID: PMC2670943 DOI: 10.1016/j.jmb.2009.01.030] [Citation(s) in RCA: 67] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/05/2008] [Revised: 01/16/2009] [Accepted: 01/19/2009] [Indexed: 11/27/2022]
Abstract
We report the X-ray crystal structure of human potassium channel tetramerization domain-containing protein 5 (KCTD5), the first member of the family to be so characterized. Four findings were unexpected. First, the structure reveals assemblies of five subunits while tetramers were anticipated; pentameric stoichiometry is observed also in solution by scanning transmission electron microscopy mass analysis and analytical ultracentrifugation. Second, the same BTB (bric-a-brac, tramtrack, broad complex) domain surface mediates the assembly of five KCTD5 and four voltage-gated K(+) (Kv) channel subunits; four amino acid differences appear crucial. Third, KCTD5 complexes have well-defined N- and C-terminal modules separated by a flexible linker that swivels by approximately 30 degrees; the C-module shows a new fold and is required to bind Golgi reassembly stacking protein 55 with approximately 1 microM affinity, as judged by surface plasmon resonance and ultracentrifugation. Fourth, despite the homology reflected in its name, KCTD5 does not impact the operation of Kv4.2, Kv3.4, Kv2.1, or Kv1.2 channels.
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Affiliation(s)
- Irina S. Dementieva
- Department of Pediatrics and Institute of Molecular Pediatric Sciences, University of Chicago, Chicago, Illinois, 60637, USA
| | - Valentina Tereshko
- Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois, 60637, USA
| | - Zoe A. McCrossan
- Department of Pediatrics and Institute of Molecular Pediatric Sciences, University of Chicago, Chicago, Illinois, 60637, USA
| | - Elena Solomaha
- Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois, 60637, USA
| | - Daniel Araki
- Department of Pediatrics and Institute of Molecular Pediatric Sciences, University of Chicago, Chicago, Illinois, 60637, USA
| | - Chen Xu
- Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, Massachusetts, 02454, USA
| | - Nikolaus Grigorieff
- Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, Massachusetts, 02454, USA
- Howard Hughes Medical Institute, Brandeis University, Waltham, Massachusetts, 02454, USA
| | - Steve A. N. Goldstein
- Department of Pediatrics and Institute of Molecular Pediatric Sciences, University of Chicago, Chicago, Illinois, 60637, USA
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Harvey M, Karolat J, Sakai Y, Sokolowski B. PPTX, a pentraxin domain-containing protein, interacts with the T1 domain of K v 4. J Neurosci Res 2009; 87:1841-7. [PMID: 19185023 DOI: 10.1002/jnr.22016] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2022]
Abstract
Voltage-gated K(+) (K(v)) channels reside as tetramers in the membrane. The events that coordinate folding, trafficking, and tetramerization are mediated by an array of associated proteins and phospholipids whose identification is vital to understanding the dynamic nature of channel expression and activity. An interaction between an A-type K(+) channel, K(v)4.2, and a protein containing a pentraxin domain (PPTX) was demonstrated in the cochlea (Duzhyy et al. [ 2005] J. Biol. Chem. 280:15165-15172). Here, we present results based on fold recognition and homology modeling that revealed the tetramerization (T1) domain of K(v)4.2 as a potential docking site for interacting proteins such as PPTX. By using this model, putative sites were experimentally tested with the yeast two-hybrid system to assay interactions between PPTX and the T1 domain of K(v)4.2 wild type (wt) and mutants (mut). Results showed that amino acid residues 86 and 118 in the T1 domain are essential for interaction, because replacing these negatively charged with neutrally charged amino acids inhibits interactions. Cotransfections of Chinese hamster ovary cells with PPTX and K(v)4.2wt further revealed that PPTX increases K(v)4.2 wt expression in vitro when analyzing total lysates, whereas interactions with K(v)4.2 microt resulted in a decrease. These studies suggest that portions of the T1 domain can act as docking sites for proteins such as PPTX, further underscoring the significance of this domain.
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Affiliation(s)
- Margaret Harvey
- Department of Otolaryngology-HNS, Otology Laboratory, University of South Florida College of Medicine, Tampa, Florida 33612, USA
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The desensitization gating of the MthK K+ channel is governed by its cytoplasmic amino terminus. PLoS Biol 2008; 6:e223. [PMID: 18959476 PMCID: PMC2573919 DOI: 10.1371/journal.pbio.0060223] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/08/2008] [Accepted: 07/29/2008] [Indexed: 01/04/2023] Open
Abstract
The RCK-containing MthK channel undergoes two inactivation processes: activation-coupled desensitization and acid-induced inactivation. The acid inactivation is mediated by the C-terminal RCK domain assembly. Here, we report that the desensitization gating is governed by a desensitization domain (DD) of the cytoplasmic N-terminal 17 residues. Deletion of DD completely removes the desensitization, and the process can be fully restored by a synthetic DD peptide added in trans. Mutagenesis analyses reveal a sequence-specific determinant for desensitization within the initial hydrophobic segment of DD. Proton nuclear magnetic resonance (1H NMR) spectroscopy analyses with synthetic peptides and isolated RCK show interactions between the two terminal domains. Additionally, we show that deletion of DD does not affect the acid-induced inactivation, indicating that the two inactivation processes are mutually independent. Our results demonstrate that the short N-terminal DD of MthK functions as a complete moveable module responsible for the desensitization. Its interaction with the C-terminal RCK domain may play a role in the gating process. Nerve cells use ion channels, pores in the cell membrane, to send messages in the form of electrical signals between cells. Most ion channels have evolved several elaborate mechanisms that allow the channels to close quickly after opening to prevent wasteful leakage of the electrochemical potential—the currency of neuron communication—across the cell membrane. The process is known as inactivation or desensitization. Previous study on the model RCK-containing MthK K+ channel in the enlarged Escherichia coli membrane has shown that this archaeon channel also undergoes desensitization. Using the same method, we demonstrate that the desensitization is indeed an intrinsic molecular property of the MthK protein. We show that a specific region of MthK, the short N terminus of the protein, functions as a structurally independent domain and is entirely responsible for the desensitization gating process. Moreover, we show that this N-terminal domain interacts with the C-terminal RCK domain as part of the desensitization mechanism. This unique desensitization mechanism, by interaction between the two cytoplasmic termini, is distinct from those traditional mechanisms known as N- and C-type inactivation found in many voltage-gated Na+ and K+ channels or as the desensitization observed in the glutamate receptors. Since the KTN/RCK domain is found in a large number of prokaryotic K+ channels and transporters, this unique mechanism may be common to these transport systems for regulating the K+ flux through the cell membrane. The N terminus of the ion channel MthK functions as a structurally independent domain and is entirely responsible for the desensitization gating process required for neuron-to-neuron communication.
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Mederos Y Schnitzler M, Rinné S, Skrobek L, Renigunta V, Schlichthörl G, Derst C, Gudermann T, Daut J, Preisig-Müller R. Mutation of histidine 105 in the T1 domain of the potassium channel Kv2.1 disrupts heteromerization with Kv6.3 and Kv6.4. J Biol Chem 2008; 284:4695-704. [PMID: 19074135 DOI: 10.1074/jbc.m808786200] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
The voltage-activated K(+) channel subunit Kv2.1 can form heterotetramers with members of the Kv6 subfamily, generating channels with biophysical properties different from homomeric Kv2.1 channels. The N-terminal tetramerization domain (T1) has been shown previously to play a role in Kv channel assembly, but the mechanisms controlling specific heteromeric assembly are still unclear. In Kv6.x channels the histidine residue of the zinc ion-coordinating C3H1 motif of Kv2.1 is replaced by arginine or valine. Using a yeast two-hybrid assay, we found that substitution of the corresponding histidine 105 in Kv2.1 by valine (H105V) or arginine (H105R) disrupted the interaction of the T1 domain of Kv2.1 with the T1 domains of both Kv6.3 and Kv6.4, whereas interaction of the T1 domain of Kv2.1 with itself was unaffected by this mutation. Using fluorescence resonance energy transfer (FRET), interaction could be detected between the subunits Kv2.1/Kv2.1, Kv2.1/Kv6.3, and Kv2.1/Kv6.4. Reduced FRET signals were obtained after co-expression of Kv2.1(H105V) or Kv2.1(H105R) with Kv6.3 or Kv6.4. Wild-type Kv2.1 but not Kv2.1(H105V) could be co-immunoprecipitated with Kv6.4. Co-expression of dominant-negative mutants of Kv6.3 reduced the current produced Kv2.1, but not of Kv2.1(H105R) mutants. Co-expression of Kv6.3 or Kv6.4 with wt Kv2.1 but not with Kv2.1(H105V) or Kv2.1(H105R) changed the voltage dependence of activation of the channels. Our results suggest that His-105 in the T1 domain of Kv2.1 is required for functional heteromerization with members of the Kv6 subfamily. We conclude from our findings that Kv2.1 and Kv6.x subunits have complementary T1 domains that control selective heteromerization.
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