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Cryo-EM structures of the human cation-chloride cotransporter KCC1.
Liu S, Chang S, Han B, Xu L, Zhang M, Zhao C, Yang W, Wang F, Li J, Delpire E, Ye S, Bai XC, Guo J
(2019) Science 366: 505-508
MeSH Terms: Amino Acid Sequence, Animals, Binding Sites, Cryoelectron Microscopy, HEK293 Cells, Humans, Ion Transport, Mice, Molecular Dynamics Simulation, Oocytes, Protein Domains, Protein Multimerization, Protein Structure, Quaternary, Sequence Alignment, Sodium-Potassium-Chloride Symporters, Symporters, Xenopus laevis
Show Abstract · Added March 18, 2020
Cation-chloride cotransporters (CCCs) mediate the coupled, electroneutral symport of cations with chloride across the plasma membrane and are vital for cell volume regulation, salt reabsorption in the kidney, and γ-aminobutyric acid (GABA)-mediated modulation in neurons. Here we present cryo-electron microscopy (cryo-EM) structures of human potassium-chloride cotransporter KCC1 in potassium chloride or sodium chloride at 2.9- to 3.5-angstrom resolution. KCC1 exists as a dimer, with both extracellular and transmembrane domains involved in dimerization. The structural and functional analyses, along with computational studies, reveal one potassium site and two chloride sites in KCC1, which are all required for the ion transport activity. KCC1 adopts an inward-facing conformation, with the extracellular gate occluded. The KCC1 structures allow us to model a potential ion transport mechanism in KCCs and provide a blueprint for drug design.
Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.
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17 MeSH Terms
SLC12A ion transporter mutations in sporadic and familial human congenital hydrocephalus.
Jin SC, Furey CG, Zeng X, Allocco A, Nelson-Williams C, Dong W, Karimy JK, Wang K, Ma S, Delpire E, Kahle KT
(2019) Mol Genet Genomic Med 7: e892
MeSH Terms: Animals, Cerebral Aqueduct, Genetic Diseases, X-Linked, Humans, Hydrocephalus, Male, Mutation, Neural Cell Adhesion Molecule L1, Symporters, Whole Exome Sequencing, Xenopus laevis
Show Abstract · Added March 18, 2020
BACKGROUND - Congenital hydrocephalus (CH) is a highly morbid disease that features enlarged brain ventricles and impaired cerebrospinal fluid homeostasis. Although early linkage or targeted sequencing studies in large multigenerational families have localized several genes for CH, the etiology of most CH cases remains unclear. Recent advances in whole exome sequencing (WES) have identified five new bona fide CH genes, implicating impaired regulation of neural stem cell fate in CH pathogenesis. Nonetheless, in the majority of CH cases, the pathological etiology remains unknown, suggesting more genes await discovery.
METHODS - WES of family members of a sporadic and familial form of severe L1CAM mutation-negative CH associated with aqueductal stenosis was performed. Rare genetic variants were analyzed, prioritized, and validated. De novo copy number variants (CNVs) were identified using the XHMM algorithm and validated using qPCR. Xenopus oocyte experiments were performed to access mutation impact on protein function and expression.
RESULTS - A novel inherited protein-damaging mutation (p.Pro605Leu) in SLC12A6, encoding the K -Cl cotransporter KCC3, was identified in both affected members of multiplex kindred CHYD110. p.Pro605 is conserved in KCC3 orthologs and among all human KCC paralogs. The p.Pro605Leu mutation maps to the ion-transporting domain, and significantly reduces KCC3-dependent K transport. A novel de novo CNV (deletion) was identified in SLC12A7, encoding the KCC3 paralog and binding partner KCC4, in another family (CHYD130) with sporadic CH.
CONCLUSION - These findings identify two novel, related genes associated with CH, and implicate genetically encoded impairments in ion transport for the first time in CH pathogenesis.
© 2019 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.
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11 MeSH Terms
Developmental regulation of Wnt signaling by Nagk and the UDP-GlcNAc salvage pathway.
Neitzel LR, Spencer ZT, Nayak A, Cselenyi CS, Benchabane H, Youngblood CQ, Zouaoui A, Ng V, Stephens L, Hann T, Patton JG, Robbins D, Ahmed Y, Lee E
(2019) Mech Dev 156: 20-31
MeSH Terms: Animals, Body Patterning, Drosophila, Embryonic Development, Evolution, Molecular, Gene Expression Regulation, Developmental, Glycosylation, Humans, Phosphotransferases (Alcohol Group Acceptor), Wnt Signaling Pathway, Xenopus laevis, Zebrafish
Show Abstract · Added April 10, 2019
In a screen for human kinases that regulate Xenopus laevis embryogenesis, we identified Nagk and other components of the UDP-GlcNAc glycosylation salvage pathway as regulators of anteroposterior patterning and Wnt signaling. We find that the salvage pathway does not affect other major embryonic signaling pathways (Fgf, TGFβ, Notch, or Shh), thereby demonstrating specificity for Wnt signaling. We show that the role of the salvage pathway in Wnt signaling is evolutionarily conserved in zebrafish and Drosophila. Finally, we show that GlcNAc is essential for the growth of intestinal enteroids, which are highly dependent on Wnt signaling for growth and maintenance. We propose that the Wnt pathway is sensitive to alterations in the glycosylation state of a cell and acts as a nutritional sensor in order to couple growth/proliferation with its metabolic status. We also propose that the clinical manifestations observed in congenital disorders of glycosylation (CDG) in humans may be due, in part, to their effects on Wnt signaling during development.
Copyright © 2019 Elsevier B.V. All rights reserved.
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12 MeSH Terms
Mistargeting of a truncated Na-K-2Cl cotransporter in epithelial cells.
Koumangoye R, Omer S, Delpire E
(2018) Am J Physiol Cell Physiol 315: C258-C276
MeSH Terms: Animals, Cell Membrane, Cells, Cultured, Colon, Cytoplasm, Dogs, Epithelial Cells, Female, Madin Darby Canine Kidney Cells, Male, Mice, Oocytes, Salivary Glands, Sodium-Potassium-Chloride Symporters, Sodium-Potassium-Exchanging ATPase, Solute Carrier Family 12, Member 2, Xenopus laevis
Show Abstract · Added May 4, 2018
We recently reported the case of a young patient with multisystem failure carrying a de novo mutation in SLC12A2, the gene encoding the Na-K-2Cl cotransporter-1 (NKCC1). Heterologous expression studies in nonepithelial cells failed to demonstrate dominant-negative effects. In this study, we examined expression of the mutant cotransporter in epithelial cells. Using Madin-Darby canine kidney (MDCK) cells grown on glass coverslips, permeabilized support, and Matrigel, we show that the fluorescently tagged mutant cotransporter is expressed in cytoplasm and at the apical membrane and affects epithelium integrity. Expression of the mutant transporter at the apical membrane also results in the mislocalization of some of the wild-type transporter to the apical membrane. This mistargeting is specific to NKCC1 as the Na-K-ATPase remains localized on the basolateral membrane. To assess transporter localization in vivo, we created a mouse model using CRISPR/cas9 that reproduces the 11 bp deletion in exon 22 of Slc12a2. Although the mice do not display an overt phenotype, we show that the colon and salivary gland expresses wild-type NKCC1 abundantly at the apical pole, confirming the data obtained in cultured epithelial cells. Enough cotransporter must remain, however, on the basolateral membrane to participate in saliva secretion, as no significant decrease in saliva production was observed in the mutant mice.
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17 MeSH Terms
Functional features of the "finger" domain of the DEG/ENaC channels MEC-4 and UNC-8.
Matthewman C, Johnson CK, Miller DM, Bianchi L
(2018) Am J Physiol Cell Physiol 315: C155-C163
MeSH Terms: Amino Acid Sequence, Animals, Calcium, Cell Death, Cell Membrane Permeability, Epithelial Sodium Channels, Magnesium, Membrane Proteins, Mutation, Oocytes, Protein Transport, Sodium, Xenopus laevis
Show Abstract · Added March 26, 2019
UNC-8 and MEC-4 are two members of the degenerin/epithelial Na channel (DEG/ENaC) family of voltage-independent Na channels that share a high degree of sequence homology and functional similarity. For example, both can be hyperactivated by genetic mutations [UNC-8(d) and MEC-4(d)] that induce neuronal death by necrosis. Both depend in vivo on chaperone protein MEC-6 for function, as demonstrated by the finding that neuronal death induced by hyperactive UNC-8 and MEC-4 channels is prevented by null mutations in mec-6. UNC-8 and MEC-4 differ functionally in three major ways: 1) MEC-4 is calcium permeable, whereas UNC-8 is not; 2) UNC-8, but not MEC-4, is blocked by extracellular calcium and magnesium in the micromolar range; and 3) MEC-6 increases the number of MEC-4 channels at the cell surface in oocytes but does not have this effect on UNC-8. We previously reported that Capermeability of MEC-4 is conferred by the second transmembrane domain. We show here that the extracellular "finger" domain of UNC-8 is sufficient to mediate inhibition by divalent cations and that regulation by MEC-6 also depends on this region. Thus, our work confirms that the finger domain houses residues involved in gating of this channel class and shows for the first time that the finger domain also mediates regulation by chaperone protein MEC-6. Given that the finger domain is the most divergent region across the DEG/ENaC family, we speculate that it influences channel trafficking and function in a unique manner depending on the channel subunit.
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13 MeSH Terms
Structural Mechanism of Functional Modulation by Gene Splicing in NMDA Receptors.
Regan MC, Grant T, McDaniel MJ, Karakas E, Zhang J, Traynelis SF, Grigorieff N, Furukawa H
(2018) Neuron 98: 521-529.e3
MeSH Terms: Animals, Cell Line, Female, HEK293 Cells, Humans, Insecta, Protein Splicing, Protein Structure, Secondary, Protein Structure, Tertiary, Receptors, N-Methyl-D-Aspartate, Xenopus laevis
Show Abstract · Added April 10, 2019
Alternative gene splicing gives rise to N-methyl-D-aspartate (NMDA) receptor ion channels with defined functional properties and unique contributions to calcium signaling in a given chemical environment in the mammalian brain. Splice variants possessing the exon-5-encoded motif at the amino-terminal domain (ATD) of the GluN1 subunit are known to display robustly altered deactivation rates and pH sensitivity, but the underlying mechanism for this functional modification is largely unknown. Here, we show through cryoelectron microscopy (cryo-EM) that the presence of the exon 5 motif in GluN1 alters the local architecture of heterotetrameric GluN1-GluN2 NMDA receptors and creates contacts with the ligand-binding domains (LBDs) of the GluN1 and GluN2 subunits, which are absent in NMDA receptors lacking the exon 5 motif. The unique interactions established by the exon 5 motif are essential to the stability of the ATD/LBD and LBD/LBD interfaces that are critically involved in controlling proton sensitivity and deactivation.
Copyright © 2018 Elsevier Inc. All rights reserved.
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11 MeSH Terms
Chemical Screening Using Cell-Free Egg Extract.
Broadus MR, Lee E
(2018) Cold Spring Harb Protoc 2018:
MeSH Terms: Animals, Cell-Free System, Drug Evaluation, Preclinical, Ovum, Xenopus laevis
Show Abstract · Added April 8, 2018
Most drug screening methods use purified proteins, cultured cells, and/or small model organisms such as , zebrafish, flies, or nematodes. These systems have proven successes in drug discovery, but they also have weaknesses. Although purified cellular components allow for identification of compounds with activity against specific targets, such systems lack the complex biological interactions present in cellular and organismal screens. In vivo systems overcome these weaknesses, but the lack of cellular permeability, efflux by cellular pumps, and/or toxicity can be major limitations. egg extract, a concentrated and biologically active cytosol, can potentially overcome these weaknesses. Drug interactions occur in a near-physiological milieu, thereby functioning in a "truer" endogenous manner than purified components. Also, egg extract is a cell-free system that lacks intact plasma membranes that could restrict drug access to potential targets. Finally, egg extract is readily manipulated at the protein level: Proteins are easily depleted or added to the system, an important feature for analyzing drug effects in disease states. Thus, egg extract offers an attractive media for screening drugs that merges strengths of both in vitro and in vivo systems.
© 2018 Cold Spring Harbor Laboratory Press.
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5 MeSH Terms
Differential abundance of CK1α provides selectivity for pharmacological CK1α activators to target WNT-dependent tumors.
Li B, Orton D, Neitzel LR, Astudillo L, Shen C, Long J, Chen X, Kirkbride KC, Doundoulakis T, Guerra ML, Zaias J, Fei DL, Rodriguez-Blanco J, Thorne C, Wang Z, Jin K, Nguyen DM, Sands LR, Marchetti F, Abreu MT, Cobb MH, Capobianco AJ, Lee E, Robbins DJ
(2017) Sci Signal 10:
MeSH Terms: Animals, Antineoplastic Agents, Benzoates, Casein Kinase Ialpha, Enzyme Activation, Enzyme Activators, Gene Expression Regulation, Neoplastic, HCT116 Cells, Humans, Mice, Mice, Inbred BALB C, Mice, Inbred C57BL, Mice, Nude, Neoplasm Metastasis, Neoplasms, Organ Culture Techniques, Phosphorylation, Pyrvinium Compounds, Signal Transduction, Surface Plasmon Resonance, Wnt Proteins, Wnt Signaling Pathway, Xenograft Model Antitumor Assays, Xenopus laevis
Show Abstract · Added July 18, 2017
Constitutive WNT activity drives the growth of various human tumors, including nearly all colorectal cancers (CRCs). Despite this prominence in cancer, no WNT inhibitor is currently approved for use in the clinic largely due to the small number of druggable signaling components in the WNT pathway and the substantial toxicity to normal gastrointestinal tissue. We have shown that pyrvinium, which activates casein kinase 1α (CK1α), is a potent inhibitor of WNT signaling. However, its poor bioavailability limited the ability to test this first-in-class WNT inhibitor in vivo. We characterized a novel small-molecule CK1α activator called SSTC3, which has better pharmacokinetic properties than pyrvinium, and found that it inhibited the growth of CRC xenografts in mice. SSTC3 also attenuated the growth of a patient-derived metastatic CRC xenograft, for which few therapies exist. SSTC3 exhibited minimal gastrointestinal toxicity compared to other classes of WNT inhibitors. Consistent with this observation, we showed that the abundance of the SSTC3 target, CK1α, was decreased in WNT-driven tumors relative to normal gastrointestinal tissue, and knocking down CK1α increased cellular sensitivity to SSTC3. Thus, we propose that distinct CK1α abundance provides an enhanced therapeutic index for pharmacological CK1α activators to target WNT-driven tumors.
Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.
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24 MeSH Terms
Molecular Basis for Subtype Specificity and High-Affinity Zinc Inhibition in the GluN1-GluN2A NMDA Receptor Amino-Terminal Domain.
Romero-Hernandez A, Simorowski N, Karakas E, Furukawa H
(2016) Neuron 92: 1324-1336
MeSH Terms: 2-Hydroxyphenethylamine, Animals, Binding Sites, Blotting, Western, Crystallography, Hydrogen Bonding, Piperidines, Protein Structure, Quaternary, Receptors, N-Methyl-D-Aspartate, Sf9 Cells, Spodoptera, Xenopus laevis, Zinc
Show Abstract · Added April 3, 2018
Zinc is vastly present in the mammalian brain and controls functions of various cell surface receptors to regulate neurotransmission. A distinctive characteristic of N-methyl-D-aspartate (NMDA) receptors containing a GluN2A subunit is that their ion channel activity is allosterically inhibited by a nano-molar concentration of zinc that binds to an extracellular domain called an amino-terminal domain (ATD). Despite physiological importance, the molecular mechanism underlying the high-affinity zinc inhibition has been incomplete because of the lack of a GluN2A ATD structure. Here we show the first crystal structures of the heterodimeric GluN1-GluN2A ATD, which provide the complete map of the high-affinity zinc-binding site and reveal distinctive features from the ATD of the GluN1-GluN2B subtype. Perturbation of hydrogen bond networks at the hinge of the GluN2A bi-lobe structure affects both zinc inhibition and open probability, supporting the general model in which the bi-lobe motion in ATD regulates the channel activity in NMDA receptors.
Copyright © 2016 Elsevier Inc. All rights reserved.
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MeSH Terms
KCNE1 induces fenestration in the Kv7.1/KCNE1 channel complex that allows for highly specific pharmacological targeting.
Wrobel E, Rothenberg I, Krisp C, Hundt F, Fraenzel B, Eckey K, Linders JT, Gallacher DJ, Towart R, Pott L, Pusch M, Yang T, Roden DM, Kurata HT, Schulze-Bahr E, Strutz-Seebohm N, Wolters D, Seebohm G
(2016) Nat Commun 7: 12795
MeSH Terms: Adamantane, Allosteric Regulation, Animals, Binding Sites, Cross-Linking Reagents, Humans, Ion Channel Gating, KCNQ1 Potassium Channel, Models, Molecular, Mutagenesis, Mutation, Oocytes, Potassium Channel Blockers, Potassium Channels, Voltage-Gated, Tandem Mass Spectrometry, Xenopus laevis
Show Abstract · Added March 24, 2020
Most small-molecule inhibitors of voltage-gated ion channels display poor subtype specificity because they bind to highly conserved residues located in the channel's central cavity. Using a combined approach of scanning mutagenesis, electrophysiology, chemical ligand modification, chemical cross-linking, MS/MS-analyses and molecular modelling, we provide evidence for the binding site for adamantane derivatives and their putative access pathway in Kv7.1/KCNE1 channels. The adamantane compounds, exemplified by JNJ303, are highly potent gating modifiers that bind to fenestrations that become available when KCNE1 accessory subunits are bound to Kv7.1 channels. This mode of regulation by auxiliary subunits may facilitate the future development of potent and highly subtype-specific Kv channel inhibitors.
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MeSH Terms