FMP Publications

Our publications are recorded in a searchable database since 2010, updates will be added regularly.

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References
From mice to man: chloride transport in leukoencephalopathy
Jentsch, T. J.
Lancet Neurol, 12:626-628
(2013)

Tags: Physiology and Pathology of Ion Transport (Jentsch)

Common gating of both CLC transporter subunits underlies voltage-dependent activation of the 2Cl-/1H+ exchanger ClC-7/Ostm1
Ludwig, C. F., Ullrich, F., Leisle, L., Stauber, T.; Jentsch, T. J.
J Biol Chem, 288:28611-28619
(2013)

Tags: Physiology and Pathology of Ion Transport (Jentsch)

Abstract: CLC anion transporters form dimers that function either as Cl(-) channels or as electrogenic Cl(-)/H(+) exchangers. CLC channels display two different types of "gates," "protopore" gates that open and close the two pores of a CLC dimer independently of each other and common gates that act on both pores simultaneously. ClC-7/Ostm1 is a lysosomal 2Cl(-)/1H(+) exchanger that is slowly activated by depolarization. This gating process is drastically accelerated by many CLCN7 mutations underlying human osteopetrosis. Making use of some of these mutants, we now investigate whether slow voltage activation of plasma membrane-targeted ClC-7/Ostm1 involves protopore or common gates. Voltage activation of wild-type ClC-7 subunits was accelerated by co-expressing an excess of ClC-7 subunits carrying an accelerating mutation together with a point mutation rendering these subunits transport-deficient. Conversely, voltage activation of a fast ClC-7 mutant could be slowed by co-expressing an excess of a transport-deficient mutant. These effects did not depend on whether the accelerating mutation localized to the transmembrane part or to cytoplasmic cystathionine-beta-synthase (CBS) domains of ClC-7. Combining accelerating mutations in the same subunit did not speed up gating further. No currents were observed when ClC-7 was truncated after the last intramembrane helix. Currents and slow gating were restored when the C terminus was co-expressed by itself or fused to the C terminus of the beta-subunit Ostm1. We conclude that common gating underlies the slow voltage activation of ClC-7. It depends on the CBS domain-containing C terminus that does not require covalent binding to the membrane domain of ClC-7.

Vestibular role of KCNQ4 and KCNQ5 K+ channels revealed by mouse models
Spitzmaul, G., Tolosa(*), L., Winkelman(*), B. H., Heidenreich, M., Frens(*), M. A., Chabbert(*), C., De Zeeuw(*), C. I.; Jentsch, T. J.
J Biol Chem, 288:9334-9344
(2013)

Tags: Physiology and Pathology of Ion Transport (Jentsch)

Abstract: The function of sensory hair cells of the cochlea and vestibular organs depends on an influx of K(+) through apical mechanosensitive ion channels and its subsequent removal over their basolateral membrane. The KCNQ4 (Kv7.4) K(+) channel, which is mutated in DFNA2 human hearing loss, is expressed in the basal membrane of cochlear outer hair cells where it may mediate K(+) efflux. Like the related K(+) channel KCNQ5 (Kv7.5), KCNQ4 is also found at calyx terminals ensheathing type I vestibular hair cells where it may be localized pre- or postsynaptically. Making use of Kcnq4(-/-) mice lacking KCNQ4, as well as Kcnq4(dn/dn) and Kcnq5(dn/dn) mice expressing dominant negative channel mutants, we now show unambiguously that in adult mice both channels reside in postsynaptic calyx-forming neurons, but cannot be detected in the innervated hair cells. Accordingly, whole cell currents of vestibular hair cells did not differ between genotypes. Neither Kcnq4(-/-), Kcnq5(dn/dn) nor Kcnq4(-/-)/Kcnq5(dn/dn) double mutant mice displayed circling behavior found with severe vestibular impairment. However, a milder form of vestibular dysfunction was apparent from altered vestibulo-ocular reflexes in Kcnq4(-/-)/Kcnq5(dn/dn) and Kcnq4(-/-) mice. The larger impact of KCNQ4 may result from its preferential expression in central zones of maculae and cristae, which are innervated by phasic neurons that are more sensitive than the tonic neurons present predominantly in the surrounding peripheral zones where KCNQ5 is found. The impact of postsynaptic KCNQ4 on vestibular function may be related to K(+) removal and modulation of synaptic transmission.

Chloride in vesicular trafficking and function
Stauber, T.; Jentsch, T. J.
Annu Rev Physiol, 75:453-477
(2013)

Tags: Physiology and Pathology of Ion Transport (Jentsch)

Abstract: Luminal acidification is of pivotal importance for the physiology of the secretory and endocytic pathways and its diverse trafficking events. Acidification by the proton-pumping V-ATPase requires charge compensation by counterion currents that are commonly attributed to chloride. The molecular identification of intracellular chloride transporters and the improvement of methodologies for measuring intraorganellar pH and chloride have facilitated the investigation of the physiology of vesicular chloride transport. New data question the requirement of chloride for pH regulation of various organelles and furthermore ascribe functions to chloride that are beyond merely electrically shunting the proton pump. This review surveys the currently established and proposed intracellular chloride transporters and gives an overview of membrane-trafficking steps that are affected by the perturbation of chloride transport. Finally, potential mechanisms of membrane-trafficking modulation by chloride are discussed and put into the context of organellar ion homeostasis in general.

Exome sequencing reveals new causal mutations in children with epileptic encephalopathies
Veeramah(*), K. R., Johnstone(*), L., Karafet(*), T. M., Wolf(*), D., Sprissler(*), R., Salogiannis(*), J., Barth-Maron(*), A., Greenberg(*), M. E., Stuhlmann, T., Weinert, S., Jentsch, T. J., Pazzi(*), M., Restifo(*), L. L., Talwar(*), D., Erickson(*), R. P.; Hammer(*), M. F.
Epilepsia, 54:1270-1281
(2013)

Tags: Physiology and Pathology of Ion Transport (Jentsch)

Abstract: Purpose: The management of epilepsy in children is particularly challenging when seizures are resistant to antiepileptic medications, or undergo many changes in seizure type over time, or have comorbid cognitive, behavioral, or motor deficits. Despite efforts to classify such epilepsies based on clinical and electroencephalographic criteria, many children never receive a definitive etiologic diagnosis. Whole exome sequencing (WES) is proving to be a highly effective method for identifying de novo variants that cause neurologic disorders, especially those associated with abnormal brain development. Herein we explore the utility of WES for identifying candidate causal de novo variants in a cohort of children with heterogeneous sporadic epilepsies without etiologic diagnoses. Methods: We performed WES (mean coverage approximately 403) on 10 trios comprised of unaffected parents and a child with sporadic epilepsy characterized by difficult-to-control seizures and some combination of developmental delay, epileptic encephalopathy, autistic features, cognitive impairment, or motor deficits. Sequence processing and variant calling were performed using standard bioinformatics tools. A custom filtering system was used to prioritize de novo variants of possible functional significance for validation by Sanger sequencing. Key Findings: In 9 of 10 probands, we identified one or more de novo variants predicted to alter protein function, for a total of 15. Four probands had de novo mutations in genes previously shown to harbor heterozygous mutations in patients with severe, early onset epilepsies (two in SCN1A, and one each in CDKL5 and EEF1A2). In three children, the de novo variants were in genes with functional roles that are plausibly relevant to epilepsy (KCNH5, CLCN4, and ARHGEF15). The variant in KCNH5 alters one of the highly conserved arginine residues of the voltage sensor of the encoded voltage-gated potassium channel. In vitro analyses using cell-based assays revealed that the CLCN4 mutation greatly impaired ion transport by the ClC-4 2Cl(-)/H+-exchanger and that the mutation in ARHGEF15 reduced GEF exchange activity of the gene product, Ephexin5, by about 50%. Of interest, these seven probands all presented with seizures within the first 6 months of life, and six of these have intractable seizures. Significance: The finding that 7 of 10 children carried de novo mutations in genes of known or plausible clinical significance to neuronal excitability suggests that WES will be of use for the molecular genetic diagnosis of sporadic epilepsies in children, especially when seizures are of early onset and difficult to control.

The mechanism of denaturation and the unfolded state of the alpha-helical membrane-associated protein Mistic
Jacso, T., Bardiaux, B., Broecker(*), J., Fiedler(*), S., Baerwinkel(*), T., Mainz, A., Fink, U., Vargas(*), C., Oschkinat, H., Keller(*), S.; Reif, B.
J Am Chem Soc, 135:18884-18891
(2013)

Tags: Solid-State NMR Spectroscopy (Reif), NMR-Supported Structure Biology (Oschkinat)

Abstract: In vitro protein-folding studies using chemical denaturants such as urea are indispensible in elucidating the forces and mechanisms determining the stability, structure, and dynamics of water-soluble proteins. By contrast, alpha-helical membrane-associated proteins largely evade such approaches because they are resilient to extensive unfolding. We have used optical and NMR spectroscopy to provide an atomistic-level dissection of the effects of urea on the structure and dynamics of the alpha-helical membrane-associated protein Mistic as well as its interactions with detergent and solvent molecules. In the presence of the zwitterionic detergent lauryl dimethylamine oxide, increasing concentrations of urea result in a complex sequence of conformational changes that go beyond simple two-state unfolding. Exploiting this finding, we report the first high-resolution structural models of the urea denaturation process of an alpha-helical membrane-associated protein and its completely unfolded state, which contains almost no regular secondary structure but nevertheless retains a topology close to that of the folded state.

What's in a name? Why these proteins are intrinsically disordered: Why these proteins are intrinsically disordered
Dunker(*), A. K., Babu(*), M. M., Barbar(*), E., Blackledge(*), M., Bondos(*), S. E., Dosztanyi(*), Z., Dyson(*), H. J., Forman-Kay(*), J., Fuxreiter(*), M., Gsponer(*), J., Han(*), K. H., Jones(*), D. T., Longhi(*), S., Metallo(*), S. J., Nishikawa(*), K., Nussinov(*), R., Obradovic(*), Z., Pappu(*), R. V., Rost(*), B., Selenko, P., Subramaniam(*), V., Sussman(*), J. L., Tompa(*), P.; Uversky(*), V. N.
Intrinsically disordered proteins, 1:e24157
(2013)

Tags: In-Cell NMR (Selenko)

Abstract: "What's in a name? That which we call a rose By any other name would smell as sweet." From "Romeo and Juliet", William Shakespeare (1594) This article opens a series of publications on disambiguation of the basic terms used in the field of intrinsically disordered proteins. We start from the beginning, namely from the explanation of what the expression "intrinsically disordered protein" actually means and why this particular term has been chosen as the common denominator for this class of proteins characterized by broad structural, dynamic and functional characteristics.

Mapping discontinuous protein-binding sites via structure-based peptide libraries: combining in silico and in vitro approaches
Jaeger(*), I. S., Kretzschmar(*), I., Körner, J., Weiser(*), A. A., Mahrenholz(*), C. C., Potty(*), A., Kourentzi(*), K., Willson(*), R. C., Volkmer(*), R.; Preissner(*), R.
J Mol Recognit, 26:23-31
(2013)

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: To perform their various functions, protein surfaces often have to interact with each other in a specific way. Usually, only parts of a protein are accessible and can act as binding sites. Because proteins consist of polypeptide chains that fold into complex three-dimensional shapes, binding sites can be divided into two different types: linear sites that follow the primary amino acid sequence and discontinuous binding sites, which are made up of short peptide fragments that are adjacent in spatial proximity. Such discontinuous binding sites dominate proteinprotein interactions, but are difficult to identify. To meet this challenge, we combined a computational, structure-based approach and an experimental, high-throughput method. SUPERFICIAL is a program that uses protein structures as input and generates peptide libraries to represent the protein's surface. A large number of the predicted peptides can be simultaneously synthesised applying the SPOT technology. The results of a binding assay subsequently help to elucidate proteinprotein interactions; the approach is applicable to any kind of protein. The crystal structure of the complex of hen egg lysozyme with the well-characterised murine IgG1 antibody HyHEL-5 is available, and the complex is known to have a discontinuous binding site. Using SUPERFICIAL, the entire surface of lysozyme was translated into a peptide library that was synthesised on a cellulose membrane using the SPOT technology and tested against the HyHEL-5 antibody. In this way, it was possible to identify two peptides (longest common sequence and peptide 19) that represented the discontinuous epitope of lysozyme. Copyright (c) 2012 John Wiley & Sons, Ltd.

A Floquet description of phase alternated sequences for efficient homonuclear recoupling in solid perdeuterated systems
Jayanthi(*), S., Akbey, Ü., Uluca(*), B., Oschkinat, H.; Vega(*), S.
Journal of Magnetic Resonance, 234:10-20
(2013)

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: A Floquet description of a phase alternated homonuclear recoupling scheme for perdeuterated systems is presented. As a result, we demonstrate improvements in the recoupling efficiency of the DOuble Nucleus Enhanced Recoupling [DONER; J. Am. Chem. Soc. 131 (2009) 170541 technique by utilizing Phase Alternated Recoupling Irradiation Schemes [PARIS; Chem. Phys. Lett. 469 (2009) 342]. The effect of proton and deuterium radio frequency irradiation during recoupling has been systematically studied and theoretical observations have been verified experimentally using a deuterated model compound, L-Alanine, at 10 and 20 kHz magic angle spinning frequency. Experimental results are well in agreement with theoretical observations, thereby significantly increasing the recoupling efficiency of conventional DONER in perdeuterated systems. (C) 2013 Elsevier Inc. All rights reserved.

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Leibniz-Forschungsinstitut für Molekulare Pharmakologie im Forschungsverbund Berlin e.V. (FMP)
Campus Berlin-Buch
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13125 Berlin, Germany
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info(at)fmp-berlin.de

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