56 results on '"Günther Schmalzing"'
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2. Amyloid beta peptide (Aβ 1–42 ) antagonizes nicotinic acetylcholine receptors of monocytes and enables ATP‐mediated release of interleukin‐1β
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Alisa Agné, Anna I Chaveiro, Klaus-Dieter Schlüter, Matthias Hecker, Ivan Manzini, Sigrid Wilker, Andreas Hecker, Martin Reichert, Veronika Grau, Günther Schmalzing, J. M. McIntosh, Raymond Ogiemwonyi-Schaefer, Winfried Padberg, Anca-Laura Amati, and Katrin Richter
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chemistry.chemical_classification ,biology ,Chemistry ,Amyloid beta ,Peptide ,Biochemistry ,Cell biology ,Interleukin 1β ,Nicotinic agonist ,Genetics ,biology.protein ,Molecular Biology ,Biotechnology ,Acetylcholine receptor - Published
- 2021
3. LncRNA UC.360+ shRNA Improves Diabetic Cardiac Sympathetic Dysfunction Mediated by the P2X4 Receptor in the Stellate Ganglion
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Minghao Sun, Guodong Li, Xiumei Xu, Runan Yang, Hong Nie, Zijing Li, Shuangmei Liu, Lin Li, Guilin Li, Shangdong Liang, Xinlu Ren, Günther Schmalzing, and Liran Shi
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medicine.medical_specialty ,Physiology ,Cognitive Neuroscience ,Stellate Ganglion ,In situ hybridization ,Biochemistry ,Diabetes Mellitus, Experimental ,Small hairpin RNA ,Rats, Sprague-Dawley ,03 medical and health sciences ,0302 clinical medicine ,Downregulation and upregulation ,Diabetes mellitus ,Internal medicine ,medicine ,Animals ,Humans ,RNA, Small Interfering ,Receptor ,030304 developmental biology ,0303 health sciences ,business.industry ,Cell Biology ,General Medicine ,medicine.disease ,Long non-coding RNA ,Rats ,Endocrinology ,medicine.anatomical_structure ,Diabetes Mellitus, Type 2 ,Stellate ganglion ,Tumor necrosis factor alpha ,RNA, Long Noncoding ,business ,Receptors, Purinergic P2X4 ,030217 neurology & neurosurgery - Abstract
Diabetic cardiac autonomic neuropathy (DCAN) is a complication that affects more than 60% of diabetic patients. There is evidence for the involvement of P2X4 receptor in DCAN. This study showed that the expression of the long noncoding RNA (lncRNA) UC.360+ was increased in the stellate ganglion (SG) of type 2 diabetes mellitus (DM) rats, and in situ hybridization revealed a clear presence of UC.360+ in SG neurons. The potential roles of UC.360+ in DCAN and its relationship with P2X4 receptor in SG were further explored via application of the short hairpin RNA (shRNA) against lncRNA UC.360+ in DM rats. The abnormal cardiac sympathetic changes in diabetic rats were improved after treatment with lncRNA UC.360+ shRNA. In the SG of these shRNA-treated DM rats, the upregulation of P2X4, tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β), and phosphorylated ERK1/2 was inhibited. Thus, lncRNA UC.360+ shRNA treatment may improve DCAN mediated by the P2X4 receptor in SG.
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- 2021
4. Flexible subunit stoichiometry of functional human P2X2/3 heteromeric receptors
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Anke Dopychai, Peter Illes, Maria Kowalski, Günther Schmalzing, Julia Schmid, Gabriele Stephan, Yong Tang, Patrizia Rubini, and Ralf Hausmann
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Patch-Clamp Techniques ,Purinergic P2X Receptor Antagonists ,Cations, Divalent ,Protein subunit ,Allosteric regulation ,Gi alpha subunit ,Biology ,Transfection ,Membrane Potentials ,Interleukin 10 receptor, alpha subunit ,Purinergic P2X Receptor Agonists ,Xenopus laevis ,Cellular and Molecular Neuroscience ,Adenosine Triphosphate ,Allosteric Regulation ,Phenols ,Heterotrimeric G protein ,Animals ,Humans ,Polycyclic Compounds ,Receptor ,Pharmacology ,Dose-Response Relationship, Drug ,Cell Membrane ,Wild type ,body regions ,HEK293 Cells ,Biochemistry ,Competitive antagonist ,Mutation ,Oocytes ,Calcium ,Receptors, Purinergic P2X3 ,Receptors, Purinergic P2X2 - Abstract
The aim of the present work was to clarify whether heterotrimeric P2X2/3 receptors have a fixed subunit stoichiometry consisting of one P2X2 and two P2X3 subunits as previously suggested, or a flexible stoichiometry containing also the inverse subunit composition. For this purpose we transfected HEK293 cells with P2X2 and P2X3 encoding cDNA at the ratios of 1:2 and 4:1, and analysed the biophysical and pharmacological properties of the generated receptors by means of the whole-cell patch-clamp technique. The concentration-response curves for the selective agonist α,β-meATP did not differ from each other under the two transfection ratios. However, co-expression of an inactive P2X2 mutant and the wild type P2X3 subunit and vice versa resulted in characteristic distortions of the α,β-meATP concentration–response relationships, depending on which subunit was expressed in excess, suggesting that HEK293 cells express mixtures of (P2X2)1/(P2X3)2 and (P2X2)2/(P2X3)1 receptors. Whereas the allosteric modulators H+ and Zn2+ failed to discriminate between the two possible heterotrimeric receptor variants, the α,β-meATP-induced responses were blocked more potently by the competitive antagonist A317491, when the P2X2 subunit was expressed in deficit of the P2X3 subunit. Furthermore, blue-native PAGE analysis of P2X2 and P2X3 subunits co-expressed in Xenopus laevis oocytes and HEK293 cells revealed that plasma membrane-bound P2X2/3 receptors appeared in two clearly distinct heterotrimeric complexes: a (P2X2-GFP)2/(P2X3)1 complex and a (P2X2-GFP)1/(P2X3)2 complex. These data strongly indicate that the stoichiometry of the heteromeric P2X2/3 receptor is not fixed, but determined in a permutational manner by the relative availability of P2X2 and P2X3 subunits.
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- 2015
5. Key Sites for P2X Receptor Function and Multimerization: Overview of Mutagenesis Studies on a Structural Basis
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Ralf Hausmann, Günther Schmalzing, and Achim Kless
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Models, Molecular ,Ion flow ,Protein Conformation ,Computational biology ,P2X quaternary structure ,Biochemistry ,Article ,Protein structure ,Complementary DNA ,Drug Discovery ,mutational P2X receptor analysis ,P2X assembly domains ,Animals ,Humans ,Binding site ,P2X receptor function ,Receptor ,Zebrafish ,Ion channel ,P2X ATP binding pocket ,Pharmacology ,Binding Sites ,biology ,Organic Chemistry ,biology.organism_classification ,Structure and function ,Receptors, Purinergic P2X ,Mutagenesis, Site-Directed ,Molecular Medicine - Abstract
P2X receptors constitute a seven-member family (P2X1-7) of extracellular ATP-gated cation channels of widespread expression. Because P2X receptors have been implicated in neurological, inflammatory and cardiovascular diseases, they constitute promising drug targets. Since the first P2X cDNA sequences became available in 1994, numerous site-directed mutagenesis studies have been conducted to disclose key sites of P2X receptor function and oligomerization. The publication of the 3-A crystal structures of the zebrafish P2X4 (zfP2X4) receptor in the homotrimeric apo-closed and ATP-bound open states in 2009 and 2012, respectively, has ushered a new era by allowing for the interpretation of the wealth of molecular data in terms of specific three-dimensional models and by paving the way for designing more-decisive experiments. Thanks to these structures, the last five years have provided invaluable insight into our understanding of the structure and function of the P2X receptor class of ligandgated ion channels. In this review, we provide an overview of mutagenesis studies of the pre- and post-crystal structure eras that identified amino acid residues of key importance for ligand binding, channel gating, ion flow, formation of the pore and the channel gate, and desensitization. In addition, the sites that are involved in the trimerization of P2X receptors are reviewed based on mutagenesis studies and interface contacts that were predicted by the zfP2X4 crystal structures.
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- 2015
6. A hydrophobic residue in position 15 of the rP2X3 receptor slows desensitization and reveals properties beneficial for pharmacological analysis and high-throughput screening
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Gregor Bahrenberg, Michaela Schumacher, Daniel Kuhlmann, Ursula Braam, Ralf Hausmann, Ilka Schlusche, Dagmar Bieler, and Günther Schmalzing
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Patch-Clamp Techniques ,Purinergic P2X Receptor Antagonists ,medicine.medical_treatment ,Molecular Sequence Data ,Mutant ,Membrane Potentials ,Xenopus laevis ,Cellular and Molecular Neuroscience ,Adenosine Triphosphate ,Cell Line, Tumor ,Homologous desensitization ,medicine ,Animals ,Humans ,Amino Acid Sequence ,Receptor ,Glucagon-like peptide 1 receptor ,Protease-activated receptor 2 ,Desensitization (medicine) ,Pharmacology ,Sigma-1 receptor ,Chemistry ,Optical Imaging ,High-Throughput Screening Assays ,Rats ,Cell biology ,body regions ,Biochemistry ,Mechanism of action ,Mutation ,Calcium ,medicine.symptom ,Hydrophobic and Hydrophilic Interactions ,Receptors, Purinergic P2X3 ,Receptors, Purinergic P2X2 - Abstract
The homotrimeric P2X3 subtype, one of the seven members of the ATP-gated P2X receptor family, plays a role in sensory neurotransmission, including nociception. To overcome the bias resulting from fast desensitization of the P2X3 receptor in dose-response analyses, a non-desensitizing P2X2-X3 receptor chimera has been repeatedly used as a surrogate for the P2X3 receptor for functional analysis. Here, we show that only three of the P2X2-specific amino acid residues of the P2X2-X3 chimera, (19)P(21)V(22)I, are needed to confer a slowly desensitizing phenotype to the P2X3 receptor. The strongest delay in desensitization of the P2X3 receptor by a single residue was observed when (15)Ser was replaced by Val or another hydrophobic residue. Pharmacologically, the S(15)V-rP2X3 mutant behaved similarly to the wt-P2X3 receptor. Analysis of the S(15)V-rP2X3 receptor in 1321N1 astrocytoma cells by a common calcium-imaging-based assay showed 10-fold higher calcium transients relative to those of the wt-rP2X3 receptor. The S(15)V-rP2X3 cell line enabled reliable analysis of antagonistic potencies and correctly reported the mechanism of action of the P2X3 receptor antagonists A-317491 and TNP-ATP by a calcium-imaging assay. Together, these data suggest that the S(15)V-rP2X3 mutant may be suitable not only for automated fluorescence-based screening of molecule libraries for identification of lead compounds but also for facilitated pharmacological characterization of specific P2X3 receptor ligands. We suggest that the mechanism of desensitization of the P2X3 receptor may involve the movement of an N-terminal inactivation particle, in analogy to the "hinged-lid" or "ball and chain" mechanisms of voltage-gated NaV and Shaker KV channels, respectively.
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- 2014
7. The effect of anions on the human P2X7 receptor
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Christoph Kubick, Fritz Markwardt, and Günther Schmalzing
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Anions ,Agonist ,Stereochemistry ,medicine.drug_class ,Iodide ,Inorganic chemistry ,Anion ,Biophysics ,Chloride ,Biochemistry ,Xenopus laevis ,medicine ,Extracellular ,Animals ,Humans ,Patch clamp ,Anion binding ,Ion channel ,chemistry.chemical_classification ,biology ,Glutamate receptor ,Single channel ,Cell Biology ,P2 purinergic receptor ,P2X receptor ,chemistry ,P2X7 receptor ,Mutation ,biology.protein ,Receptors, Purinergic P2X7 ,medicine.drug ,Organic anion - Abstract
P2X7 receptors (P2X7Rs) are nonselective cation channels that are opened by the binding of extracellular ATP and are involved in the modulation of epithelial secretion, inflammation and nociception. Here, we investigated the effect of extracellular anions on channel gating and permeation of human P2X7Rs (hP2X7Rs) expressed in Xenopus laevis oocytes. Two-microelectrode voltage-clamp recordings showed that ATP-induced hP2X7R-mediated currents increased when extracellular chloride was substituted by the organic anions glutamate or aspartate and decreased when chloride was replaced by the inorganic anions nitrate, sulfate or iodide. ATP concentration-response comparisons revealed that substitution of chloride by glutamate decreased agonist efficacy, while substitution by iodide increased agonist efficacy at high ATP concentrations. Meanwhile, the ATP potency remained unchanged. Activation of the hP2X7R at low ATP concentrations via the high-affinity ATP effector site was not affected by the replacement of chloride by glutamate or iodide. To analyze the anion effect on the hP2X7R at the single-molecule level, we performed single-channel current measurements using the patch-clamp technique in the outside-out configuration. Chloride substitution did not affect the single-channel conductance, but the probability that the P2X7R channel was open increased when chloride was replaced by glutamate and decreased when chloride was replaced by iodide. This effect was due to an influence of the anions on the mean closed times of the hP2X7R channel. We conclude that hP2X7R channels are not anion-permeable in physiological Na+-based media and that external anions allosterically affect ion channel opening in the fully ATP4--liganded P2X7R through an extracellular anion binding site.This work was supported by Deutsche Forschungsgemeinschaft Grants Ma1581/15-1 (to F.M.) and Schm536/9-1 (to G. S.) and by the Medical Faculty of the Martin-Luther-University Roux Program Grant 22/18 (to F. M.).
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- 2011
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8. A Single Phenylalanine Residue in the Main Intracellular Loop of α1 γ-Aminobutyric Acid Type A and Glycine Receptors Influences Their Sensitivity to Propofol
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Gustavo Moraga-Cid, Luis G. Aguayo, Robert W. Peoples, Gonzalo E. Yévenes, and Günther Schmalzing
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Patch-Clamp Techniques ,Allosteric modulator ,Phenylalanine ,Molecular Sequence Data ,Transfection ,Aminobutyric acid ,Article ,gamma-Aminobutyric acid ,Receptors, Glycine ,Extracellular ,Humans ,Medicine ,Amino Acid Sequence ,Receptor ,Propofol ,Glycine receptor ,Alanine ,business.industry ,DNA ,Receptors, GABA-A ,Electrophysiological Phenomena ,Kinetics ,HEK293 Cells ,Anesthesiology and Pain Medicine ,Amino Acid Substitution ,Biochemistry ,Mutation ,Extracellular Space ,business ,Anesthetics, Intravenous ,Intracellular ,medicine.drug - Abstract
Background The intravenous anesthetic propofol acts as a positive allosteric modulator of glycine (GlyRs) and γ-aminobutyric acid type A (GABAARs) receptors. Although the role of transmembrane residues is recognized, little is known about the involvement of other regions in the modulatory effects of propofol. Therefore, the influence of the large intracellular loop in propofol sensitivity of both receptors was explored. Methods The large intracellular loop of α1 GlyRs and α1β2 GABAARs was screened using alanine replacement. Sensitivity to propofol was studied using patch-clamp recording in HEK293 cells transiently transfected with wild type or mutant receptors. Results Alanine mutation of a conserved phenylalanine residue within the α1 large intracellular loop significantly reduced propofol enhancement in both GlyRs (360 ± 30 vs. 75 ± 10%, mean ± SEM) and GABAARs (361 ± 49% vs. 80 ± 23%). Remarkably, propofol-hyposensitive mutant receptors retained their sensitivity to other allosteric modulators such as alcohols, etomidate, trichloroethanol, and isoflurane. At the single-channel level, the ability of propofol to increase open probability was significantly reduced in both α1 GlyR (189 ± 36 vs. 22 ± 13%) and α1β2 GABAAR (279 ± 29 vs. 29 ± 11%) mutant receptors. Conclusion In this study, it is demonstrated that the large intracellular loop of both GlyR and GABAAR has a conserved single phenylalanine residue (F380 and F385, respectively) that influences its sensitivity to propofol. Results suggest a new role of the large intracellular loop in the allosteric modulation of two members of the Cys-loop superfamily. Thus, these data provide new insights into the molecular framework behind the modulation of inhibitory ion channels by propofol.
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- 2011
9. TMEM16A(a)/anoctamin-1 Shares a Homodimeric Architecture with CLC Chloride Channels
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Ursula Braam, Ghada Fallah, Silvia Detro-Dassen, Thomas Römer, Fritz Markwardt, and Günther Schmalzing
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Patch-Clamp Techniques ,Regular Issue ,Anoctamins ,Xenopus ,Biology ,Biochemistry ,Analytical Chemistry ,Mice ,Xenopus laevis ,Chloride Channels ,Animals ,Humans ,Denaturation (biochemistry) ,Protein Structure, Quaternary ,Molecular Biology ,Anoctamin-1 ,Models, Genetic ,Research ,HEK 293 cells ,Native Polyacrylamide Gel Electrophoresis ,Exons ,biology.organism_classification ,Electrophysiology ,Cross-Linking Reagents ,HEK293 Cells ,Oocytes ,Chloride channel ,Biophysics ,Protein quaternary structure ,Dimerization - Abstract
TMEM16A/anoctamin-1 has been identified as a protein with the classic properties of a Ca(2+)-activated chloride channel. Here, we used blue native polyacrylamide gel electrophoresis (BN-PAGE) and chemical cross-linking to assess the quaternary structure of the mouse TMEM16A(a) and TMEM16A(ac) splice variants as well as a genetically concatenated TMEM16A(a) homodimer. The constructs carried hexahistidyl (His) tags to allow for their purification using a nondenaturing metal affinity resin. Neither His-tagging nor head-to-tail concatenation of two copies of TMEM16A(a) noticeably affected Ca(2+)-induced measured macroscopic Cl(-) currents compared with the wild-type TMEM16A(a) channel. The digitonin-solubilized, nondenatured TMEM16A(a) protein migrated in the BN-PAGE gel as a homodimer, as judged by comparison with the concatenated TMEM16A(a) homodimer and channel proteins of known oligomeric structures (e.g. the voltage-gated Cl(-) channel CLC-1). Cross-linking with glutaraldehyde corroborated the homodimeric structure of TMEM16A(a). The TMEM16A(a) homodimer detected in Xenopus laevis oocytes and HEK 293 cells dissociated into monomers following denaturation with SDS, and reducing versus nonreducing SDS-PAGE provided no evidence for the presence of intersubunit disulfide bonds. Together, our data demonstrate that the Ca(2+)-activated chloride channel member TMEM16A shares an obligate homodimeric architecture with the hCLC-1 channel.
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- 2011
10. Amino Acid Residues Constituting the Agonist Binding Site of the Human P2X3 Receptor
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Marcus Grohmann, Mandy Bodnar, Peter Illes, Ghada Fallah, Rashid Giniatullin, Erzsébet Kató, Haihong Wang, Patrizia Rubini, Ralf Hausmann, Thomas Riedel, Helke Gröger-Arndt, Stefan Hintze, and Günther Schmalzing
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Agonist ,medicine.drug_class ,Protein subunit ,Mutation, Missense ,Biology ,Peptide Mapping ,Biochemistry ,Purinergic P2X Receptor Agonists ,Xenopus laevis ,Neurobiology ,medicine ,Animals ,Humans ,Homomeric ,Patch clamp ,Binding site ,Receptor ,Molecular Biology ,Alanine ,chemistry.chemical_classification ,Binding Sites ,Cell Biology ,Molecular biology ,Amino acid ,Protein Subunits ,HEK293 Cells ,Amino Acid Substitution ,chemistry ,Oocytes ,Receptors, Purinergic P2X3 - Abstract
Homomeric P2X3 receptors are present in sensory ganglia and participate in pain perception. Amino acid (AA) residues were replaced in the four supposed nucleotide binding segments (NBSs) of the human (h) P2X3 receptor by alanine, and these mutants were expressed in HEK293 cells and Xenopus laevis oocytes. Patch clamp and two-electrode voltage clamp measurements as well as the Ca(2+) imaging technique were used to compare the concentration-response curves of the selective P2X1,3 agonist α,β-methylene ATP obtained at the wild-type P2X3 receptor and its NBS mutants. Within these NBSs, certain Gly (Gly-66), Lys (Lys-63, Lys-176, Lys-284, Lys-299), Asn (Asn-177, Asn-279), Arg (Arg-281, Arg-295), and Thr (Thr-172) residues were of great importance for a full agonist response. However, the replacement of further AAs in the NBSs by Ala also appeared to modify the amplitude of the current and/or [Ca(2+)](i) responses, although sometimes to a minor degree. The agonist potency decrease was additive after the simultaneous replacement of two adjacent AAs by Ala (K65A/G66A, F171A/T172A, N279A/F280A, F280A/R281A) but was not altered after Ala substitution of two non-adjacent AAs within the same NBS (F171A/N177A). SDS-PAGE in the Cy5 cell surface-labeled form demonstrated that the mutants appeared at the cell surface in oocytes. Thus, groups of AAs organized in NBSs rather than individual amino acids appear to be responsible for agonist binding at the hP2X3 receptor. These NBSs are located at the interface of the three subunits forming a functional receptor.
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- 2011
11. Conserved Dimeric Subunit Stoichiometry of SLC26 Multifunctional Anion Exchangers
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Ina Martin, Christoph Fahlke, Heike Lauks, Sonja Meyer zu Berstenhorst, Delany Torres-Salazar, Günther Schmalzing, Michael Schänzler, Patricia Hidalgo, Silvia Detro-Dassen, and Doreen Nothmann
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Gene isoform ,biology ,Protein subunit ,Anion Transport Proteins ,Xenopus ,Cell Biology ,biology.organism_classification ,Biochemistry ,Rats ,Transport protein ,Motor protein ,Xenopus laevis ,Chloride Channels ,Sulfate Transporters ,biology.protein ,Animals ,Humans ,Gene family ,Electrophoresis, Polyacrylamide Gel ,Protein quaternary structure ,sense organs ,Prestin ,Dimerization ,Molecular Biology - Abstract
The SLC26 gene family encodes multifunctional transport proteins in numerous tissues and organs. Some paralogs function as anion exchangers, others as anion channels, and one, prestin (SLC26A5), represents a membrane-bound motor protein in outer hair cells of the inner ear. At present, little is known about the molecular basis of this functional diversity. We studied the subunit stoichiometry of one bacterial, one teleost, and two mammalian SLC26 isoforms expressed in Xenopus laevis oocytes or in mammalian cells using blue native PAGE and chemical cross-linking. All tested SLC26s are assembled as dimers composed of two identical subunits. Co-expression of two mutant prestins with distinct voltage-dependent capacitances results in motor proteins with novel electrical properties, indicating that the two subunits do not function independently. Our results indicate that an evolutionarily conserved dimeric quaternary structure represents the native and functional state of SLC26 transporters.
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- 2008
12. Characterization of the Interleukin (IL)-6 Inhibitor IL-6-RFP
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Silke Metz, Heike Lauks, Monique Y. Wiesinger, Michael Vogt, Peter C. Heinrich, Günther Schmalzing, and Gerhard Müller-Newen
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Receptor complex ,biology ,fungi ,Oncostatin M ,Cell Biology ,Interleukin-17 receptor ,Glycoprotein 130 ,Biochemistry ,Fusion protein ,biology.protein ,Cytokine binding ,Cytokine receptor ,Molecular Biology ,Common gamma chain - Abstract
Although fusion proteins of the extracellular parts of receptor subunits termed cytokine traps turned out to be promising cytokine inhibitors for anti-cytokine therapies, their mode of action has not been analyzed. We developed a fusion protein consisting of the ligand binding domains of the IL-6 receptor subunits IL-6Rα and gp130 that acts as a highly potent IL-6 inhibitor. Gp130 is a shared cytokine receptor also used by the IL-6-related cytokines oncostatin M and leukemia inhibitory factor. In this study, we have shown that the IL-6 receptor fusion protein (IL-6-RFP) is a specific IL-6 inhibitor that does not block oncostatin M or leukemia inhibitory factor. We characterized the complex of IL-6-RFP and fluorescently labeled IL-6 (YFPIL-6) by blue native PAGE and gel filtration. A 2-fold molar excess of IL-6-RFP over IL-6 was sufficient to entirely bind IL-6 in a complex with IL-6-RFP. As shown by treatment with urea and binding competition experiments, the complex of IL-6 and IL-6-RFP is more stable than the complex of IL-6, soluble IL-6Rα, and soluble gp130. By live cell imaging, we have demonstrated that YFP-IL-6 bound to the surface of cells expressing gp130-CFP is removed from the plasma membrane upon the addition of IL-6-RFP. The apparent molecular mass of the IL-6·IL-6-RFP complex determined by blue native PAGE and gel filtration suggests that IL-6 is trapped in a structure analogous to the native hexameric IL-6 receptor complex. Thus, fusion of the ligand binding domains of heteromeric receptors leads to highly specific cytokine inhibitors with superior activity compared with the separate soluble receptors.
- Published
- 2007
13. Synthesis and Structure−Activity Relationships of Suramin-Derived P2Y11 Receptor Antagonists with Nanomolar Potency
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Christian Wolf, Jean-Marie Boeynaems, Peter Nickel, Michael Wiese, Ralf Hausmann, Heiko Ullmann, Claudia Marzian, Sabine Meis, Matthias U. Kassack, Didier Communi, Darunee Hongwiset, and Günther Schmalzing
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medicine.drug_class ,Stereochemistry ,Suramin ,Quantitative Structure-Activity Relationship ,Carboxamide ,Transfection ,Chemical synthesis ,Cell Line ,Xenopus laevis ,Drug Discovery ,Purinergic P2 Receptor Antagonists ,medicine ,Animals ,Humans ,Potency ,Receptor ,Suramin Sodium ,Receptors, Purinergic P2 ,Reverse Transcriptase Polymerase Chain Reaction ,Chemistry ,Antagonist ,Rats ,Electrophysiology ,Biochemistry ,Oocytes ,Molecular Medicine ,Calcium ,Selectivity ,medicine.drug - Abstract
Selective and potent P2Y(11) receptor antagonists have yet to be developed, thus impeding an evaluation of this G protein-coupled receptor mainly expressed on immune cells. Taking suramin with moderate inhibitory potency as a template, 18 ureas with variations of the methyl groups of suramin and their precursors were functionally tested at P2Y(11), P2Y(1), and P2Y(2) receptors. Fluorine substitution of the methyl groups of suramin led to the first nanomolar P2Y(11) antagonist (8f, NF157, pK(i): 7.35). For selectivity, 8f was also tested at various P2X receptors. 8f displayed selectivity for P2Y(11) over P2Y(1) (>650-fold), P2Y(2) (>650-fold), P2X(2) (3-fold), P2X(3) (8-fold), P2X(4) (>22-fold), and P2X(7) (>67-fold) but no selectivity over P2X(1). QSAR studies confirm that residues with favored resonance and size parameters in the aromatic linker region can indeed lead to an increased potency as is the case for 8f. A symmetric structure linking two anionic clusters seems to be required for bioactivity. 8f may be helpful for studies evaluating the physiological role of P2Y(11) receptors.
- Published
- 2005
14. Trimeric Architecture of Homomeric P2X2 and Heteromeric P2X1+2 Receptor Subtypes
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Armaz Aschrafi, Sven Sadtler, Jürgen Rettinger, Cristina Niculescu, and Günther Schmalzing
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Xenopus ,Xenopus laevis ,Protein structure ,Structural Biology ,Animals ,Humans ,Homomeric ,Protein Structure, Quaternary ,Receptor ,Molecular Biology ,Ion channel ,biology ,Receptors, Purinergic P2 ,Chemistry ,Cell Membrane ,biology.organism_classification ,Rats ,Protein Subunits ,Biochemistry ,Receptors, Purinergic P2X ,Oocytes ,Biophysics ,Ligand-gated ion channel ,Protein quaternary structure ,Receptors, Purinergic P2X5 ,Receptors, Purinergic P2X4 ,Receptors, Purinergic P2X3 ,Receptors, Purinergic P2X2 ,Ionotropic effect - Abstract
Of the three major classes of ligand-gated ion channels, nicotinic receptors and ionotropic glutamate receptors are known to be organized as pentamers and tetramers, respectively. The architecture of the third class, P2X receptors, is under debate, although evidence for a trimeric assembly is accumulating. Here we provide biochemical evidence that in addition to the rapidly desensitising P2X1 and P2X3 receptors, the slowly desensitising subtypes P2X2, P2X4, and P2X5 are trimers of identical subunits. Similar (heteromeric) P2X subunits also formed trimers, as shown for co-expressed P2X1 and P2X2 subunits, which assembled efficiently to a P2X1+2 receptor that was exported to the plasma membrane. In contrast, P2X6 subunits, which are incapable of forming functional homomeric channels in Xenopus oocytes, were retained in the ER as apparent tetramers and high molecular mass aggregates. Altogether, we conclude from these data that a trimeric architecture is the structural hallmark of functional homomeric and heteromeric P2X receptors.
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- 2004
15. A Trimeric Quaternary Structure Is Conserved in Bacterial and Human Glutamate Transporters
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Delany Torres-Salazar, Christoph Fahlke, Stephan Voswinkel, H. Heidtmann, Silvia Detro-Dassen, Niklas Lang, Sandra Gendreau, Günther Schmalzing, and Patricia Hidalgo
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Glycosylation ,Protein subunit ,Molecular Sequence Data ,Population ,Xenopus ,Gene Expression ,Kidney ,Biochemistry ,Evolution, Molecular ,Xenopus laevis ,Animals ,Humans ,Amino Acid Sequence ,Protein Structure, Quaternary ,education ,Molecular Biology ,Cells, Cultured ,education.field_of_study ,biology ,Escherichia coli Proteins ,Metabotropic glutamate receptor 7 ,Metabotropic glutamate receptor 6 ,Transporter ,Cell Biology ,biology.organism_classification ,Cross-Linking Reagents ,Excitatory Amino Acid Transporter 2 ,Oocytes ,Metabotropic glutamate receptor 1 ,Electrophoresis, Polyacrylamide Gel ,Protein quaternary structure ,Protein Processing, Post-Translational - Abstract
Neuronal and glial glutamate transporters play a central role in the termination of synaptic transmission and in extracellular glutamate homeostasis in the mammalian central nervous system. They are known to be multimers; however, the number of subunits forming a functional transporter is controversial. We studied the subunit stoichiometry of two distantly related glutamate transporters, the human glial glutamate transporter hEAAT2 and a bacterial glutamate transporter from Escherichia coli, ecgltP. Using blue native polyacrylamide gel electrophoresis, analysis of concatenated transporters, and chemical cross-linking, we demonstrated that human and prokaryotic glutamate transporters expressed in Xenopus laevis oocytes or in mammalian cells are assembled as trimers composed of three identical subunits. In an inducible mammalian cell line expressing hEAAT2 the glutamate uptake currents correlate to the amount of trimeric transporters. Overexpression and purification of ecgltP in E. coli resulted in a homogenous population of trimeric transporters that were functional after reconstitution in lipid vesicles. Our results indicate that an evolutionarily conserved trimeric quaternary structure represents the sole native and functional state of glutamate transporters.
- Published
- 2004
16. Desensitization Masks Nanomolar Potency of ATP for the P2X1 Receptor
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Günther Schmalzing and Juergen Rettinger
- Subjects
Agonist ,Time Factors ,Stereochemistry ,medicine.drug_class ,Recombinant Fusion Proteins ,Xenopus ,medicine.medical_treatment ,Suramin ,Binding, Competitive ,Models, Biological ,Biochemistry ,Adenosine Triphosphate ,Homologous desensitization ,medicine ,Animals ,Receptor ,Molecular Biology ,Desensitization (medicine) ,Dose-Response Relationship, Drug ,Receptors, Purinergic P2 ,Chemistry ,Antagonist ,Affinity Labels ,Cell Biology ,Thionucleotides ,Adenosine ,Electrophysiology ,Kinetics ,Models, Chemical ,Ectodomain ,Receptors, Purinergic P2X ,Oocytes ,Biophysics ,Plasmids ,Protein Binding ,medicine.drug - Abstract
ATP-gated P2X1 receptors feature fast activation and fast desensitization combined with slow recovery from desensitized states. Here, we exploited a non-desensitizing P2X2/P2X1 chimera that includes the entire P2X1 ectodomain (Werner, P., Seward, E. P., Buell, G. N., and North, R. A. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 15485-15490) to obtain a macroscopic representation of intrinsic receptor kinetics without bias arising from the overlap of channel activation and desensitization. From the stationary currents made amenable to analysis by this chimera, an EC50 for ATP of 3.3 nM was derived, representing a >200- and >7000-fold higher ATP potency than observed for the parental P2X1 and P2X2A receptors, respectively. Also, other agonists activated the P2X2/P2X1 chimera with nanomolar EC50 values ranging from 3.5 to 73 nM in the following rank order: 2-methylthio-ATP, 2',3'-O-(4-benzoylbenzoyl)-ATP, alpha,beta-methylene-ATP, adenosine 5'-O-(3-thiotriphosphate). Upon washout, the P2X2/P2X1 chimera deactivated slowly with a time constant (ranging from 63 to 2.5 s) that is inversely related to the EC50 value for the corresponding agonist. This suggests that deactivation time courses reflect unbinding rates, which by themselves define agonist potencies. The P2X2/P2X1 chimera and the P2X1 receptor possess virtually identical sensitivity to inhibition by the P2X1 receptor-selective antagonist NF279, a suramin analog. These results suggest that the P2X1 ectodomain confers nanomolar ATP sensitivity, which, within the wild-type P2X1 receptor, is obscured by desensitization such that only a micromolar ATP potency can be deduced from peak current measurements, representing an amalgam of activation and desensitization.
- Published
- 2004
17. Oligomerization States of LRRC8A and LRRC8B, Essential Components of the Volume-Regulated Anion Channel VRAC
- Author
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Fritz Markwardt, Silvia Detro-Dassen, Günther Schmalzing, and Christoph Fahlke
- Subjects
Gene isoform ,biology ,Chemistry ,Biophysics ,Xenopus ,Native Polyacrylamide Gel Electrophoresis ,Protomer ,biology.organism_classification ,Membrane protein ,Biochemistry ,biology.protein ,Homomeric ,Protein quaternary structure ,Organic anion - Abstract
Cellular volume regulation plays an important role in many cellular processes. VRAC is crucial for regulating volume decrease by allowing a net efflux of chloride and organic anions. Its molecular nature remained unclear until 2014 when Qiu et al. (Cell 157, 447-458) and Voss et al. (Science 344, 634-638) identified leucine-rich repeat-containing 8 (LRRC8) membrane proteins as subunits of VRAC. LRRC8 proteins, which occur in five isoforms (LRRC8A-E), have been suggested to share a common ancestor and accordingly a hexameric quaternary structure with pannexins (Abascal & Zardoya, BioEssays 34, 551-560, 2012). Here, we expressed two human isoforms, hLRRC8A and hLRRC8B, in Xenopus laevis oocytes, purified the LRRC8 proteins by Ni-NTA chromatography in dodecyl maltoside, and assessed their oligomeric structure by blue native polyacrylamide gel electrophoresis (BN-PAGE). Detergent screening revealed that maltoside detergents with alkyl chain length from 10 to 12 were similarly well suited to purify hLRRC8A as a stable homomultimeric protein complex. The non-denatured homomeric hLRRC8A protein and the heteromeric hLRRC8A+B protein (with calculated non-glycosylated protomer masses of 95 and 92 kDa, respectively) approximately co-migrated in the BN-PAGE gel with the GFP-tagged tetrameric hTRPV1 channel (calculated mass close to 500 kDa). Slight denaturing treatments of the heteromeric hLRRC8A+B protein complex resulted in the appearance of lower order oligomers in the BN-PAGE gel, which we could tentatively identify as dimers, tetramers and hexamers by comparison with the migration patterns of concatenated hLRRC8A/A and A/B dimers and A/A/A trimers. We conclude that hLRRC8A and hLRR8A+B assemble as homo- and heterohexamers, respectively, in X. laevis oocytes. In contrast to the hLRRC8A isoform, hLRRC8B requires co-assembly with hLRRC8A to achieve a defined (hexameric) assembly state and plasma membrane localization.
- Published
- 2016
18. A Basic Cluster Determines Topology of the Cytoplasmic M3-M4 Loop of the Glycine Receptor α1 Subunit
- Author
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Sven Sadtler, Annette Nicke, Bodo Laube, Günther Schmalzing, Heinrich Betz, and Alhassan Lashub
- Subjects
Protein Conformation ,Chemistry ,Xenopus ,Protein subunit ,Endoplasmic reticulum ,Cell Membrane ,Molecular Sequence Data ,Cell Biology ,Topology ,Biochemistry ,Transmembrane protein ,Structure-Activity Relationship ,Receptors, Glycine ,Ectodomain ,Neurotransmitter receptor ,Cytoplasm ,Mutation ,Animals ,Humans ,Amino Acid Sequence ,Molecular Biology ,Glycine receptor ,Ion channel - Abstract
The inhibitory glycine receptor is a member of the ligand-gated ion channel superfamily of neurotransmitter receptors, which are composed of homologous subunits with four transmembrane segments (M1-M4), each. Here, we demonstrate that the correct topology of the glycine receptor alpha1 subunit depends critically on six positively charged residues within a basic cluster, RFRRKRR, located in the large cytoplasmic loop (designated M3-M4 loop) following the C-terminal end of M3. Neutralization of one or more charges of this cluster, but not of other charged residues in the M3-M4 loop, led to an aberrant translocation into the endoplasmic reticulum lumen of the M3-M4 loop. However, when two of the three basic charges located in the ectodomain linking M2 and M3 were neutralized, in addition to two charges of the basic cluster, endoplasmic reticulum disposition of the M3-M4 loop was prevented. We conclude that a high density of basic residues C-terminal to M3 is required to compensate for the presence of positively charged residues in the M2-M3 ectodomain, which otherwise impair correct membrane integration of the M3 segment.
- Published
- 2003
19. Identification of a tubulin binding motif on the P2X2 receptor
- Author
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Sandra Gendreau, Günther Schmalzing, and Jörg Schirmer
- Subjects
DNA, Complementary ,Recombinant Fusion Proteins ,Protein subunit ,Amino Acid Motifs ,Detergents ,Molecular Sequence Data ,Clinical Biochemistry ,Biochemistry ,Mass Spectrometry ,Analytical Chemistry ,Tubulin binding ,Rats, Sprague-Dawley ,Tubulin ,Microtubule ,Animals ,Amino Acid Sequence ,Receptor ,chemistry.chemical_classification ,Sequence Homology, Amino Acid ,biology ,Receptors, Purinergic P2 ,Cell Biology ,General Medicine ,Fusion protein ,Rats ,Amino acid ,Myelin basic protein ,chemistry ,biology.protein ,Electrophoresis, Polyacrylamide Gel ,Receptors, Purinergic P2X3 ,Protein Binding ,Synaptosomes - Abstract
To isolate proteins interacting with P2X receptors, GST fusion proteins containing the intracellular C terminal tail of P2X(2), P2X(5), or P2X(7) were used as bait to screen detergent extracts of rat brain synaptosomes. By SDS-PAGE combined with mass spectrometry, two interacting proteins were identified: betaIII tubulin and myelin basic protein. While myelin basic protein bound to all three P2X subunits, betaIII tubulin interacted exclusively with the P2X(2) subunit. The tubulin binding domain could be confined to a proline-rich segment (amino acids 371-412) of the P2X(2) subunit. Our results suggest a role for microtubules in the cellular localisation of the P2X(2) receptor.
- Published
- 2003
20. Glu496Ala polymorphism of human P2X7receptor does not affect its electrophysiological phenotype
- Author
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Günther Schmalzing, W Boldt, Sven Sadtler, Manuela Klapperstück, Fritz Markwardt, and Cora Büttner
- Subjects
Cell Membrane Permeability ,Physiology ,Glutamine ,Biology ,Ion Channels ,Membrane Potentials ,Xenopus laevis ,chemistry.chemical_compound ,Adenosine Triphosphate ,Cations ,Animals ,Humans ,Amino Acid Sequence ,Neurotransmitter ,Cells, Cultured ,Loss function ,Alanine ,chemistry.chemical_classification ,Polymorphism, Genetic ,Dose-Response Relationship, Drug ,Receptors, Purinergic P2 ,Cell Membrane ,HEK 293 cells ,Glutamate receptor ,Cell Biology ,Transfection ,Molecular biology ,Phenotype ,Amino acid ,Kinetics ,Eukaryotic Cells ,chemistry ,Biochemistry ,Mutation ,Oocytes ,Female ,Receptors, Purinergic P2X7 - Abstract
A glutamate to alanine exchange at amino acid position 496 of the human P2X7receptor was recently shown to be associated with a loss of function in human B lymphocytes in terms of ATP-induced ethidium+uptake, Ba2+influx, and induction of apoptosis (Gu BJ, Zhang WY, Worthington RA, Sluyter R, Dao-Ung P, Petrou S, Barden JA, and Wiley JS. J Biol Chem 276: 11135–11142, 2001). Here we analyzed the effect of the Glu496to Ala exchange on the channel properties of the human P2X7receptor expressed in Xenopus oocytes with the two-microelectrode voltage-clamp technique. The amplitudes of ATP-induced whole cell currents characteristic of functional expression, kinetic properties including ATP concentration dependence, and permeation behavior were not altered by this amino acid exchange. Also in HEK293 cells, the Ala496mutant mediated typical P2X7receptor-dependent currents like the parent Glu496hP2X7receptor. Because the function of the P2X7receptor as an ATP-gated channel for small cations including Ba2+remained unaffected by this mutation, we conclude that Glu496plays a critical role in pore formation but does not determine the ion channel properties of the human P2X7receptor.
- Published
- 2003
21. Conformational flexibility of the agonist binding jaw of the human P2X3 receptor is a prerequisite for channel opening
- Author
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Marcus Grohmann, Heike Franke, Günther Schmalzing, Karen Nieber, Anke Dopychai, Ralf Hausmann, Peter Illes, Maria Kowalski, and Thomas Riedel
- Subjects
Pharmacology ,Agonist ,biology ,medicine.drug_class ,Protein subunit ,Xenopus ,biology.organism_classification ,Dithiothreitol ,chemistry.chemical_compound ,Biochemistry ,chemistry ,medicine ,Biophysics ,Membrane channel ,Receptor ,Adenosine triphosphate ,Cysteine - Abstract
Background and Purpose It is assumed that ATP induces closure of the binding jaw of ligand-gated P2X receptors, which eventually results in the opening of the membrane channel and the flux of cations. Immobilization by cysteine mutagenesis of the binding jaw inhibited ATP-induced current responses, but did not allow discrimination between disturbances of binding, gating, subunit assembly or trafficking to the plasma membrane. Experimental Approach A molecular model of the pain-relevant human (h)P2X3 receptor was used to identify amino acid pairs, which were located at the lips of the binding jaw and did not participate in agonist binding but strongly approached each other even in the absence of ATP. Key Results A series of cysteine double mutant hP2X3 receptors, expressed in HEK293 cells or Xenopus laevis oocytes, exhibited depressed current responses to α,β-methylene ATP (α,β-meATP) due to the formation of spontaneous inter-subunit disulfide bonds. Reducing these bonds with dithiothreitol reversed the blockade of the α,β-meATP transmembrane current. Amino-reactive fluorescence labelling of the His-tagged hP2X3 receptor and its mutants expressed in HEK293 or X. laevis oocytes demonstrated the formation of inter-subunit cross links in cysteine double mutants and, in addition, confirmed their correct trimeric assembly and cell surface expression. Conclusions and Implications In conclusion, spontaneous tightening of the binding jaw of the hP2X3 receptor by inter-subunit cross-linking of cysteine residues substituted at positions not directly involved in agonist binding inhibited agonist-evoked currents without interfering with binding, subunit assembly or trafficking.
- Published
- 2014
22. Roles of Individual N-Glycans for ATP Potency and Expression of the Rat P2X1 Receptor
- Author
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Günther Schmalzing, Jürgen Rettinger, and Armaz Aschrafi
- Subjects
Glycan ,Glycosylation ,Protein subunit ,Molecular Sequence Data ,Biology ,Biochemistry ,Structure-Activity Relationship ,Xenopus laevis ,chemistry.chemical_compound ,Adenosine Triphosphate ,Polysaccharides ,Animals ,Structure–activity relationship ,Amino Acid Sequence ,Receptor ,Molecular Biology ,Peptide sequence ,Gel electrophoresis ,Receptors, Purinergic P2 ,Cell Biology ,Ligand (biochemistry) ,Molecular biology ,Rats ,Protein Subunits ,chemistry ,biology.protein - Abstract
P2X(1) receptor subunits assemble in the ER of Xenopus oocytes to homotrimers that appear as ATP-gated cation channels at the cell surface. Here we address the extent to which N-glycosylation contributes to assembly, surface appearance, and ligand recognition of P2X(1) receptors. SDS-polyacrylamide gel electrophoresis (PAGE) analysis of glycan minus mutants carrying Gln instead of Asn at five individual NXT/S sequons reveals that Asn(284) remains unused because of a proline in the +4 position. The four other sites (Asn(153), Asn(184), Asn(210), and Asn(300)) carry N-glycans, but solely Asn(300) located only eight residues upstream of the predicted reentry loop of P2X(1) acquires complex-type carbohydrates. Like parent P2X(1), glycan minus mutants migrate as homotrimers when resolved by blue native PAGE. Recording of ATP-gated currents reveals that elimination of Asn(153) or Asn(210) diminishes or increases functional expression levels, respectively. In addition, elimination of Asn(210) causes a 3-fold reduction of the potency for ATP. If three or all four N-glycosylation sites are simultaneously eliminated, formation of P2X(1) receptors is severely impaired or abolished, respectively. We conclude that at least one N-glycan per subunit of either position is absolutely required for the formation of P2X(1) receptors and that individual N-glycans possess marked positional effects on expression levels (Asn(154), Asn(210)) and ATP potency (Asn(210)).
- Published
- 2000
23. Molecular determinants of glycine receptor subunit assembly
- Author
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Heinrich Betz, Cora Büttner, Günther Schmalzing, Annette Nicke, Nathalie Griffon, and Jochen Kuhse
- Subjects
Models, Molecular ,Xenopus ,Protein subunit ,Molecular Sequence Data ,Interleukin 5 receptor alpha subunit ,Biology ,Endoplasmic Reticulum ,Transfection ,Gamma-aminobutyric acid receptor subunit alpha-1 ,General Biochemistry, Genetics and Molecular Biology ,Cell Line ,RNA, Complementary ,Interleukin 10 receptor, alpha subunit ,Receptors, Glycine ,Animals ,Humans ,Amino Acid Sequence ,Molecular Biology ,Glycine receptor ,G alpha subunit ,Microscopy, Confocal ,Dose-Response Relationship, Drug ,Models, Genetic ,General Immunology and Microbiology ,General Neuroscience ,Endoplasmic reticulum ,Biochemistry ,Mutagenesis, Site-Directed ,Biophysics ,biology.protein ,ATP synthase alpha/beta subunits ,Research Article - Abstract
The inhibitory glycine receptor (GlyR) is a pentameric transmembrane protein composed of homologous alpha and beta subunits. Single expression of alpha subunits generates functional homo-oligomeric GlyRs, whereas the beta subunit requires a co-expressed alpha subunit to assemble into hetero-oligomeric channels of invariant stoichiometry (alpha(3)beta(2)). Here, we identified eight amino acid residues within the N-terminal region of the alpha1 subunit that are required for the formation of homo-oligomeric GlyR channels. We show that oligomerization and N-glycosylation of the alpha1 subunit are required for transit from the endoplasmic reticulum to the Golgi apparatus and later compartments, and that addition of simple carbohydrate side chains occurs prior to GlyR subunit assembly. Our data are consistent with both intersubunit surface and conformational differences determining the different assembly behaviour of GlyR alpha and beta subunits.
- Published
- 1999
24. Blue Native Page as a Useful Method for the Analysis of the Assembly of Distinct Combinations of Nicotinic Acetylcholine Receptor Subunits
- Author
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Jürgen Rettinger, Annette Nicke, Ernst Mutschler, and Günther Schmalzing
- Subjects
Protein Conformation ,Pentamer ,Stereochemistry ,Xenopus ,Trimer ,Native page ,In Vitro Techniques ,Receptors, Nicotinic ,Biochemistry ,Xenopus laevis ,chemistry.chemical_compound ,Animals ,Receptor ,Molecular Biology ,biology ,Chemistry ,Muscles ,Cell Biology ,biology.organism_classification ,Recombinant Proteins ,Rats ,Nicotinic acetylcholine receptor ,Digitonin ,Oocytes ,Electrophoresis, Polyacrylamide Gel ,Female ,Alpha-4 beta-2 nicotinic receptor - Abstract
Oligomerization of complete and incomplete combinations of rat muscle-type nicotinic acetylcholine receptor (nAChR) subunits in Xenopus oocytes was studied by blue native PAGE and compared with acetylcholine-activated current in these cells. The rank order of expression level judged by current was alpha 1 beta 1 gamma deltaalpha 1 beta 1 gammaalpha 1 beta 1 deltaalpha 1 gamma deltaalpha 1 deltaalpha 1 gamma. alpha 1 and alpha 1 beta 1 were not functional. Protein complexes incorporating a heptahistidyl-tagged alpha 1 subunit were chromatographically purified from digitonin extracts of oocytes and resolved by blue native PAGE. In the absence of any co-expressed nAChR subunit, the majority of alpha 1 formed aggregates. Co-expression of beta 1 had no effect on alpha 1 aggregation, whereas both gamma and delta diminished alpha 1 aggregation in favor of discrete oligomers: alpha 1 formed tetramers together with gamma and dimers, trimers, and tetramers together with delta. When alpha 1 gamma was complemented with beta 1 to form a functional alpha 1 beta 1 gamma receptor, a small amount of a pentamer was found besides a prominent alpha 1-His7 beta 1 gamma trimer. Expression of the functional alpha 1 beta 1 delta receptor yielded marked amounts of a pentamer besides dimers and trimers. These results are discussed in terms of the assembly model of Green and Claudio (Cell 74, 57-69, 1994), substantiating that blue native PAGE is suited for the investigation of ion channel assembly.
- Published
- 1999
25. ATP Binding Site Mutagenesis Reveals Different Subunit Stoichiometry of Functional P2X2/3 and P2X2/6 Receptors*
- Author
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Karen Nieber, Nanette Messemer, Ying Qin, Haihong Wang, Thomas Riedel, Ronja Woltersdorf, Martin Fuchs, Janka Günther, Ralf Hausmann, Patrizia Rubini, Peter Illes, Günther Schmalzing, Marcus Grohmann, and Mandy Bodnar
- Subjects
Agonist ,endocrine system ,Patch-Clamp Techniques ,medicine.drug_class ,Surface Properties ,Protein subunit ,Mutant ,Xenopus ,Biology ,Biochemistry ,Structure-Activity Relationship ,Xenopus laevis ,Adenosine Triphosphate ,Neurobiology ,medicine ,Animals ,Humans ,Binding site ,Receptor ,Molecular Biology ,Binding Sites ,urogenital system ,Receptors, Purinergic P2 ,musculoskeletal, neural, and ocular physiology ,Purinergic receptor ,Cell Biology ,respiratory system ,biology.organism_classification ,Electrophysiology ,HEK293 Cells ,Mutagenesis ,Mutation ,Calcium ,Signal transduction ,human activities ,Dimerization ,Receptors, Purinergic P2X3 ,Protein Binding ,Receptors, Purinergic P2X2 ,Signal Transduction - Abstract
The aim of the present experiments was to clarify the subunit stoichiometry of P2X2/3 and P2X2/6 receptors, where the same subunit (P2X2) forms a receptor with two different partners (P2X3 or P2X6). For this purpose, four non-functional Ala mutants of the P2X2, P2X3, and P2X6 subunits were generated by replacing single, homologous amino acids particularly important for agonist binding. Co-expression of these mutants in HEK293 cells to yield the P2X2 WT/P2X3 mutant or P2X2 mutant/P2X3 WT receptors resulted in a selective blockade of agonist responses in the former combination only. In contrast, of the P2X2 WT/P2X6 mutant and P2X2 mutant/P2X6 WT receptors, only the latter combination failed to respond to agonists. The effects of α,β-methylene-ATP and 2-methylthio-ATP were determined by measuring transmembrane currents by the patch clamp technique and intracellular Ca(2+) transients by the Ca(2+)-imaging method. Protein labeling, purification, and PAGE confirmed the assembly and surface trafficking of the investigated WT and WT/mutant combinations in Xenopus laevis oocytes. In conclusion, both electrophysiological and biochemical investigations uniformly indicate that one subunit of P2X2 and two subunits of P2X3 form P2X2/3 heteromeric receptors, whereas two subunits of P2X2 and one subunit of P2X6 constitute P2X2/6 receptors. Further, it was shown that already two binding sites of the three possible ones are sufficient to allow these receptors to react with their agonists.
- Published
- 2012
26. Robust post-translocational N-glycosylation at the extreme C-terminus of membrane and secreted proteins in Xenopus laevis oocytes and HEK293 cells
- Author
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Günther Schmalzing, Silvia Detro-Dassen, Frauke Pult, Ghada Fallah, Cristina Niculescu, Ursula Braam, and Bodo Laube
- Subjects
Glycosylation ,Patch-Clamp Techniques ,Time Factors ,Molecular Sequence Data ,Xenopus ,Gene Expression ,Endoplasmic Reticulum ,Protein Engineering ,Transfection ,Biochemistry ,chemistry.chemical_compound ,Xenopus laevis ,Species Specificity ,Animals ,Humans ,Amino Acid Sequence ,Integral membrane protein ,Membrane Glycoproteins ,biology ,Endoplasmic reticulum ,Sequon ,biology.organism_classification ,Recombinant Proteins ,Membrane glycoproteins ,Protein Transport ,Secretory protein ,HEK293 Cells ,Membrane protein ,chemistry ,biology.protein ,Oocytes ,Electrophoresis, Polyacrylamide Gel ,Female ,Asparagine ,Plasmids - Abstract
N-Glycosylation is normally a co-translational process that occurs as soon as a nascent and unfolded polypeptide chain has emerged ~12 residues into the lumen of the endoplasmic reticulum (ER). Here, we describe the efficient utilization of an N-glycosylation site engineered within the luminal extreme C-terminal residues of distinct integral membrane glycoproteins, a native ER resident protein and an engineered secreted protein. This N-glycan addition required that the acceptor asparagine within an Asn-Trp-Ser sequon be located at least four residues away from the C-terminus of the polypeptide and, in the case of membrane proteins, at least 13 residues away from the lumenal side of the transmembrane segment. Pulse-chase assays revealed that the natural N-glycans of the proteins studied were attached co-translationally, whereas C-terminal N-glycosylation occurred post-translocationally within a time frame of hours in Xenopus laevis oocytes and minutes in human embryonic kidney 293 (HEK293) cells. In oocyte and HEK cell expression systems, affinity tag-driven C-terminal N-glycosylation may facilitate the determination of orientation of the C-terminal tail of membrane proteins relative to the membrane.
- Published
- 2011
27. Trophic activity of a naturally occurring truncated isoform of the P2X7 receptor
- Author
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Patrizia Pellegatti, Simonetta Falzoni, Elena Adinolfi, Günther Schmalzing, Fritz Markwardt, Paola Chiozzi, Ronja Woltersdorf, Maria Cirillo, Doriana Sandonà, Francesco Di Virgilio, and Maria Giulia Callegari
- Subjects
Gene isoform ,Cell type ,Molecular Sequence Data ,Fluorescent Antibody Technique ,Biology ,Biochemistry ,Cell Line ,Membrane Potentials ,Adenosine Triphosphate ,purinergic receptors ,Genetics ,cell growth ,Humans ,Protein Isoforms ,Amino Acid Sequence ,Receptor ,Molecular Biology ,Transcription factor ,Ion channel ,Sequence Homology, Amino Acid ,Receptors, Purinergic P2 ,Reverse Transcriptase Polymerase Chain Reaction ,Endoplasmic reticulum ,Purinergic receptor ,HEK 293 cells ,Cell Membrane ,ion channels ,Molecular biology ,extracellular ATP ,Cell biology ,Receptors, Purinergic P2X7 ,Biotechnology - Abstract
P2X7 is the largest member of the P2X subfamily of purinergic receptors. A typical feature is the carboxyl tail, which allows formation of a large pore. Recently a naturally occurring truncated P2X7 splice variant, isoform B (P2X7B), has been identified. Here we show that P2X7B expression in HEK293 cells, a cell type lacking endogenous P2X receptors, mediated ATP-stimulated channel activity but not plasma membrane permeabilization, raised endoplasmic reticulum Ca(2+) content, activated the transcription factor NFATc1, increased the cellular ATP content, and stimulated growth. In addition, P2X7B-transfected HEK293 cells (HEK293-P2X7B), like most tumor cells, showed strong soft agar-infiltrating ability. When coexpressed with full-length P2X7 (P2X7A), P2X7B coassembled with P2X7A into a heterotrimer and potentiated all known responses mediated by this latter receptor. P2X7B mRNA was found to be widely distributed in human tissues, especially in the immune and nervous systems, and to a much higher level than P2X7A. Finally, P2X7B expression was increased on mitogenic stimulation of peripheral blood lymphocyte. Altogether, these data show that P2X7B is widely expressed in several human tissues, modulates P2X7A functions, participates in the control of cell growth, and may help understand the role of the P2X7 receptor in the control of normal and cancer cell proliferation.
- Published
- 2010
28. Up-regulation of sodium pump activity in Xenopus laevis oocytes by expression of heterologous β1 subunits of the sodium pump
- Author
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Silke Kröner, Halit Omay, Sergio M. Gloor, Wolfgang Schwarz, Günther Schmalzing, and Heribert Appelhans
- Subjects
Male ,Macromolecular Substances ,Protein subunit ,Xenopus ,Gene Expression ,Diaphragm pump ,Torpedo ,Transfection ,Biochemistry ,Ouabain ,RNA, Complementary ,law.invention ,Xenopus laevis ,Species Specificity ,law ,medicine ,Animals ,Molecular Biology ,Immunosorbent Techniques ,G alpha subunit ,biology ,Sodium ,Cell Biology ,biology.organism_classification ,Rats ,Cell biology ,Kinetics ,Oocytes ,biology.protein ,RNA ,Female ,Protein Multimerization ,Sodium-Potassium-Exchanging ATPase ,Chickens ,Rubidium Radioisotopes ,Intracellular ,ATP synthase alpha/beta subunits ,Research Article ,medicine.drug - Abstract
Recent evidence suggests that the beta subunit of the Na+ pump is essential for the alpha subunit to express catalytic activity and for assembly of the holoenzyme in the plasma membrane. We report here that injection into Xenopus laevis oocytes of cRNAs specific for beta 1 subunit isoforms of the Na+ pump of four species (Torpedo californica, chicken, mouse and rat) causes a time-dependent increase in the number of ouabain-binding sites, both in the plasma membrane and in internal membranes. Expression of the beta 1 subunit of the Na+ pump of mouse and rat in the oocytes could be substantiated by immunoprecipitation using a polyclonal antiserum against the mouse beta 1 subunit. Scatchard analysis in permeabilized cells disclosed that the affinity for ouabain is unchanged after expression of each of the beta 1 subunits. A proportional increase in ouabain-sensitive 86Rb+ uptake indicates that the additionally expressed ouabain-binding sites on the cell surface represent functional Na+ pumps. The findings support the concept of Geering. Theulaz, Verrey, Häuptle & Rossier [(1989) Am. J. Physiol. 257, C851-C858] that beta 1 subunits expressed in oocytes associate with an excess of endogenous alpha subunits of the Na+ pump to form a hybrid enzyme. In addition, all of the beta 1 isoforms investigated in the present study were also capable of combining with the co-expressed alpha 1 subunit of the Torpedo Na+ pump to produce a functional enzyme. Injection of cRNA encoding for the Torpedo alpha 1 subunit alone had no effect on the ouabain-binding capacity of the surface and intracellular membranes of the oocyte.
- Published
- 1991
29. The P2X7 carboxyl tail is a regulatory module of P2X7 receptor channel activity
- Author
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Ronja Woltersdorf, Daniel Becker, Fritz Markwardt, Ursula Braam, W Boldt, Stephan Schmitz, and Günther Schmalzing
- Subjects
DNA, Complementary ,Patch-Clamp Techniques ,Protein subunit ,Xenopus ,Gating ,Biochemistry ,Models, Biological ,Xenopus laevis ,Adenosine Triphosphate ,Animals ,Humans ,Receptor ,Molecular Biology ,biology ,Dose-Response Relationship, Drug ,Receptors, Purinergic P2 ,Cell Membrane ,Wild type ,Cell Biology ,Hydrogen-Ion Concentration ,biology.organism_classification ,Transmembrane protein ,Protein Structure, Tertiary ,Kinetics ,Cross-Linking Reagents ,Ectodomain ,Biophysics ,Oocytes ,Receptors, Purinergic P2X7 ,Intracellular - Abstract
P2X(7) receptors are ATP-gated cation channels composed of three identical subunits, each having intracellular amino and carboxyl termini and two transmembrane segments connected by a large ectodomain. Within the P2X family, P2X(7) subunits are unique in possessing an extended carboxyl tail. We expressed the human P2X(7) subunit as two complementary fragments, a carboxyl tail-truncated receptor channel core (residues 1-436 or 1-505) and a tail extension (residues 434-595) in Xenopus laevis oocytes. P2X(7) channel core subunits efficiently assembled as homotrimers that appeared abundantly at the oocyte surface, yet produced only approximately 5% of the full-length P2X(7) receptor current. Co-assembly of channel core subunits with full-length P2X(7) subunits inhibited channel current, indicating that the lack of a single carboxyl tail domain is dominant-negative for P2X(7) receptor activity. Co-expression of the tail extension as a discrete protein increased ATP-gated current amplitudes of P2X(7) channel cores 10-20-fold, fully reconstituting the wild type electrophysiological phenotype of the P2X(7) receptor. Chemical cross-linking revealed that the discrete tail extension bound with unity stoichiometry to the carboxyl tail of the P2X(7) channel core. We conclude that a non-covalent association of crucial functional importance exists between the carboxyl tail of the channel core and the tail extension. Using a slightly shorter P2X(7) subunit core and subfragments of the tail extension, this association could be narrowed down to include residues 409-436 and 434-494 of the split receptor. Together, these results identify the tail extension as a regulatory gating module, potentially making P2X(7) channel gating sensitive to intracellular regulation.
- Published
- 2008
30. Activation of protein kinase C by phorbol ester induces downregulation of the Na+/K+-ATPase in oocytes ofXenopus laevis
- Author
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Günther Schmalzing, Wolfgang Schwarz, Winfried Haase, Larisa A. Vasilets, and Kristin Mädefessel
- Subjects
Cell Membrane Permeability ,Physiology ,Biophysics ,Xenopus ,Down-Regulation ,Diaphragm pump ,Membrane Potentials ,Xenopus laevis ,chemistry.chemical_compound ,Downregulation and upregulation ,Animals ,Na+/K+-ATPase ,Ouabain ,Protein kinase A ,Protein Kinase C ,Protein kinase C ,biology ,Chemistry ,Activator (genetics) ,Cell Membrane ,Electric Conductivity ,Inulin ,Cell Biology ,biology.organism_classification ,Cell biology ,Enzyme Activation ,Kinetics ,Microscopy, Electron ,Biochemistry ,Oocytes ,Phorbol ,Tetradecanoylphorbol Acetate ,Sodium-Potassium-Exchanging ATPase - Abstract
Full-grown prophase-arrested oocytes of Xenopus laevis were treated with 50 nM phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, or with 50 nM 4 alpha-phorbol 12,13-didecanoate (4 alpha PDD) that does not activate protein kinase C. The effect on membrane currents and capacitance, inulin uptake and ouabain binding, and on membrane morphology were analyzed. (i) During application of PMA, current generated by the Na+/K+ pump decreases; in addition, Cl- and K+ channels become inhibited. This general decrease in membrane conductance reaches steady state after about 60 min. 4 alpha PDD was ineffective. (ii) Ouabain binding experiments demonstrate that PMA (K1/2 = 7 nM), but not 4 alpha PPD, induces a reduction of the number of pump molecules in the surface membrane. Permeabilization of oocytes by digitonin plus 0.02% SDS renders all binding sites present prior to PMA treatment again accessible for ouabain. The KD value for ouabain binding is not influenced. 4 alpha PDD was ineffective. (iii) Exposure of oocytes to PMA reduces membrane capacitance and stimulates uptake of inulin suggesting an increase in endocytosis. Electron micrographs show that PMA reduces the number and length of microvilli, leading finally to a smooth membrane surface with a reduced surface area. From these results we conclude that stimulation of protein kinase C leads to downregulation of the sodium pump. A major portion of this inhibition is brought about by reduction in area of surface membrane with a concomitant internalization of pump molecules. In addition to this mode of downregulation, a direct effect of stimulation of protein kinase C on the pump molecule cannot be excluded.
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- 1990
31. Molecular determinants for G protein betagamma modulation of ionotropic glycine receptors
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Svenja Haeger, Günther Schmalzing, Gustavo Moraga-Cid, Luis G. Aguayo, Laerte Oliveira, Leonardo Guzmán, Juan Olate, and Gonzalo E. Yévenes
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Models, Molecular ,G protein ,Protein subunit ,Amino Acid Motifs ,Lipid Bilayers ,Molecular Sequence Data ,Biology ,Biochemistry ,Protein Structure, Secondary ,Cell Line ,Receptors, Glycine ,GTP-Binding Proteins ,GTP-Binding Protein gamma Subunits ,Humans ,Amino Acid Sequence ,Receptor ,Molecular Biology ,Glycine receptor ,Ion channel ,GTP-Binding Protein beta Subunits ,Cell Biology ,Cell biology ,Electrophysiology ,Transmembrane domain ,Nicotinic acetylcholine receptor ,Intracellular ,Protein Binding ,Signal Transduction - Abstract
The ligand-gated ion channel superfamily plays a critical role in neuronal excitability. The functions of glycine receptor (GlyR) and nicotinic acetylcholine receptor are modulated by G protein betagamma subunits. The molecular determinants for this functional modulation, however, are still unknown. Studying mutant receptors, we identified two basic amino acid motifs within the large intracellular loop of the GlyR alpha(1) subunit that are critical for binding and functional modulation by Gbetagamma. Mutations within these sequences demonstrated that all of the residues detected are important for Gbetagamma modulation, although both motifs are necessary for full binding. Molecular modeling predicts that these sites are alpha-helixes near transmembrane domains 3 and 4, near to the lipid bilayer and highly electropositive. Our results demonstrate for the first time the sites for G protein betagamma subunit modulation on GlyRs and provide a new framework regarding the ligand-gated ion channel superfamily regulation by intracellular signaling.
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- 2006
32. P2X5 subunit assembly requires scaffolding by the second transmembrane domain and a conserved aspartate
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Wiebke Duckwitz, Ralf Hausmann, Armaz Aschrafi, and Günther Schmalzing
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Protein Folding ,Protein subunit ,Molecular Sequence Data ,Biology ,Biochemistry ,Domain (software engineering) ,Hydrophobic mismatch ,Xenopus laevis ,Homomeric ,Animals ,Humans ,Amino Acid Sequence ,Molecular Biology ,Aspartic Acid ,Sequence Homology, Amino Acid ,Receptors, Purinergic P2 ,Mutagenesis ,Cell Biology ,Exons ,Protein Structure, Tertiary ,Rats ,Transmembrane domain ,Alternative Splicing ,Ectodomain ,Helix ,Biophysics ,Oocytes ,Nucleic Acid Conformation ,Receptors, Purinergic P2X5 - Abstract
Functional homomeric and heteromeric ATP-gated P2X receptor channels have been shown to display a characteristic trimeric architecture. Of the seven different isoforms (designated P2X(1)-P2X(7)), P2X(5) occurs in humans primarily as a non-functional variant lacking the C-terminal end of the ectodomain and the outer half of the second transmembrane domain. We show that this truncated variant, which results from the splice-skipping of exon 10, is prone to subunit aggregation because the residual transmembrane domain 2 is too short to insert into the membrane. Alleviation of the negative hydrophobic mismatch by the addition of a stretch of moderately hydrophobic residues enabled formation of a second membrane-spanning domain and strictly parallel homotrimerization. Systematic mutagenesis identified only one transmembrane domain 2 residue, Asp(355), which supported homotrimerization in a side chain-specific manner. Our results indicate that transmembrane domain 2 formation contributes 2-fold to hP2X(5) homotrimerization by tethering the end of the ectodomain to the membrane, thereby topologically restricting conformational mobility, and by intramembrane positioning of Asp(355). While transmembrane domain 2 appears to favor assembly by enabling productive subunit interactions in the ectodomain, Asp(355) seems to assist by simultaneously driving intramembrane helix interactions. Overall, these results indicate a complex interplay between topology, helix-helix interactions, and oligomerization to achieve a correctly folded structure.
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- 2006
33. Disulfide Bond Connectivity and Oligomeric State of Anoctamin-1
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Manuela Klapperstueck, Silvia Detro-Dassen, Fritz Markwardt, Ursula Braam, and Günther Schmalzing
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Alanine ,chemistry.chemical_classification ,Residue (chemistry) ,Transmembrane domain ,Biochemistry ,Ectodomain ,Chemistry ,Stereochemistry ,Mutant ,Thiol ,Biophysics ,Protomer ,Cysteine - Abstract
TMEM16A(a)/anoctamin-1 (Ano1) has been identified as a Ca-activated chloride channel. Using blue native PAGE, SDS-PAGE and chemical cross-linking, we have shown that the mouse Ano1 channel (mAno1) shares an obligate homodimeric architecture with the hCLC-1 channel (Fallah et al. Molecular & Cellular Proteomics 10.1074/mcp. M110.004697, 2011). The homodimeric mAno1 dissociated completely into the protomers in non-reducing SDS buffer, indicating that the protomers are held together by non-covalent interactions and not by intersubunit disulfide bonds.The mAno1 protomer contains a total of 16 cysteine residues, of which nine are located in the predicted ectodomain and one (C357) in the first transmembrane helix, TM1. To study whether intrasubunit disulfide bonds may shape the assembly-competent conformation of mAno1, we mutated each of the 10 cysteine residue in the ectodomain or the TM individually to alanine. We found that all the C to A single mutants were well expressed and assembled as homodimers. Eight of the C to A single mutants contained a maleimide-reactive thiol in their ectodomains, whereas the wt-mAno1 and the C357A and C631A mutants lacked a free ectodomain thiol. We conclude from this finding that only eight of the cysteine residues are accessible in the ectodomain and that these eight cysteine residues are involved in the formation of four intrachain disulfide bonds per wt-mAno1 protomer. Also all the 28 C to A double mutants that comprise all pairwise combinations possible with the eight extracellularly accessible cysteine residues assembled efficiently as homodimers. However, in contrast to the single mutants, only a very few of the double mutants exposed a free thiol group. We suggest that the six ectodomain cysteines remaining in each of the double mutants can efficiently re-arrange in a manner that prevents in most cases the formation of only one disulfide bond.
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- 2013
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34. Activation and desensitization of the recombinant P2X1 receptor at nanomolar ATP concentrations
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Jürgen Rettinger and Günther Schmalzing
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Agonist ,Purinergic P2 Receptor Agonists ,Patch-Clamp Techniques ,Physiology ,medicine.drug_class ,medicine.medical_treatment ,Xenopus ,desensitization ,In Vitro Techniques ,Article ,Membrane Potentials ,chemistry.chemical_compound ,Xenopus laevis ,Adenosine Triphosphate ,Homologous desensitization ,medicine ,Purinergic P2 Receptor Antagonists ,Animals ,Magnesium ,Patch clamp ,Receptor ,Desensitization (medicine) ,Membrane potential ,biology ,Dose-Response Relationship, Drug ,Receptors, Purinergic P2 ,biology.organism_classification ,Recombinant Proteins ,Rats ,chemistry ,Biochemistry ,P2X receptor ,Receptors, Purinergic P2X ,Biophysics ,Oocytes ,Female ,activation ,Adenosine triphosphate - Abstract
Activation and desensitization kinetics of the rat P2X1 receptor at nanomolar ATP concentrations were studied in Xenopus oocytes using two-electrode voltage-clamp recording. The solution exchange system used allowed complete and reproducible solution exchange in
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- 2003
35. Ubiquitination precedes internalization and proteolytic cleavage of plasma membrane-bound glycine receptors
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Sven Sadtler, Heinrich Betz, Anne Larissa Leyendecker, Cora Büttner, Nathalie Griffon, Bodo Laube, and Günther Schmalzing
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Proteasome Endopeptidase Complex ,DNA, Complementary ,Proteolysis ,media_common.quotation_subject ,Xenopus ,Lactacystin ,Glycine ,Plasma protein binding ,Biology ,Endocytosis ,Endoplasmic Reticulum ,Biochemistry ,Models, Biological ,RNA, Complementary ,Cell membrane ,chemistry.chemical_compound ,Ubiquitin ,Multienzyme Complexes ,medicine ,Animals ,Electrophoresis, Gel, Two-Dimensional ,Histidine ,Protease Inhibitors ,Enzyme Inhibitors ,Internalization ,Molecular Biology ,Glycine receptor ,media_common ,medicine.diagnostic_test ,Lysine ,Cell Membrane ,Cell Biology ,Hydrogen-Ion Concentration ,Cell biology ,Acetylcysteine ,Anti-Bacterial Agents ,Phenylmethylsulfonyl Fluoride ,Cysteine Endopeptidases ,medicine.anatomical_structure ,chemistry ,biology.protein ,Mutagenesis, Site-Directed ,Oocytes ,Electrophoresis, Polyacrylamide Gel ,Macrolides ,Lysosomes ,Protein Binding - Abstract
The inhibitory glycine receptor (GlyR) in developing spinal neurones is internalized efficiently upon antagonist inhibition. Here we used surface labeling combined with affinity purification to show that homopentameric alpha1 GlyRs generated in Xenopus oocytes are proteolytically nicked into fragments of 35 and 13 kDa upon prolonged incubation. Nicked GlyRs do not exist at the cell surface, indicating that proteolysis occurs exclusively in the endocytotic pathway. Consistent with this interpretation, elevation of the lysosomal pH, but not the proteasome inhibitor lactacystin, prevents GlyR cleavage. Prior to internalization, alpha1 GlyRs are conjugated extensively with ubiquitin in the plasma membrane. Our results are consistent with ubiquitination regulating the endocytosis and subsequent proteolysis of GlyRs residing in the plasma membrane. Ubiquitin-conjugating enzymes thus may have a crucial role in synaptic plasticity by determining postsynaptic receptor numbers.
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- 2001
36. Functional evidence of distinct ATP activation sites at the human P2X(7) receptor
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Fritz Markwardt, Günther Schmalzing, Cora Büttner, and Manuela Klapperstück
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Agonist ,Purinergic P2 Receptor Agonists ,Time Factors ,Physiology ,medicine.drug_class ,Cations, Divalent ,Voltage clamp ,Receptor expression ,Protein subunit ,Biology ,Divalent ,Xenopus laevis ,Adenosine Triphosphate ,medicine ,Reaction Time ,Animals ,Humans ,Magnesium ,Receptor ,Sequence Tagged Sites ,chemistry.chemical_classification ,Dose-Response Relationship, Drug ,Receptors, Purinergic P2 ,Osmolar Concentration ,Electric Conductivity ,Drug Synergism ,Original Articles ,Ligand (biochemistry) ,Peptide Fragments ,Kinetics ,Biochemistry ,chemistry ,Biophysics ,Oocytes ,Female ,Receptors, Purinergic P2X7 ,Extracellular Space ,Intracellular - Abstract
The effect of the agonist ATP on whole cell currents of Xenopus oocytes expressing either the wild-type human P2X7 receptor (hP2X7), an N-terminally hexahistidyl-tagged hP2X7 receptor (His-hP2X7), or a truncated His-hP2X7 receptor (His-hP2X7ΔC) lacking the C-terminal 156 amino acids was investigated using the two-microelectrode voltage clamp technique. The activation time course of the wild-type hP2X7 receptor can be described as the sum of an exponentially growing and an additional almost linearly activating current component. The amplitude of the exponentially activating current component of the wild-type hP2X7 receptor displayed a biphasic dependence on the agonist concentration, which could be best approximated by a model of two equal high-sensitivity and two equal low-sensitivity non-cooperative activation sites with apparent dissociation constants of about 4 and 200 μm free ATP4-, respectively. The linearly activating current was monophasically dependent on the agonist concentration with an apparent dissociation constant of about 200 μm. The contribution of the low-sensitivity sites to current kinetics was reduced or almost abolished in oocytes expressing His-hP2X7 or His-hP2X7ΔC. Our data indicate that the hP2X7 receptor possesses at least two types of activation sites, which differ in ATP4- sensitivity by a factor of 50. The degree of occupation of these two sites influences both activation and deactivation kinetics. Both N- and C-terminal domains appear to be important determinants of the current elicited by activation of the sites with low ATP sensitivity, but not for that mediated by the highly ATP-sensitive sites. Many effects of extracellular ATP on cells of the immune system have been attributed to the presence of a so-called P2Z receptor. Recent work has shown that one of the members of the P2X family of ATP-gated receptors (Buell et al. 1996; Soto et al. 1997; Ralevic & Burnstock, 1998; MacKenzie et al. 1999), designated P2X7, shares many phenotypical properties with the P2Z receptor upon heterologous expression, suggesting that the two are identical. The P2X7 receptor is therefore also referred to as P2Z/P2X7 receptor (Di Virgilio, 1995; Di Virgilio et al. 1998). A peculiarity of the P2X7 subunit is its very long intracellular C-terminal tail, which is 196 or 132 amino acids longer than that of the P2X1 or P2X2 subunit isoform, respectively. Several studies attempting to characterise the recombinant P2X7 receptor have provided information about its complex function, which is not yet fully understood. During short applications of ATP lasting a few seconds, the P2X7 receptor behaves like a typical P2X family member, exhibiting permeability to small cations only. However, upon prolonged or repeated applications of ATP, large non-selective pores are formed in the plasma membrane of some cells expressing P2X7 (Surprenant et al. 1996; Rassendren et al. 1997; Virginio et al. 1999), which have been attributed to the receptor itself. On the other hand, it has also been suggested that the pores represent distinct entities, which become activated subsequent to the stimulation of P2X7 receptors (Coutinho-Silva & Persechini, 1997; Schilling et al. 1999). In native cells, ATP elicits different effects over a wide concentration range. For instance, ATP increases the cell membrane permeability in T lymphocytes, but does not cause lysis at concentrations below 100 μm (El-Moatassim et al. 1989), whereas in the millimolar range ATP induces cytolysis and the subsequent death of T lymphocytes (Di Virgilio et al. 1989; Filippini et al. 1990; Zanovello et al. 1990). Furthermore, immunomodulatory effects, such as the inhibition of human natural killer cell reactivity (Schmidt et al. 1984) and the inhibition of macrophage-mediated cytotoxicity (Cameron, 1984), have been observed to occur at < 100 μm ATP, whereas at least 500 μm ATP was necessary for ATP-induced killing of mycobacteria by human macrophages (Lammas et al. 1997). In addition to the induction of cytolytic pores in some cell types of the immune system, P2Z receptor-dependent activation of phospholipase D (Dubyak & El-Moatassim, 1993), NFkB (Ferrari et al. 1997) and interleukin-1β-converting enzyme (Di Virgilio et al. 1998) have been described, as has the P2Z receptor-dependent loss of l-selectin from human lymphocytes (Jamieson et al. 1996). The molecular basis for the distinct behaviour of immune cells over a broad ATP concentration range is still unclear and may simply reflect the modification of the internal milieu by the concentration-dependent permeabilising effect of P2X7 receptor activation. The various cellular responses may thus be triggered by quantitatively different changes in the intracellular Ca2+, Na+ or K+ concentrations due to P2X7 receptor stimulation. Thus the degree of ATP breakdown by ectoATPases, the amount of receptor expression in the investigated cells and the concentration of divalent cations reducing the concentration of the ligand, free ATP4- (Di Virgilio et al. 1998), has to be taken into account. Furthermore, the possible involvement of other receptors with different sensitivity to ATP has to be considered. Here we provide electrophysiological evidence of distinct ATP activation sites at the human P2X7 receptor. The two sites differ in their ATP sensitivity by a factor of about 50 and influence differently both the activation and deactivation kinetics and the permeation characteristics of the hP2X7 receptor. The existence of two ATP activation sites provides one possible explanation for the variable pattern of signalling of hP2X7-expressing cells at different concentrations of ATP.
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- 2001
37. Surface-localized glycine transporters 1 and 2 function as monomeric proteins in Xenopus oocytes
- Author
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Armaz Aschrafi, Jesus Gomeza, Annette Nicke, Heinrich Betz, Masahisa Horiuchi, and Günther Schmalzing
- Subjects
Neurotransmitter transporter ,Recombinant Fusion Proteins ,Xenopus ,Glycine ,Bicarbonate transporter protein ,Glycine Plasma Membrane Transport Proteins ,Glycine transporter ,Cell membrane ,Xenopus laevis ,medicine ,Animals ,Multidisciplinary ,biology ,Peripheral membrane protein ,Cell Membrane ,Membrane Proteins ,Biological Sciences ,biology.organism_classification ,medicine.anatomical_structure ,Amino Acid Transport Systems, Neutral ,Biochemistry ,Membrane protein ,Oocytes ,Carrier Proteins ,Oligopeptides - Abstract
Na + /Cl − -dependent neurotransmitter transporters form a superfamily of transmembrane proteins that share 12 membrane-spanning regions. To gain information about the quaternary structure of these transporter proteins, we heterologously expressed the glial glycine transporter GlyT1 and its neuronal homolog GlyT2 in Xenopus oocytes. By using metabolic labeling with [ 35 S]methionine or surface labeling with a plasma membrane impermeable reagent followed by affinity purification, we separately analyzed the total cellular pools of newly synthesized GlyTs and its functional plasma membrane-bound fractions. Upon blue native gel electrophoresis, the surface-localized transporter proteins were found to exist exclusively in complex-glycosylated monomeric form, whereas a significant fraction of the intracellular GlyT1 and GlyT2 was core-glycosylated and oligomeric. In contrast, even after treatment with the crosslinker glutaraldehyde, surface GlyTs failed to migrate as oligomeric proteins. These results indicate that plasma membrane-bound GlyT1 and GlyT2 are monomeric proteins. Thus, Na + /Cl − -dependent neurotransmitter transporters do not require oligomerization for substrate translocation.
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- 2001
38. A novel C3-like ADP-ribosyltransferase from Staphylococcus aureus modifying RhoE and Rnd3
- Author
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Klaus Aktories, Ingo Just, Christian Wilde, Gursgaran S. Chhatwal, and Günther Schmalzing
- Subjects
DNA, Bacterial ,Staphylococcus aureus ,RHOA ,Botulinum Toxins ,Molecular Sequence Data ,GTPase ,Biology ,medicine.disease_cause ,Biochemistry ,law.invention ,law ,medicine ,Transferase ,Amino Acid Sequence ,Cytoskeleton ,Molecular Biology ,ADP Ribose Transferases ,Base Sequence ,Sequence Homology, Amino Acid ,Rnd3 ,GTPase-Activating Proteins ,Cell Biology ,Actin cytoskeleton ,Molecular biology ,Recombinant DNA ,biology.protein ,Clostridium botulinum ,Poly(ADP-ribose) Polymerases - Abstract
Clostridium botulinum C3 is the prototype of the family of the C3-like transferases that ADP-ribosylate exclusively RhoA, -B and -C. The ADP-ribose at Asn-41 results in functional inactivation of Rho reflected by disaggregation of the actin cytoskeleton. We report on a new C3-like transferase produced by a pathogenic Staphylococcus aureus strain. The transferase designated C3(Stau) was cloned from the genomic DNA. At the amino acid level, C3(Stau) revealed an identity of 35% to C3 from C. botulinum and Clostridium limosum exoenzyme, respectively, and of 78% to EDIN from S. aureus. In addition to RhoA, which is the target of the other C3-like transferases, C3(Stau) modified RhoE and Rnd3. RhoE was ADP-ribosylated at Asn-44, which is equivalent to Asn-41 of RhoA. RhoE and Rnd3 are members of the Rho subfamily, which are deficient in intrinsic GTPase activity and possess a RhoA antagonistic cell function. The protein substrate specificity found with recombinant Rho proteins was corroborated by expression of RhoE in Xenopus laevis oocytes showing that RhoE was also modified in vivo by C3(Stau) but not by C3 from C. botulinum. The poor cell accessibility of C3(Stau) was overcome by generation of a chimeric toxin recruiting the cell entry machinery of C. botulinum C2 toxin. The chimeric C3(Stau) caused the same morphological and cytoskeletal changes as the chimeric C. botulinum C3. C3(Stau) is a new member of the family of the C3-like transferases but is also the prototype of a subfamily of RhoE/Rnd modifying transferases.
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- 2001
39. Characteristics of P2X7 receptors from human B lymphocytes expressed in Xenopus oocytes
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Cora Büttner, Günther Schmalzing, Manuela Klapperstück, Fritz Markwardt, and Thomas Böhm
- Subjects
Purinoceptor ,Kinetics ,Biophysics ,Xenopus ,In Vitro Techniques ,Biochemistry ,Ion Channels ,Membrane Potentials ,Xenopus laevis ,Adenosine Triphosphate ,Species Specificity ,Complementary DNA ,Animals ,Humans ,Receptor ,P2x7 receptor ,Gene ,Ion channel ,DNA Primers ,B-Lymphocytes ,B lymphocyte ,biology ,Base Sequence ,Receptors, Purinergic P2 ,Cell Biology ,Permeation ,biology.organism_classification ,Phenotype ,Molecular biology ,Recombinant Proteins ,Rats ,P2X receptor ,Oocytes ,Female ,Receptors, Purinergic P2X7 ,Xenopus oocyte ,Ion Channel Gating - Abstract
Human B lymphocytes express an ATP-gated ion channel (P2Z receptor), which shares similarities with the recently identified P2X7 receptor. Using gene specific primers, we have now isolated P2X7 cDNA from the total RNA of human B lymphocytes. This hP2X7 receptor subtype was expressed in Xenopus oocytes and electrophysiologically characterized. The hP2X7 receptor is similar to, but does not completely match, P2Z of human B cells. The hP2X7 receptors resemble the P2Z receptors with regard to the ATP concentration of half maximal activation, reproducibility, permeation characteristics and lack of desensitization of the ATP-evoked currents. However, in contrast to the native lymphocytic P2Z receptor, the time course of activation of hP2X7 displayed an additional linearly increasing current component. Furthermore, a second, small and slowly deactivating current component exists only in hP2X7 expressed in oocytes. The activation and deactivation kinetics as well as permeation characteristics of hP2X7 are different from rat P2X7 recently expressed in oocytes. Unlike in mammalian cells, hP2X7 expressed in Xenopus oocytes is not sufficient to induce large non-selective pores.
- Published
- 2000
40. Chapter 9 Novel ligands for P2 receptor subtypes in innervated tissues
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Susanne Damer, Jürgen Rettinger, Günter Lambrecht, Ernst Mutschler, Beate Niebel, Sittah Czeche, Peter Nickel, and Günther Schmalzing
- Subjects
P2Y receptor ,Biochemistry ,Stereochemistry ,Chemistry ,Suramin ,Functional selectivity ,Enzyme-linked receptor ,medicine ,Antagonist ,P2 receptor ,Selectivity ,Receptor ,medicine.drug - Abstract
Among suramin analogues, the properties of P2 receptor subtype blockade and ecto-nucleotidase inhibition appear to be controlled by different structural parameters (Fig. 1 and 2, Table 1; Van Rhee et al., 1994; Beukers et al., 1995; Bultmann et al., 1996; Damer et al., 1998a, 1998b; and this study): the molecular size of the compounds, the position of the sulfonic acid residues in the naphthalene rings, the substitution pattern of the benzoyl moieties and the 3'- or 4'-aminobenzoyl-linkages of the phenyl rings "1" and "2". As a result, compounds with different receptor selectivity profiles were obtained. A maximum in potency at and selectivity for P2X1 receptors is reached in NF279, which is a specific P2 receptor antagonist and the compound with the highest P2X1 vs. P2Y receptor and ecto-nucleotidase selectivity presently available.
- Published
- 1999
41. Chapter 28 Purinoceptors in human B-lymphocytes
- Author
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Cora Büttner, Fritz Markwardt, D. Riemann, Günther Schmalzing, Manuela Klapperstück, and Matthias Löhn
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Membrane potential ,Agonist ,medicine.drug_class ,Purinergic receptor ,Conductance ,Fluorescence ,chemistry.chemical_compound ,Biochemistry ,chemistry ,medicine ,Biophysics ,Patch clamp ,Adenosine triphosphate ,Intracellular - Abstract
Publisher Summary To characterize the properties of purinoceptors in human B-lymphocytes, ATPinduced membrane currents and [Ca 2+ ] i are measured simultaneously in Epstein–Barr-virus transformed and tonsillar human single B-lymphocytes by means of the tight-seal voltageclamp- and Fluo-3/Fura-red-fluorescence technique. In human B-lymphocytes, the existence of an ATPgated cation channel with a single-channel conductance of 9 pS is shown by patch clamp measurements. The channels are activated only by free ATP 4- - or BzATP 4- within less than 1 s, do not desensitize during continuous application of the agonist for several seconds and are deactivated within 1 s after agonist removal. Human B-lymphocytes are not permeabilized to the fluorescent dye Fura-red (MW 725) by an application of 1 mmo/L ATP 4- for more than 5 min. The agonist binding characteristics of purinoceptors in human B-lymphocytes are similar to cloned P2X 7 , purinoceptors. It is concluded that under physiological conditions, this purinergic receptor on B-lymphocytes does not increase the global intracellular free Ca 2+ -concentration.
- Published
- 1999
42. P2X1 and P2X3 receptors form stable trimers: a novel structural motif of ligand-gated ion channels
- Author
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Hans G. Bäumert, Annette Eichele, Jürgen Rettinger, Günter Lambrecht, Ernst Mutschler, Günther Schmalzing, and Annette Nicke
- Subjects
endocrine system ,Glycosylation ,medicine.drug_class ,Pentamer ,Stereochemistry ,Biology ,Ligands ,General Biochemistry, Genetics and Molecular Biology ,Ion Channels ,Xenopus laevis ,Glucosides ,medicine ,Purinergic P2 Receptor Antagonists ,Animals ,Histidine ,Receptors, Cholinergic ,Receptor ,Structural motif ,Molecular Biology ,Ion channel ,General Immunology and Microbiology ,Receptors, Purinergic P2 ,General Neuroscience ,Membrane Proteins ,Receptor antagonist ,Nicotinic acetylcholine receptor ,Cross-Linking Reagents ,Hexosaminidases ,Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase ,Biochemistry ,Receptors, Purinergic P2X ,Pyridoxal Phosphate ,Oocytes ,Ligand-gated ion channel ,Protein quaternary structure ,Electrophoresis, Polyacrylamide Gel ,Dimerization ,Receptors, Purinergic P2X3 ,Research Article - Abstract
P2X receptors are cation channels gated by extracellular ATP. The seven known P2X isoforms possess no sequence homology with other proteins. Here we studied the quaternary structure of P2X receptors by chemical cross-linking and blue native PAGE. P2X1 and P2X3 were N-terminally tagged with six histidine residues to allow for non-denaturing receptor isolation from cRNA-injected, [35S]methionine-labeled oocytes. The His-tag did not change the electrophysiological properties of the P2X1 receptor. His-P2X1 was found to carry four N-glycans per polypeptide chain, only one of which acquired Endo H resistance en route to the plasma membrane. 3, 3'-Dithiobis(sulfosuccinimidylpropionate) (DTSSP) and two of three bifunctional analogues of the P2X receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) cross-linked digitonin-solubilized His-P2X1 and His-P2X3 quantitatively to homo-trimers. Likewise, when analyzed by blue native PAGE, P2X receptors purified in digitonin or dodecyl-beta-D-maltoside migrated entirely as non-covalently linked homo-trimers, whereas the alpha2 beta gamma delta nicotinic acetylcholine receptor (used as a positive control) migrated as the expected pentamer. P2X monomers remained undetected soon after synthesis, indicating that trimerization occurred in the endoplasmic reticulum. The plasma membrane form of His-P2X1 was also identified as a homo-trimer. If n-octylglucoside was used for P2X receptor solubilization, homo-hexamers were observed, suggesting that trimers can aggregate to form larger complexes. We conclude that trimers represent an essential element of P2X receptor structure. Keywords: blue native PAGE/cross-linking/P2X receptor/quaternary structure.
- Published
- 1998
43. Isoform-specific interactions of Na,K-ATPase subunits are mediated via extracellular domains and carbohydrates
- Author
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Günther Schmalzing, Sergio M. Gloor, and Karina Ruhl
- Subjects
Gene isoform ,Models, Molecular ,Immunoprecipitation ,Protein Conformation ,Protein subunit ,Xenopus ,Plasma protein binding ,Mice ,Protein structure ,Enzyme Stability ,Animals ,Enzyme Inhibitors ,Ouabain ,G alpha subunit ,Glycoproteins ,Multidisciplinary ,biology ,Cell Polarity ,Biological Sciences ,biology.organism_classification ,Rats ,Isoenzymes ,Ectodomain ,Biochemistry ,Sodium-Potassium-Exchanging ATPase ,Protein Binding - Abstract
The functional unit of the Na,K-ATPase consists of a catalytic α subunit noncovalently linked with a glycoprotein subunit, β. Using ouabain binding assays and immunoprecipitation of rodent α/β complexes, we show here that all six possible isozymes between three α and two β isoforms can be formed inXenopusoocytes. Two isoform-specific differences in α/β interactions are observed: (i) α1/β1 and α2/β2 complexes, in contrast to α1/β2 complexes, are stable against Triton X-100-mediated dissociation, and (ii) β2 subunits must carryN-glycans to combine with α1 but not with α2. The interacting surfaces are mainly exposed to the extracellular side because coexpression of a truncated β1 subunit comprising the ectodomain results in assembly with α1 and α2, but not with α3; the β2 ectodomain combines with α2 only. A chimera consisting of 81% and 19% of the α1 N terminus and α2 C terminus, respectively, behaves like α2 and coprecipitates with the β2 ectodomain. In contrast, the reciprocal chimera does not coprecipitate with the β2 ectodomain. These results provide evidence for a selective interaction of Na,K-ATPase α and β subunits.
- Published
- 1997
44. Molecular characterization of human and bovine endothelin converting enzyme (ECE-1)
- Author
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Heinz Hillen, Rainer Otter, Burkhard Kröger, Thomas Subkowski, Martin Schmidt, Günther Schmalzing, Elard Jacob, Harald Seulberger, and Thomas F. Meyer
- Subjects
DNA, Complementary ,Endothelin converting enzyme 1 ,medicine.medical_treatment ,Molecular Sequence Data ,Biophysics ,Biology ,Endothelin-Converting Enzymes ,Biochemistry ,Polymerase Chain Reaction ,Structural Biology ,Complementary DNA ,Genetics ,medicine ,Animals ,Aspartic Acid Endopeptidases ,Humans ,Trypsin ,Amino Acid Sequence ,Cloning, Molecular ,education ,Molecular Biology ,Peptide sequence ,DNA Primers ,chemistry.chemical_classification ,education.field_of_study ,Metalloproteinase ,Protease ,Endothelin-1 ,Base Sequence ,Sequence Homology, Amino Acid ,Metalloendopeptidases ,Cell Biology ,Blotting, Northern ,Endothelin 1 ,Molecular biology ,Recombinant Proteins ,Kinetics ,Enzyme ,chemistry ,Organ Specificity ,Cattle ,Electrophoresis, Polyacrylamide Gel ,Endothelium, Vascular ,Endothelin converting enzyme ,DNA Probes ,medicine.drug - Abstract
A membrane-bound protease activity that specifically converts Big endothelin-1 has been purified from bovine endothelial cells (FBHE) The enzyme was cleaved with trypsin and the peptide sequencing analysis confirmed it to be a zinc chelating metalloprotease containing the typical HEXXH (HELTH) motif. RT-PCR and cDNA screens were employed to isolate the complete cDNAs of the bovine and human enzymes. This human metalloprotease was expressed heterologously in cell culture oocytes. The catalytic activity of the recombinant enzyme is the same as that determined for the natural enzyme. The data suggest that the characterized enzyme represents the functional human endothelin converting enzyme ECE-1.
- Published
- 1994
45. The adhesion molecule on glia (AMOG/beta 2) and alpha 1 subunits assemble to functional sodium pumps in Xenopus oocytes
- Author
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Melitta Schachner, Günther Schmalzing, Sergio M. Gloor, and Silke Kröner
- Subjects
Protein subunit ,Cell Adhesion Molecules, Neuronal ,Xenopus ,Alpha (ethology) ,Diaphragm pump ,Digitonin ,Nerve Tissue Proteins ,Biology ,Torpedo ,Biochemistry ,law.invention ,RNA, Complementary ,chemistry.chemical_compound ,Mice ,law ,medicine ,Animals ,Ouabain ,Molecular Biology ,Cation Transport Proteins ,Cells, Cultured ,Adenosine Triphosphatases ,Extracellular Matrix Proteins ,Cell adhesion molecule ,Cell Biology ,biology.organism_classification ,Rubidium ,Molecular biology ,Precipitin Tests ,Isoenzymes ,medicine.anatomical_structure ,chemistry ,Oocytes ,RNA ,Electrophoresis, Polyacrylamide Gel ,Sodium-Potassium-Exchanging ATPase ,Neuroglia ,Astrocyte ,Plasmids - Abstract
The adhesion molecule on glia, AMOG, an integral cell surface glycoprotein highly expressed by cerebellar astrocytes and involved in neuron to astrocyte adhesion and granule neuron migration (Antonicek, H., Persohn, E., and Schachner, M. (1987) J. Cell Biol. 104, 1587-1595) has been identified as a beta 2 subunit isoform of the mouse sodium pump (Gloor, S., Antonicek, H., Sweadner, K.J., Pagliusi, S., Frank, R., Moos, M., and Schachner, M. (1990) J. Cell Biol. 110, 165-174). Here we demonstrate that AMOG/beta 2 expressed by cRNA injection in Xenopus oocytes is capable of combining with endogenous Xenopus alpha 1 subunits or coexpressed Torpedo alpha 1 subunits to yield a functional alpha 1/AMOG sodium pump isozyme. Determinations of the number of ouabain binding sites and ouabain-sensitive 86Rb+ uptake suggest that the alpha 1/AMOG isozyme has slightly lower maximum transport rate and apparent affinity for external K+ than the alpha 1/beta 1 isozyme. Immunoprecipitation of alpha 1/AMOG complexes from digitonin extracts of [35S]methionine-labeled oocytes with a monoclonal anti-AMOG antibody provides direct evidence for a stable association between AMOG and the alpha 1 subunits of Xenopus and Torpedo.
- Published
- 1992
46. Micromolar free calcium exposes ouabain-binding sites in digitonin-permeabilized Xenopus laevis oocytes
- Author
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Silke Kröner and Günther Schmalzing
- Subjects
Intracellular Fluid ,GTP' ,Sodium ,chemistry.chemical_element ,Guanosine ,Digitonin ,Wasp Venoms ,Biology ,Biochemistry ,Ouabain ,Exocytosis ,Sodium Channels ,chemistry.chemical_compound ,Xenopus laevis ,Adenosine Triphosphate ,Calmodulin ,medicine ,Animals ,Protease Inhibitors ,Molecular Biology ,Ovum ,Polymyxin B ,Manganese ,Binding Sites ,Ionophores ,Nucleotides ,Cell Membrane ,Neomycin ,Cell Biology ,Adenosine ,Stimulation, Chemical ,beta-N-Acetylhexosaminidases ,Culture Media ,chemistry ,Mastoparan ,Ionomycin ,Oocytes ,Potassium ,Intercellular Signaling Peptides and Proteins ,Calcium ,Peptides ,medicine.drug ,Research Article - Abstract
As demonstrated previously, digitonin-permeabilized Xenopus oocytes have a large internal pool of sodium pumps which are inaccessible to cytosolic ouabain [Schmalzing, Kröner & Passow (1989) Biochem. J. 260, 395-399]. Access to internal ouabain-binding sites required permeabilization of inner membranes with SDS. In the present study, micromolar free Ca2+ was found to stimulate ouabain binding in the digitonin-permeabilized cells (K0.5 0.5 microM-Ca2+, h 1.9, average of seven experiments) without disrupting intracellular membranes. Sustained incubation at 9 microM-Ca2+ was as effective as SDS in inducing access to the ouabain-binding sites of the internal sodium pumps. Omission of either Mg2+ or ATP completely abolished the Ca2+ effect. Half-maximal stimulation by Ca2+ required approx. 0.4 mM-MgATP. Of a variety of nucleotides tested, none was as effective as ATP (rank order ATP greater than ADP greater than ATP[S] (adenosine 5′-[gamma-thio]triphosphate) greater than CTP greater than UTP greater than ITP = XTP greater than GTP). Pi, AMP, cyclic AMP, cyclic GMP, GTP[S] (guanosine 5′-[gamma-thio]triphosphate) and a stable ATP analogue p[NH]ppA (adenosine 5′-[beta gamma-imido]triphosphate), were ineffective. The metalloendoproteinase inhibitor carbobenzoxy-Gly-Phe-amide reduced the Ca2+ effect by some 50%. Inhibitors of chymotrypsin and the Ca2+ proteinase calpain had no effect. Ca2+ ionophores (A23187 and ionomycin) and the polycations neomycin and polymixin B blocked the Ca2+ response entirely. Neomycin also abolished a Ca2(+)-independent stimulation of ouabain binding by the wasp venom mastoparan. The requirements for increasing the accessibility of ouabain-binding sites are remarkably similar to those for exocytosis in secretory cells, suggesting that oocytes and eggs possess a Ca2(+)-regulated pathway for the plasma membrane insertion of sodium pumps.
- Published
- 1990
47. Downregulation of surface sodium pumps by endocytosis during meiotic maturation of Xenopus laevis oocytes
- Author
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Silke Kröner, Peter Eckard, Günther Schmalzing, and Hermann Passow
- Subjects
Cell Membrane Permeability ,Physiology ,Sodium ,chemistry.chemical_element ,Ouabain ,chemistry.chemical_compound ,Xenopus laevis ,Adenosine Triphosphate ,medicine ,Animals ,Homeostasis ,Sodium dodecyl sulfate ,Ion transporter ,Cell Membrane ,Cell Biology ,Membrane transport ,Endocytosis ,Kinetics ,Meiosis ,Membrane ,Digitonin ,chemistry ,Biochemistry ,Biophysics ,Oocytes ,Autoradiography ,Female ,Sodium-Potassium-Exchanging ATPase ,Phosphorus Radioisotopes ,Intracellular ,medicine.drug - Abstract
During meiotic maturation, plasma membranes of Xenopus laevis oocytes completely lose the capacity to transport Na and K and to bind ouabain. To explore whether the downregulation might be due to an internalization of the sodium pump molecules, the intracellular binding of ouabain was determined. Selective permeabilization of the plasma membrane of mature oocytes (eggs) by digitonin almost failed to disclose ouabain binding sites. However, when the eggs were additionally treated with 0.02% sodium dodecyl sulfate (SDS) to permeabilize inner membranes, all sodium pumps present before maturation were recovered. Phosphorylation by [gamma-32P]ATP combined with SDS-polyacrylamide gel electrophoresis (PAGE) and autoradiography showed that sodium pumps were greatly reduced in isolated plasma membranes of eggs. According to sucrose gradient fractionation, maturation induced a shift of sodium pumps from the plasma membrane fraction to membranes of lower buoyant density with a protein composition different from that of the plasma membrane. Endocytosed sodium pumps identified on the sucrose gradient from [3H]ouabain bound to the cell surface before maturation could be phosphorylated with inorganic [32P]phosphate. The findings suggest that downregulation of sodium pumps during maturation is brought about by translocation of surface sodium pumps to an intracellular compartment, presumably endosomes. This contrasts the mechanism of downregulation of Na-dependent cotransport systems, the activities of which are reduced as a consequence of a maturation-induced depolarization of the membrane without a removal of the corresponding transporter from the plasma membrane.
- Published
- 1990
48. Kinetics of [3H] Trifluoperazine in Bile Fistula Rats
- Author
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Ursula Breyer and Günther Schmalzing
- Subjects
Male ,Tail ,medicine.medical_specialty ,Time Factors ,Fistula ,Health, Toxicology and Mutagenesis ,Metabolite ,Kinetics ,Portal vein ,Trifluoperazine ,Toxicology ,Biochemistry ,Excretion ,chemistry.chemical_compound ,Biliary excretion ,Internal medicine ,medicine ,Animals ,Bile ,Tissue Distribution ,Biliary Tract ,Pharmacology ,Portal Vein ,General Medicine ,medicine.disease ,Rats ,Biliary Tract Surgical Procedures ,Endocrinology ,chemistry ,Injections, Intravenous ,Glucuronide ,medicine.drug - Abstract
1. Anaesthetized male rats with a bile fistula received 12-3 micron mol/kg [9-3H]tri-fluoperazine into the tail vein, and the biliary excretion of total radioactivity, unchanged drug and phenolic glucuronides was followed for 8 h. 2. About half of the administered radioactivity apeared in bile within 8 h;80% of the biliary metabolites were unextractable even after beta-glucuronidasearylsulphatase hydrolysis; about 10% were glucuronides of 7-hydroxytrifluoperazine and its N-demethylated analogue; approx. 0-6% of the excreted radioactivity was unchanged drug. 3. A more rapid excretion, but a similar metabolite pattern, was observed when the drug was administered into the portal vein and bile was collected for 2 h. 4. Rats pre-treated with trifluoperazine per os for 3 weeks and then given the radioactive dose into the tail vein excreted increased quantities of the demethylated phenol glucuronide, while the other metabolities remained unchanged.
- Published
- 1978
49. Modulation of ATPase activities of human erythrocyte membranes by free fatty acids or phospholipase A2
- Author
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Petra Kutschera and Günther Schmalzing
- Subjects
Erythrocytes ,Physiology ,ATPase ,Biophysics ,Calcium-Transporting ATPases ,Fatty Acids, Nonesterified ,Phospholipase ,Phospholipases A ,Membrane Lipids ,Structure-Activity Relationship ,chemistry.chemical_compound ,Phospholipase A2 ,Humans ,Phospholipids ,Adenosine Triphosphatases ,chemistry.chemical_classification ,biology ,Erythrocyte Membrane ,Fatty acid ,Cell Biology ,Kinetics ,Phospholipases A2 ,Membrane ,chemistry ,Biochemistry ,Phospholipases ,Fatty Acids, Unsaturated ,biology.protein ,Free fatty acid receptor ,Ca(2+) Mg(2+)-ATPase ,Stearic acid ,Polyunsaturated fatty acid - Abstract
The artificial insertion of increasing amounts of unsaturated fatty acids into human erythrocyte membranes modulated ATPase activities in a biphasic manner, depending on the number and position of double bonds, their configuration, and the chain length. Uncharged long-chain fatty acid derivatives with double bonds and short-chain fatty acids were ineffective. Stearic acid stimulated Na+ K+-ATPase only. Anionic and non-ionic detergents and alpha-lysophosphatidylcholine failed to stimulate ATPase activities at low, and inhibited them at high concentrations. Mg2+-AtPase activity was maximally enhanced by a factor of 2 in the presence of monoenoic fatty acids; half-maximal stimulation was achieved at a molar ratio of cis(trans)-configurated C18 acids/membrane phospholipid of 0.16 (0.26). Na+K+-ATPase activity was maximally augmented by 20% in the presence of monoenoic C18 fatty acids at 37 degrees C. Half-maximal effects were attained at a molar ratio oleic (elaidic) acid/phospholipid of 0.032 (0.075). Concentrations of free fatty acids which inhibited ATPases activities at 37 degrees C were most stimulatory at reduced temperatures. At 10 degrees C, oleic acid increased Na+K+-ATPase activity fivefold (molar ratio 0.22). Unsaturated fatty acids simulated the effects of calmodulin on Ca2+-ATPase of native erythrocyte membranes (i.e., increase of Vmax from 1.6 to 5 mumol PO43- . phospholipid-1 . hr-1, decrease of K'Ca from 6 microM to 1.4-1.8 microM). Stearic acid decreased K'Ca (2 microM) only, probably due to an increase of negative surface charges. A stimulation of Mg2+-ATPase, Na+K+-ATPase, and Ca2+-ATPase could be achieved by incubation of the membranes with phospholipase A2. An electrostatic segregation of free fatty acids by ATPases with ensuing alterations of surface charge densities and disordering of the hydrophobic environment of the enzymes provides an explanation of the results.
- Published
- 1982
50. Assembly of nicotinic α7 subunits in Xenopus oocytes is partially blocked at the tetramer level
- Author
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Jürgen Rettinger, Annette Nicke, Heike Thurau, Sven Sadtler, and Günther Schmalzing
- Subjects
Glycosylation ,Patch-Clamp Techniques ,alpha7 Nicotinic Acetylcholine Receptor ,Blue native PAGE ,Biophysics ,Xenopus ,Receptors, Nicotinic ,Biology ,α7 Nicotinic acetylcholine receptor subunit ,Biochemistry ,Xenopus laevis ,Ganglion type nicotinic receptor ,5-Hydroxytryptamine receptor subunit ,Tetramer ,Polysaccharides ,Structural Biology ,Genetics ,Sucrose density centrifugation ,Animals ,Quaternary structure ,Protein Structure, Quaternary ,Molecular Biology ,Cell Biology ,biology.organism_classification ,Rats ,Protein Subunits ,Nicotinic agonist ,G12/G13 alpha subunits ,Mutagenesis, Site-Directed ,Oocytes ,Protein quaternary structure ,Receptors, Serotonin, 5-HT3 ,Alpha-4 beta-2 nicotinic receptor ,Protein Processing, Post-Translational ,Cys-loop receptors - Abstract
The assembly of nicotinic alpha1beta1gammadelta, alpha3beta4, and alpha7 receptors and 5-hydroxytryptamine 3A (5HT3A) receptors was comparatively evaluated in Xenopus oocytes by blue native PAGE analysis. While alpha1betagammadelta subunits, alpha3beta4 subunits, and 5HT3A subunits combined efficiently to pentamers, alpha7 subunits existed in various assembly states including trimers, tetramers, pentamers, and aggregates. Only alpha7 subunits that completed the assembly process to homopentamers acquired complex-type carbohydrates and appeared at the cell surface. We conclude that Xenopus oocytes have a limited capacity to guide the assembly of alpha7 subunits, but not 5HT3A subunits to homopentamers. Accordingly, ER retention of imperfectly assembled alpha7 subunits rather than inefficient routing of fully assembled alpha7 receptors to the cell surface limits surface expression levels of alpha7 nicotinic acetylcholine receptors.
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