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Featured researches published by Ralf Hausmann.


Purinergic Signalling | 2012

Molecular and functional properties of P2X receptors—recent progress and persisting challenges

Karina Kaczmarek-Hájek; Éva Lörinczi; Ralf Hausmann; Annette Nicke

ATP-gated P2X receptors are trimeric ion channels that assemble as homo- or heteromers from seven cloned subunits. Transcripts and/or proteins of P2X subunits have been found in most, if not all, mammalian tissues and are being discovered in an increasing number of non-vertebrates. Both the first crystal structure of a P2X receptor and the generation of knockout (KO) mice for five of the seven cloned subtypes greatly advanced our understanding of their molecular and physiological function and their validation as drug targets. This review summarizes the current understanding of the structure and function of P2X receptors and gives an update on recent developments in the search for P2X subtype-selective ligands. It also provides an overview about the current knowledge of the regulation and modulation of P2X receptors on the cellular level and finally on their physiological roles as inferred from studies on KO mice.


Journal of Laryngology and Otology | 2002

Survival in second primary malignancies of patients with head and neck cancer.

Ercole Di Martino; Bernd Sellhaus; Ralf Hausmann; Ralf Minkenberg; Melanie Lohmann; Martin W Esthofen

Second primary tumours occur frequently in patients with a history of head and neck malignancies. Delays in making an early and correct diagnosis can seriously affect the therapy management and survival. This was a retrospective study of 120 patients with a history of head and neck cancer, presenting with a second primary tumour. Current follow-up strategies and the use of routine sonographic imaging of the head and neck regions were evaluated, and the impact that tumour chronology, the tumour site and the various treatment modalities have on the survival were assessed. Forty-two per cent of patients developed a metachronous second malignancy more than five years after diagnosis of the index tumour. The accuracy of colour-duplex sonography in detection of second primaries in the head and neck was 82.3 per cent. First and second primary tumours located in the larynx were observed to have the highest five-year survival rate. Patients who developed metachronous tumours had a five-year survival rate of 68.9 per cent for the index tumours, and a 26 per cent five-year survival rate with the occurrence of a second neoplasm. With synchronous tumours a mean survival time of 18 months and a five-year survival rate of 11.9 per cent was found (p < 0.0001). Where clinically appropriate an aggressive treatment strategy was employed and yielded the most favourable results with a five-year survival rate of 66.8 per cent and 35.9 per cent for index tumours and second primary malignancies, respectively. Since more than 40 per cent of the metachronous second primaries in patients with a history of head and neck malignancy occur beyond the five-year follow-up period, an extended protocol with individually adjusted close monitoring of high-risk patients seems appropriate. Colour-duplex sonography is a valuable screening investigation for the early detection of second primary tumours. The treatment of a second primary is often less successful than for the same malignancy occurring primarily. The prognosis of synchronous tumours is significantly lower when compared to malignancies of a metachronous nature, despite some encouraging individual results. Only the early implementation of aggressive treatment methods for second primaries is successful in terms of survival.


Journal of Pharmacology and Experimental Therapeutics | 2010

NF546 [4,4′-(Carbonylbis(imino-3,1-phenylene-carbonylimino-3,1-(4-methyl-phenylene)-carbonylimino))-bis(1,3-xylene-α,α′-diphosphonic Acid) Tetrasodium Salt] Is a Non-Nucleotide P2Y11 Agonist and Stimulates Release of Interleukin-8 from Human Monocyte-Derived Dendritic Cells

Sabine Meis; Alexandra Hamacher; Darunee Hongwiset; Claudia Marzian; Michael Wiese; Niels Eckstein; Hans-Dieter Royer; Didier Communi; Jean-Marie Boeynaems; Ralf Hausmann; Günther Schmalzing; Matthias U. Kassack

The G protein-coupled P2Y11 receptor is involved in immune system modulation. In-depth physiological evaluation is hampered, however, by a lack of selective and potent ligands. By screening a library of sulfonic and phosphonic acid derivatives at P2Y11 receptors recombinantly expressed in human 1321N1 astrocytoma cells (calcium and cAMP assays), the selective non-nucleotide P2Y11 agonist NF546 [4,4′-(carbonylbis(imino-3,1-phenylene-carbonylimino-3,1-(4-methyl-phenylene)carbonylimino))-bis(1,3-xylene-α,α′-diphosphonic acid) tetrasodium salt] was identified. NF546 had a pEC50 of 6.27 and is relatively selective for P2Y11 over P2Y1, P2Y2, P2Y4, P2Y6, P2Y12, P2X1, P2X2, and P2X2-X3. Adenosine-5′-O-(3-thio)triphosphate (ATPγS), a nonhydrolyzable analog of the physiological P2Y11 agonist ATP, and NF546 use a common binding site as suggested by molecular modeling studies and their competitive behavior toward the nanomolar potency antagonist NF340 [4,4′-(carbonylbis(imino-3,1-(4-methyl-phenylene)carbonylimino))bis(naphthalene-2,6-disulfonic acid) tetrasodium salt] in Schild analysis. The pA2 of NF340 was 8.02 against ATPγS and 8.04 against NF546 (calcium assays). NF546 was further tested for P2Y11-mediated effects in monocyte-derived dendritic cells. Similarly to ATPγS, NF546 led to thrombospondin-1 secretion and inhibition of lipopolysaccharide-stimulated interleukin-12 release, whereas NF340 inhibited these effects. Further, for the first time, it was shown that ATPγS or NF546 stimulation promotes interleukin 8 (IL-8) release from dendritic cells, which could be inhibited by NF340. In conclusion, we have described the first selective, non-nucleotide agonist NF546 for P2Y11 receptors in both recombinant and physiological expression systems and could show a P2Y11-stimulated IL-8 release, further supporting the immunomodulatory role of P2Y11 receptors.


Molecular Pharmacology | 2006

The Suramin Analog 4,4′,4″,4″′-(Carbonylbis(imino-5,1,3-benzenetriylbis (carbonylimino)))tetra-kis-benzenesulfonic Acid (NF110) Potently Blocks P2X3 Receptors: Subtype Selectivity Is Determined by Location of Sulfonic Acid Groups

Ralf Hausmann; Jürgen Rettinger; Zoltan Gerevich; Sabine Meis; Matthias U. Kassack; Peter Illes; Günter Lambrecht; Günther Schmalzing

We have previously identified the suramin analog 4,4′,4″,4″′-(carbonylbis(imino-5,1,3-benzenetriylbis(carbonylimino)))tetrakis-benzene-1,3-disulfonic acid (NF449) as a low nanomolar potency antagonist of recombinant P2X1 receptors. Here, we characterize, by two-electrode voltage-clamp electrophysiology, three isomeric suramin analogs designated para-4,4′,4″,4″″-(carbonylbis(imino-5,1,3-benzenetriylbis (carbonylimino)))tetrakis-benzenesulfonic acid (NF110), meta-(3,3′,3″,3″″-(carbonylbis(imino-5,1,3-benzenetriylbis (carbonylimino)))tetra-kis-benzenesulfonic acid (NF448), and ortho-(2,2′,2″,2″″-(carbonylbis(imino-5,1,3-benzenetriylbis (carbonylimino)))tetra-kis-benzenesulfonic acid (MK3) with respect to their potency in antagonizing rat P2X receptor-mediated inward currents in Xenopus laevis oocytes. Meta, para, and ortho refer to the position of the single sulfonic acid group relative to the amide bond linking the four symmetrically oriented benzenesulfonic acid moieties to the central, invariant suramin core. NF448, NF110, and MK3 were >200-fold less potent in blocking P2X1 receptors than NF449, from which they differ structurally only by having one instead of two sulfonic acid residues per benzene ring. Although the meta- and ortho-isomers retained P2X1 receptor selectivity, the para-isomer NF110 exhibited a significantly increased activity at P2X3 receptors (Ki ∼ 36 nM) and displayed the following unique selectivity profile among suramin derivatives: P2X2+3 = P2X3 > P2X1 > P2X2 >> P2X4 > P2X7. The usefulness of NF110 as a P2X3 receptor antagonist in native tissues could be demonstrated by showing that NF110 blocks αβ-methylene-ATP-induced currents in rat dorsal root ganglia neurons with similar potency as recombinant rat P2X3 receptors. Together, these data highlight the importance of both the number and exact location of negatively charged groups for P2X subtype potency and selectivity.


Journal of Biological Chemistry | 2006

P2X5 Subunit Assembly Requires Scaffolding by the Second Transmembrane Domain and a Conserved Aspartate

Wiebke Duckwitz; Ralf Hausmann; Armaz Aschrafi; Günther Schmalzing

Functional homomeric and heteromeric ATP-gated P2X receptor channels have been shown to display a characteristic trimeric architecture. Of the seven different isoforms (designated P2X1-P2X7), P2X5 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, Asp355, which supported homotrimerization in a side chain-specific manner. Our results indicate that transmembrane domain 2 formation contributes 2-fold to hP2X5 homotrimerization by tethering the end of the ectodomain to the membrane, thereby topologically restricting conformational mobility, and by intramembrane positioning of Asp355. While transmembrane domain 2 appears to favor assembly by enabling productive subunit interactions in the ectodomain, Asp355 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.


Journal of The American Society of Nephrology | 2010

Electrical Forces Determine Glomerular Permeability

Ralf Hausmann; Christoph Kuppe; Herbert Egger; Frank Schweda; Volker Knecht; Marlies Elger; Sylvia Menzel; Douglas Somers; Gerald S. Braun; Astrid Fuss; Sandra Uhlig; Wilhelm Kriz; George A. Tanner; Jürgen Floege; Marcus J. Moeller

There is ongoing controversy about the mechanisms that determine the characteristics of the glomerular filter. Here, we tested whether flow across the glomerular filter generates extracellular electrical potential differences, which could be an important determinant of glomerular filtration. In micropuncture experiments in Necturus maculosus, we measured a potential difference across the glomerular filtration barrier that was proportional to filtration pressure (-0.045 mV/10 cm H₂O). The filtration-dependent potential was generated without temporal delay and was negative within Bowmans space. Perfusion with the cationic polymer protamine abolished the potential difference. We propose a mathematical model that considers the relative contributions of diffusion, convection, and electrophoretic effects on the total flux of albumin across the filter. According to this model, potential differences of -0.02 to -0.05 mV can induce electrophoretic effects that significantly influence the glomerular sieving coefficient of albumin. This model of glomerular filtration has the potential to provide a mechanistic theory, based on experimental data, about the filtration characteristics of the glomerular filtration barrier. It provides a unique approach to the microanatomy of the glomerulus, renal autoregulation, and the pathogenesis of proteinuria.


Journal of Biological Chemistry | 2011

Amino Acid Residues Constituting the Agonist Binding Site of the Human P2X3 Receptor

Mandy Bodnar; Haihong Wang; Thomas Riedel; Stefan Hintze; Erzsébet Kató; Ghada Fallah; Helke Gröger-Arndt; Rashid Giniatullin; Marcus Grohmann; Ralf Hausmann; Günther Schmalzing; Peter Illes; Patrizia Rubini

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 Ca2+ 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 [Ca2+]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.


Molecular Pharmacology | 2011

Molecular Determinants of Potent P2X2 Antagonism Identified by Functional Analysis, Mutagenesis, and Homology Docking

Christian Wolf; Christiane Rosefort; Ghada Fallah; Matthias U. Kassack; Alexandra Hamacher; Mandy Bodnar; Haihong Wang; Peter Illes; Achim Kless; Gregor Bahrenberg; Günther Schmalzing; Ralf Hausmann

P2X2 receptors are members of the ATP-gated P2X family of cation channels, and they participate in neurotransmission in sympathetic ganglia and interneurons. Here, we identified 7,7′-(carbonylbis(imino-3,1-phenylenecarbonylimino-3,1-(4-methyl-phenylene)carbonylimino))bis(1-methoxy-naphthalene-3,6-disulfonic acid) tetrasodium salt (NF770) as a nanomolar-potent competitive P2X2 receptor antagonist within a series of 139 suramin derivatives. Three structural determinants contributed to the inhibition of P2X2 receptors by NF770: 1) a “large urea” structure with two symmetric phenylenecarbonylimino groups; 2) attachment of the naphthalene moiety in position 7,7′; and 3) the specific position of two sulfonic acid groups (3,3′; 6,6′) and of one methoxy group (1,1′) at the naphthalene moiety. This structure-activity relationship was interpreted using a rat P2X2 homology model based on the crystal structure of the closed zebrafish P2X4 receptor. Docking of the suramin derivatives into the modeled ATP-binding pocket provides a uniform explanation for the observed differences in inhibitory potencies. Changes in the chemical structure that increase the inhibitory potency of the suramin derivatives improved the spatial orientation within the ATP-binding pocket to allow for stronger polar interactions of functional groups with Gly72, Glu167, or Arg290. Gly72 is responsible for the orientation of the methoxy group close to Arg290 or Glu167. Combined mutational and functional analysis confirmed that residues Gly72 and Glu167 are as important for ATP binding as Arg290, the ATP-binding role of which has been shown in previous studies. The in silico prediction of Gly72 and Glu167 as ATP-binding residues strongly supports the validity of our homology docking.


Frontiers in Cellular Neuroscience | 2013

Heteromeric assembly of P2X subunits

Anika Saul; Ralf Hausmann; Achim Kless; Annette Nicke

Transcripts and/or proteins of P2X receptor (P2XR) subunits have been found in virtually all mammalian tissues. Generally more than one of the seven known P2X subunits have been identified in a given cell type. Six of the seven cloned P2X subunits can efficiently form functional homotrimeric ion channels in recombinant expression systems. This is in contrast to other ligand-gated ion channel families, such as the Cys-loop or glutamate receptors, where homomeric assemblies seem to represent the exception rather than the rule. P2XR mediated responses recorded from native tissues rarely match exactly the biophysical and pharmacological properties of heterologously expressed homomeric P2XRs. Heterotrimerization of P2X subunits is likely to account for this observed diversity. While the existence of heterotrimeric P2X2/3Rs and their role in physiological processes is well established, the composition of most other P2XR heteromers and/or the interplay between distinct trimeric receptor complexes in native tissues is not clear. After a description of P2XR assembly and the structure of the intersubunit ATP-binding site, this review summarizes the distribution of P2XR subunits in selected mammalian cell types and the biochemically and/or functionally characterized heteromeric P2XRs that have been observed upon heterologous co-expression of P2XR subunits. We further provide examples where the postulated heteromeric P2XRs have been suggested to occur in native tissues and an overview of the currently available pharmacological tools that have been used to discriminate between homo- and heteromeric P2XRs.


Molecular Pharmacology | 2013

Salt bridge switching from Arg290/Glu167 to Arg290/ATP promotes the closed-to-open transition of the P2X2 receptor

Ralf Hausmann; Janka Günther; Achim Kless; Daniel Kuhlmann; Matthias U. Kassack; Gregor Bahrenberg; Fritz Markwardt; Günther Schmalzing

P2X receptors are trimeric adenosine-5’-triphosphate (ATP)-gated cation channels involved in fast signal transduction in many cell types. In this study, we used homology modeling of the rat P2X2 receptor with the zebrafish P2X4 X-ray template to determine that the side chains of the Glu167 and Arg290 residues are in close spatial vicinity within the ATP-binding pocket when the rat P2X2 channel is closed. Through charge reversal mutation analysis and mutant cycle analysis, we obtained evidence that Glu167 and Arg290 form an electrostatic interaction. In addition, disulfide trapping indicated the close proximity of Glu167 and Arg290 when the channel is in the closed state, but not in the ATP-bound open state. Consistent with a gating-induced movement that disrupts the Glu167/Arg290 salt bridge, a comparison of the closed and open rat P2X2 receptor models revealed a significant rearrangement of the protein backbone and the side chains of the Glu167 and Arg290 residues during the closed-to-open transition. The associated release of the Glu167/Arg290 salt bridge during channel opening allows a strong ionic interaction between Arg290 and a γ-phosphate oxygen of ATP. We conclude from these results that the state-dependent salt bridge switching from Arg290/Glu167 to Arg290/ATP fulfills a dual role: to destabilize the closed state of the receptor and to promote the ionic coordination of ATP in the ATP-binding pocket.

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Bernd Nowak

RWTH Aachen University

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