Structure and function of purinergic P2X receptors

Purinergic P2X receptors are cationic channels in the cell membrane which are opened by extracellular ATP. They are widely expressed in different tissues like skeletal muscle, endothelial cells, neurons and leukocytes. ATP is released from nerve endings into the extracellular space as a neurotransmitter and from several other cells during pathophysiological conditions like hypoxia, thrombosis and inflammation. We investigate the function of P2X receptors in lymphocytes and macrophages by means of the whole-cell and single-channel voltage clamp technique and by measurement of P2X-dependent changes in the membrane potential and the intracellular Ca2+ and Na+-concentration in single cells by voltage clamp fluorimetry and in cell populations using flow cytometry (FACS). In collaboration with molecular biologists (Prof. Günther Schmalzing, RWTH Aachen) mutated P2X receptor constructs are expressed in Xenopus oocytes and mammalian cells. Structure-function relationships of the receptors and the interaction of P2X receptors with other ion channels (pannexin, anion channels,trp channels) are investigated by the measurement of whole cell and single channel P2X-dependent ionic currents and fluorescence measurements.

Supported by the DFG and the Roux programme of the Medical Faculty of the Martin Luther University.


Mechanism of sphingosine phosphate-induced ATP secretion

Macrophyges are versatile players coordinating inflammatory and regenerative processes under pathological conditions in which sphingosine-1-phosphate (S1P)-mediated migration is essential. We investigated the involved signaling cascade by means of voltage clamp, measurement of ATP secretion, and wound healing assay.

Staff

M.sc. Malte Berthold (Ph.D. student)

Amelie Spilker

Doctoral candidates:

Mira Trang
Eike Schön
Danyal Zahiri
Markus Schneider
Hannah Dentler
Ida Schiller
Thomas Kendzierski

Publications (selection)

  1. Modulation of Calcium channel current in guinea-pig single ventricular heart cells by the dihydropyridine Bay k 8644
    F. Markwardt, B. Nilius
    J. Physiol. (Lond.) 399 (1988), 559-575
    PubMed
  2. Thrombin stimulates Ca-channel currents in isolated frog ventricular cells
    F. Markwardt, R. Albitz, T. Franke, B. Nilius
    Pflügers Arch. 412 (1988), 668-670 
    PubMed
  3. Effects of thrombin on single calcium channels in frog ventricular cells
    F. Markwardt, T. Franke, R. Albitz, B. Nilius
    Pflügers Arch. 415 (1990), 547-553
    PubMed
  4. Gating of maxi K+ channels studied by Ca2+ concentration jumps in excised inside-out multi-channel patches (myocytes from guinea-pig urinary bladder)
    F. Markwardt, G. Isenberg
    J. Gen. Physiol. 99 (1992), 841-862 
    PubMed
  5. Nonselective cationic currents elicited by extracellular ATP in human B-lymphocytes
    F. Bretschneider, M. Klapperstück, M. Löhn, F. Markwardt
    Pflügers Arch. 429 (1995), 691-698 
    PubMed
  6. Purinoceptor-operated cationic channels in human B lymphocytes
    F. Markwardt, M. Löhn, T. Böhm, M. Klapperstück
    J. Physiol. Lond. 498 (1997), 143-151 
    PubMed
  7. Acetylcholine-induced K+-currents in smooth muscle cells of intact rat small arteries
    T. Weidelt, W. Boldt, F. Markwardt
    J. Physiol. Lond. 500 (1997), 617-630
    PubMed
  8. Antagonism by the suramin analogue NF279 on human P2X1 and P2X7 receptors
    M. Klapperstück, C. Büttner, P. Nickel, G. Schmalzing, G. Lambrecht, F. Markwardt
    Eur. J. Pharmacol. 387 (2000), 245-252
    PubMed
  9. Block by extracellular Mg2+ of single human purinergic P2X4 receptor channels expressed in human embryonic kidney cells
    Y.A. Negulyaev, F. Markwardt
    Neuroscience Lett. 279 (2000), 165-168
    PubMed
  10. Characteristics of P2X7 receptors from human B lymphocytes expressed in Xenopus oocytes
    M. Klapperstück, C. Büttner, T. Böhm, G. Schmalzing, F. Markwardt
    Biochim. Biophys. Acta 1467 (2000), 444-456
    PubMed
  11. Sodium block and depolarisation diminish P2Z-dependent Ca2+ entry in human B lymphocytes
    M. Löhn, M. Klapperstück, D. Riemann, F. Markwardt
    Cell Calcium 29 (2001), 395-408
    PubMed
  12. Functional evidence of distinct ATP activation sites at the human P2X7 receptor
    M. Klapperstück, C. Büttner, G. Schmalzing, F. Markwardt
    J. Physiol. Lond. 534 (2001), 25-35
    PubMed
  13. Desynchronizing effect of the endothelium on intracellular Ca2+ concentration dynamics in vascular smooth muscle cells of rat mesenteric arteries
    M. Sell, W. Boldt, F. Markwardt
    Cell Calcium 32 (2002), 105-120
    PubMed
  14. The Glu496Ala polymorphism of the human P2X7 receptor does not affect its electrophysiological phenotype
    W. Boldt, M. Klapperstück, C. Büttner, S. Sadtler, G. Schmalzing, F. Markwardt
    Am. J. Physiol. Cell Physiol. 284 (2003), C749-C756
    PubMed
  15. NF449, a novel picomolar potency antagonist at human P2X1 receptors
    M. Hülsmann, P. Nickel, M. Kassack, G. Schmalzing, G. Lambrecht, F. Markwardt
    Eur. J. Pharmacol. 470 (2003), 1-7
    PubMed
  16. Kinetics of P2X7 receptor-operated single channels currents
    T. Riedel, I. Lozinsky, G. Schmalzing, F. Markwardt
    Biophys. J. 92 (2007), 2377-2391
    PubMed
  17. Influence of extracellular monovalent cations on pore and gating properties of P2X7 receptor-operated single channels currents
    T. Riedel, G. Schmalzing, F. Markwardt
    Biophys. J. 93 (2007), 846-858
    PubMed
  18. The P2X7 carboxyl tail is a regulatory module of P2X7 receptor channel activity
    D. Becker, R. Woltersdorf, W. Boldt, S. Schmitz, U. Braam, G. Schmalzing, F. Markwardt
    J. Biol. Chem. 283 (2008), 25725-25734
    PubMed
  19. Sphingosine-1-phosphate receptors stimulate macrophage plasma membrane actin assembly via ADP release, ATP synthesis and P2X7 receptor activation
    M.P. Kuhnel, M. Reiss, P. Anand, I. Treede, D. Holzer, E. Hoffmann, M. Klapperstueck, T. Steinberg, F. Markwardt, G. Griffiths
    J. Cell Sci. (2009), 122, 505-512
    PubMed
  20. Effects of protons on macroscopic and single-channel currents mediated by the human P2X7 receptor
    B. Flittiger, M. Klapperstück, G. Schmalzing, F. Markwardt
    Biochim. Biophys. Acta Biomembranes 1798 (2010), 947-957
    PubMed
  21. Trophic activity of a naturally occurring truncated isoform of the P2X7 receptor
    E. Adinolfi, M. Cirillo, R. Woltersdorf, S. Falzoni, P. Chiozzi, P. Pellegatti, M.G. Callegari, D. Sandonà, F. Markwardt, G. Schmalzing, F. Di Virgilio
    FASEB J. 24 (2010), 3393-3404
    PubMed
  22. TMEM16A(a)/anoctamin-1 shares a homodimeric architecture with ClC chloride channels
    G. Fallah, T. Römer, U. Braam, S. Detro-Dassen, F. Markwardt, G. Schmalzing
    Mol. Cell. Proteomics 10 (2011), M110.004697
    PubMed
  23. The effect of anions on the human P2X7 receptor
    C. Kubick, G. Schmalzing, F. Markwardt
    Biochim. Biophys. Acta Biomembranes 1808 (2011), 2913-22
    PubMed
  24. Salt bridge switching from Arg290/Glu167 to Arg290/ATP promotes the closed-to-open transition of the P2X2 receptor
    R. Hausmann, J. Gunther, A. Kless, D. Kuhlmann, M.U. Kassack, G. Bahrenberg, F. Markwardt, G. Schmalzing
    Mol. Pharmacol. 83 (2013), 73-84
    PubMed
  25. Calibration procedures for the quantitative determination of membrane potential in human cells using anionic dyes
    T. Klapperstück, D. Glanz, S. Hanitsch, M. Klapperstück, F. Markwardt, J. Wohlrab
    Cytometry A 83 (2013), 612-626
    PubMed
  26. Activation of ATP secretion via volume-regulated anion channels by sphingosine-1-phosphate in RAW macrophages
    P. Burow, M. Klapperstück, F. Markwardt
    Pflügers Arch. (2015), 467, 1215-1226
    PubMed
  27. Homodimeric anoctamin-1, but not homodimeric anoctamin-6, is activated by calcium increases mediated by the P2Y1 and P2X7 receptors
    M. Stolz, M. Klapperstück, T. Kendzierski, S. Detro-Dassen, A. Panning, G. Schmalzing, F. Markwardt
    Pflügers Arch. 467 (2015), 2121-2140
    PubMed
  28. Inhibition of antigen receptor-dependent Ca2+ signals and NF-AT activation by P2X7 receptors in human B lymphocytes
    A. Pippel, B. Beßler, M. Klapperstück, F. Markwardt
    Cell Calcium  57 (2015), 275-289
    PubMed
  29. Localization of the gate and selectivity filter of the full-length P2X7 receptor
    A. Pippel, M. Stolz, R. Woltersdorf, A. Kless, G. Schmalzing, F. Markwardt
    Proc Natl Acad Sci U S A. 114 (2017), E2156-E2165
    PubMed
  30. Interaction of purinergic P2X4 and P2X7 receptor subunits
    M. Schneider, K. Prudic, A. Pippel, M. Klapperstück, U. Braam, C.E. Müller, G. Schmalzing, F. Markwardt
    Front. Pharmacol. 8 (2017), 860, doi: 10.3389/fphar.2017.00860
    PubMed
  31. The elusive P2X7 macropore
    F. Di Virgilio, G. Schmalzing, F. Markwardt
    Trends Cell Biol. 28 (2018), 392-404, 15,3
    PubMed
  32. Dissection of P2X4 and P2X7 receptor current components in BV-2 microglia
    M. Trang, G. Schmalzing, C.E. Müller, F. Markwardt
    Int.J.Mol.Sci. 21 (2020), 8489
    https://doi.org/10.3390/ijms21228489
  33. Sphingosine-1-phosphate induces migration of microglial cells via activation of volume-sensitive anion channels, ATP secretion and activation of purinergic receptors
    D. Zahiri, P. Burow, C. Großmann, C.E. Müller, M. Klapperstück, F. Markwardt
    BBA - Molecular Cell Research (2020) 1868(2):118915
    https://doi.org/10.1016/j.bbamcr.2020.118915
  34. Human P2X7 receptors - properties of single ATP-gated ion channels
    F. Markwardt
    Biochemical Pharmacology (2020) 114307
    PubMed