A15

A15 Project movie

A15

In the 1st funding period, project A15 carefully validated its P2X7-EGFP overexpressing mouse model compared it with a BAC transgenic P2X7 reporter mouse from the Gene Expression Nervous System Atlas (GENSAT) project, and analyzed P2X7 expression in intestinal cells and effects of P2X7 overexpression on enteric neurons. Using their mouse model, project A15 then focused on how P2X7 induces diverse cellular responses, such as formation of large membrane pores, changes in plasma membrane composition and morphology, ectodomain shedding, cytokine release, apoptosis, and gene transcription. Since the large intracellular domain that is not found in other P2X family members is crucial for these functions, project A15 has used two approaches to investigate its involvement: 1) in situ cross-linking and subsequent analysis of purified P2X7-EGFP complexes by mass spectrometry to identify interacting proteins and 2) structure function analysis by voltage clamp fluorometry. As a proof of concept, project A15 confirmed previously identified P2X7 interactions partners and in addition, identified a range of new potential interactors, most of them with predicted immune functions and including proteins involved in leukocyte migration. Further, project A15 created functional P2X7 constructs labelled with a fluorescent unnatural amino acid and a genetically encoded Ca2+ sensor and established a set-up to analyze P2X7 interactions and dynamics by dual wavelength voltage-clamp fluorometry (VCF)/Ca2+ imaging and Förster resonance energy transfer (FRET) measurements.

Prof. Dr. Annette Nicke

Selected publications

 

  1. Ramírez-Fernández A, Urbina-Treviño L, Conte G, Alves M, Rissiek B, Durner A, Scalbert N, Zhang J, Magnus T, Koch-Nolte F, Plesnila N, Deussing JM, Engel T, Kopp R, Nicke A (2020) Deviant reporter expression and P2X4 passenger gene overexpression in the soluble EGFP BAC transgenic P2X7 reporter mouse model. Sci Rep 10:19876
  2. Kaczmarek-Hajek K, Zhang J, Kopp R, Grosche A, Rissiek B, Saul A, Bruzzone S, Engel T, Jooss T, Krautloher A, Schuster S, Magnus T, Stadelmann C, Sirko S, Koch-Nolte F, Eulenburg V, Nicke A (2018) Re-evaluation of neuronal P2X7 expression using novel mouse models and a P2X7-specific nanobody. Elife 7:e36217
  3. Lörinczi É, Bhargava Y, Marino SF, Taly A, Kaczmarek-Hájek K, Barrantes-Freer A, Dutertre S, Grutter T, Rettinger J, Nicke A (2012) Involvement of the cysteine-rich “head” domain in activation and desensitization of the P2X1 receptor. Proc Natl Acad Sci USA 109:11396-11401.
  4. Beissner M*, Dutertre S*, Schemm R*, Danker T, Sporning A, Grubmüller H, Nicke A(2012) Efficient binding of 4/7 α-conotoxins to nicotinic α4β2 receptors is prevented by R185 and P195 in the α4 subunit Mol Pharmacol 82, 711-718
  5. Nicke A, Kuan YH, Masin M, Rettinger J, Marquez-Klaka B. Bender O, Gorecki D, Murrell-Lagnado R, Soto F (2009) A functional P2X7 splice variant with an alternative transmembrane domain 1 escapes gene inactivation in P2X7 KO mice. J Biol Chem 284:25813-25822.
  6. Marquez-Klaka, B, Rettinger J, Bhargava Y, Eisele, T, Nicke A (2007) Identification of an inter-subunit cross-link between substituted cysteine residues located in the putative ATP binding site of the P2X1 receptor. J Neurosci 27:1456-1466.
  7. Grudzinska J, Schemm R, Haeger S, Nicke A, Schmalzing G, Betz H, Laube B (2005) The β subunit determines the ligand binding properties of synaptic glycine Receptors. Neuron 45:727-739.
  8. Nicke A, Loughnan M, Millard E, Alewood PF, Adams DJ, Daly N, Craik DJ, Lewis RJ (2003) Isolation, structure, and activity of GID, a novel α4/7-conotoxin with an additional N-terminal sequence. J Biol Chem 278:3137-3144.
  9. Horiuchi M*, Nicke A*, Gomeza J, Ashrafi A, Schmalzing G, Betz H. Surface-localized glycine transporters 1 and 2 function as monomeric proteins in Xenopus oocytes. Proc Natl Acad Sci USA  98, 1448-1453.
  10. Nicke A, Bäumert HG, Rettinger J, Eichele A, Lambrecht G, Mutschler E, Schmalzing G (1998) P2X1 and P2X3 receptors form stable trimers: a novel structural motif of ligand-gated ion channels EMBO J 17, 3016-3028.

OUR TEAM

Prof. Dr. Annette Nicke

LMU Munich
Walther Straub Institute of Pharmacology and Toxicology
 

Dr. Béla Zimmer

LMU Munich
Walther Straub Institute of Pharmacology and Toxicology

M.Sc. Lukas Sassenbach

LMU Munich
Walther Straub Institute of Pharmacology and Toxicology

Jonas Seidler

LMU Munich
Walther Straub Institute of Pharmacology and Toxicology

Stefan Fuchs

LMU Munich
Walther Straub Institute of Pharmacology and Toxicology

Alumni

Dr. Robin Kopp

LMU Munich
Walther Straub Institute of Pharmacology and Toxicology

Dr. Anna Durner

LMU Munich
Walther Straub Institute of Pharmacology and Toxicology

M.Sc. Isabel Müller

LMU Munich
Walther Straub Institute of Pharmacology and Toxicology

Contact

University Medical Center Hamburg-Eppendorf 
Department of Biochemistry and Molecular Cell Biology 
Martinistrasse 52
20246 Hamburg

Scientific Coordinator

Dr. Björn-Philipp Diercks
Fon: +49  (0) 40 7410 54338
E-Mail: b.diercks©uke.de

Administration

Laura Mitsching
Fon: +49  (0) 40 7410 50301
E-Mail: l.mitsching©uke.de