Nicke Lab

Head of Lab

Prof. Dr. phil. nat. Annette Nicke

Research

• Structure and molecular function of P2X receptors and other ion channels
• Physiological and pathophysiological functions of P2X7 receptors
• P2X7 receptor signaling
• Structure-activity relations of peptide toxins

Adenosine 5’-triphosphate (ATP) is an essential molecule for all life forms. It is not only a principal energy source and component of nucleic acids inside the cell, but also plays a crucial role as an extracellular signaling molecule that mediates fast and slow communication between cells. Metabotropic P2Y or ionotropic P2X receptors (Illes et al. 2021) for ATP and its metabolites have been identified in all mammalian tissues. The purinergic P2X7 receptor subtype is involved in pro-inflammatory processes and represents a promising drug target (Kaczmarek-Hájek et al. 2012). Its activation induces diverse cellular responses, such as large membrane pore formation, changes in the plasma membrane composition and morphology, ectodomain shedding, cytokine release, apoptosis, and induction of gene transcription. The underlying signaling pathways are poorly understood but are associated with a large intracellular domain that is not found in other P2X family members (Kopp, Durner et al. 2019) and has no homology to known proteins. A recent breakthrough was the determination of the cryo-EM structure of the full-length rat P2X7 receptor which revealed an unexpected novel GTP/GDP binding motif and a dinuclear Zn2+ binding site in the intracellular domain.

Current projects

1) P2X receptors (P2XRs) are ATP-activated, Ca2+-permeable cation channels. We previously determined their trimeric structure (Nicke et al. 1998) and localized the ATP binding site at the interface of neighboring P2X subunits (Marquez-Klaka et al. 2007). Recent work focuses on the molecular functions of the P2X7 receptor and its enigmatic C-terminal tail. To investigate molecular movements that are associated with channel activation, we use voltage clamp fluorometry. We recently optimized the introduction of an fluorescent unnatural amino acid (UAA) into proteins expressed in Xenopus laevis and established a set-up to analyse molecular interactions and dynamics by dual wavelength voltage-clamp fluorometry (VCF)/Ca2+ imaging (Durner et al. 2013; Lörinczi et al. 2012)

2) To better understand the physiological and pathophysiological functions of the P2X7 receptor, we generated and validated a bacterial artificial chromosome (BAC) transgenic reporter mouse model in which and fluorescently labelled P2X7 receptor (P2X7-EGFP) is moderately overexpressed under the control of the endogenous P2X7 promoter. This mouse model allows visualization and determination of the cell type-specific and subcellular P2X7 localization (Kaczmarek-Hájek, Zhang, Kopp et al. 2018; Ramírez-Fernández et al. 2019; Jooss, Zhang et al. 2023). In addition, we investigate consequences of altered receptor function by comparing these P2X7 overexpressing mice with P2X7 ko mouse models.

3) To investigate P2X7 signaling we performed in situ cross-linking and subsequent analysis of purified P2X7-EGFP complexes by mass spectrometry. As a proof of concept, we confirmed some previously found P2X7 interactions partners and identified a range of new potential interactors, most of them with predicted immune functions. These are currently further validated.

4) In several further projects, we identify novel α-conotoxins, small peptides from the venom of marine snails (Dutertre, Nicke and Tsetlin 2017) and study their subtype selectivity and structure-function relationships. This work aims to develop optimized selective peptide ligands as pharmacological tools for the subtype differentiation of nAChRs and to better understand receptor ligand interactions. So far, we isolated various novel peptides and determined relations between their sequence and post-translational modifications, and their potency and selectivity (e.g. Nicke et al. J. Biol. Chem. 2003, Loughnan et al 2006). By rational mutagenesis based on computational docking studies, we could identify amino acid residues that contribute to the specific interactions with α-conotoxins (Dutertre et al. 2008; Beissner et al. 2012; Leffler et al. 2017, Haufe et al 2023).

Methods

• Protein biochemistry (e.g. cross-linking mass spectrometry, BN-PAGE analysis )
• Molecular biology (mutagenesis and introduction of unnatural amino acids)
• Functional analysis of ion channels (uptake measurements, 2-Electrode voltage-clamp, patch-clamp, voltage clamp fluorometry)
• Generation and characterization of transgenic mice (immunofluorescence staining, confocal microscopy)