City University of New York (CUNY) CUNY Academic Works Publications and Research CUNY Graduate Center 2009 Manipulating neuronal circuits with endogenous and recombinant cell-surface tethered modulators Mande Holford CUNY Graduate Center Sebastian Auer Max-Delbrück Center for Molecular Medicine Martin Laqua Max-Delbrück Center for Molecular Medicine Ines Ibanez-Tallon Max-Delbrück Center for Molecular Medicine How does access to this work benefit ou?y Let us know! More information about this work at: https://academicworks.cuny.edu/gc_pubs/574 Discover additional works at: https://academicworks.cuny.edu This work is made publicly available by the City University of New York (CUNY). Contact: [email protected] REVIEW ARTICLE published: 30 October 2009 MOLECULAR NEUROSCIENCE doi: 10.3389/neuro.02.021.2009 Manipulating neuronal circuits with endogenous and recombinant cell-surface tethered modulators Mandë Holford1, Sebastian Auer 2, Martin Laqua 2 and Ines Ibañez-Tallon 2* 1 York College and The Graduate Center, The American Museum of Natural History, The City University of New York, New York, NY, USA 2 Max-Delbrück Center for Molecular Medicine, Berlin, Germany Edited by: Neuronal circuits depend on the precise regulation of cell-surface receptors and ion channels. An William Wisden, Imperial College, UK ongoing challenge in neuroscience research is deciphering the functional contribution of specifi c Reviewed by: receptors and ion channels using engineered modulators. A novel strategy, termed “tethered Peer Wulff, University of Aberdeen, UK Sheriar Hormuzdi, University of toxins”, was recently developed to characterize neuronal circuits using the evolutionary derived Dundee, UK selectivity of venom peptide toxins and endogenous peptide ligands, such as lynx1 prototoxins. William Wisden, Imperial College, UK Herein, the discovery and engineering of cell-surface tethered peptides is reviewed, with *Correspondence: particular attention given to their cell-autonomy, modular composition, and genetic targeting in Ines Ibañez-Tallon, Molecular different model organisms. The relative ease with which tethered peptides can be engineered, Neurobiology Group, Max-Delbrück Center for Molecular Medicine, coupled with the increasing number of neuroactive venom toxins and ligand peptides being Helmoltz Gemeinschaft, Robert-Rössle discovered, imply a multitude of potentially innovative applications for manipulating neuronal Str. 10, Berlin-Buch, Berlin 13125, circuits and tissue-specifi c cell networks, including treatment of disorders caused by malfunction Germany. of receptors and ion channels. e-mail: [email protected] Keywords: tethered-toxins, cell-surface modulators, lynx1, receptors, ion channels INTRODUCTION of the ligand or blocker peptide, and permits the engineering of Understanding complex processes such as neuronal activity or a large variety of t-peptides (e.g., including use of fl uorescent cell signaling malfunctions that result in human disorders or markers, viral vectors and point mutation variants). diseases relies on extensive knowledge about the function, struc- This focused review describes the identifi cation of lynx-1 ture and precision of ion channels, receptors and modulators. and related endogenous cell surface modulators, the develop- As a result of this, ion channels are at present the third biggest ment of the t-peptide technology, and the application of the target class in drug discovery; yet still remain underexploited t- peptide strategy to basic research, cell-based therapies, and as drug targets. Recent reviews describe the increasing interest drug discovery. in peptide venom toxins for the development of drug therapies directed towards ion channels and receptors (Blumenthal and ENDOGENOUS CELL-SURFACE MODULATORS OF Seibert, 2003; Phui Yee et al., 2004; Lynch et al., 2006; Han et al., LIGAND-GATED ION CHANNELS: THE LY6 SUPERFAMILY 2008; Twede et al., 2009). Specifi c areas in which peptide toxins Cell-surface receptors and ion channels are modulated by a rich have demonstrated their potential include Alzheimer’s disease variety of peptide neurotransmitters, hormones and ligands, but (candoxin) (Nirthanan et al., 2002), chronic pain (MVIIA) there are few examples of membrane-anchored modulators in (Miljanich, 2004) and myasthenic autoimmune response (α- nature. The Ly6 superfamily which includes lynx1-and slurp- Bgtx) (Drachman, 1981; Mebs, 1989). For instance, snake neu- 1 cholinergic modulators, elapid snake venom toxins, and Ly6 rotoxins bind to nicotinic acetylcholine receptors (nAChRs) with molecules of the immune system, constitutes a unique class of affi nities within the pico and nanomolar range (Chiappinelli, short proteins that are either tethered to the cell surface via a 1991), which indicates that these would be among the best probes glycosylphosphatidylinositol (GPI) anchor, like lynx1, or secreted for investigating potential therapeutics that affect nAChR activ- as venom toxins (Figure 1). Members of this superfamily share ity. The unique homologies of endogenous lynx1 prototoxins the characteristic 8–10 cysteine motif that determines their com- with venom toxins provided a biological scaffold for developing pact three-fi nger structure. Examples of the GPI-anchor sub- recombinant molecules to selectively modulate ion channels and group include molecules of the immune system such as CD59 receptors. Thus, based on the characteristics and mode of action (Davies et al., 1989), ly6A-E (Rock et al., 1989), ly6G (Mallya of lynx1 cell-surface modulators, new classes of “tethered toxins” et al., 2006), and ly6K (de Nooij-van Dalen et al., 2003), the neu- and “tethered ligands” were created as probes to characterize ion ronal proteins lypd6 (Darvas et al., 2009), ly6H (Dessaud et al., channels and receptors (Ibañez-Tallon et al., 2004; Fortin et al., 2006) and the urokinase plasminogen activator receptor (uPAR) 2009; Auer et al., 2009; Stürzebecher et al., 2009). Tethering pep- (Blasi and Carmeliet, 2002). Members lacking the GPI-anchor tide toxins or ligands close to their point of activity in the cell comprise the cholinergic modulators SLURP-1 (Adermann plasma membrane provides a new approach to the study of cell et al., 1999; Chimienti et al., 2003) and SLURP-2 (Tsuji et al., networks and neuronal circuits, as it allows selective targeting 2003), cobra toxins, and other three fi nger venom toxins (Tsetlin, of specifi c cell populations, enhances the working concentration 1999) (Figure 1). Frontiers in Molecular Neuroscience www.frontiersin.org October 2009 | Volume 2 | Article 21 | 1 Holford et al. Neuronal regulation using tethered peptides FIGURE 1 | Schematic representation of Ly-6/uPAR channel modifi ers and homology of lynx1 with αBgtx gave rise to the tethered-peptide strategy of engineered tethered toxins (t-toxins). (A) Examples of the Ly-6/uPAR using the biological scaffold of lynx1 (secretory signal and GPI signal) to superfamily include soluble Slurp-1, snake α-bungarotoxin (αBgtx) and the GPI- generate recombinant membrane-bound toxins and peptide ligands such as the anchored cell-membrane bound lynx1. The schematic below the drawing of the illustrated t-αBgtx. The schematic below the drawing of the channel indicates channel indicates the coding sequences associated with lynx1, namely an N- the coding regions that were conserved from lynx1 (shown in A), and those that terminal secretory signal region, followed by the amino acid residues that were altered to accommodate the αBgtx (the toxins sequence, fl ag tag, and correspond to the lynx1 peptide, and a C-terminal GPI anchor. (B) The structural linker regions). ALLOSTERIC MODULATION OF NACHRS BY Ly6 SUPERFAMILY Ly6 SPECIES DIVERSITY MOLECULES Lynx1-like molecules are well conserved across species, both in Lynx1 was the fi rst identifi ed member of the Ly6 superfamily structure and function, suggesting the importance of cell-sur- capable of cell-surface modulatory action on a neurotransmitter face modulators of nicotinic receptors in nature. Examples of receptor (Miwa et al., 1999). Lynx1 assembles and colocalizes with Ly6 superfamily species diversity include molecules found in C. nAChR in the brain (Ibañez-Tallon et al., 2002) and in the lung elegans (Odr-2; Chou et al., 2001), fi refl ies (Pr-lynx1; Choo et al., (Sekhon et al., 2005). nAChRs stably associated with lynx1 are less 2008), Drosophila (Hijazi et al., 2009) and chicken (recently iden- sensitive to their ligand agonists acetylcholine and nicotine, display tifi ed prostate stem cell antigen PSCA; Hruska et al., 2009). Pr- more rapid desensitization, and show a shift in the distribution of lynx1 and PSCA are of particular importance as Pr-lynx1 is the channel openings toward a faster inactivating species with more fi rst modulator of nAChRs in an insect species (Choo et al., 2008), uniform, larger amplitude currents (Ibañez-Tallon et al., 2002). and PSCA appears to prevent programmed cell death of neurons by These fi ndings, along with studies showing enhanced nicotine- antagonizing nAChRs (Hruska et al., 2009). The lynx1-like family mediated calcium infl ux and synaptic effi cacy in lynx1 null mutant of allosteric modulators of nAChRs constitutes a unique example mice, are strong indicators that lynx1 is an allosteric modulator of of cell-surface channel modifi ers that have evolved for fi ne-tuning nAChR function in vivo (Miwa et al., 2006). Other lynx-like mol- of neurotransmitter receptor function in vivo. ecules recently identifi ed have similar properties. Lynx2 and ly6H, which are