Tethering Naturally Occurring Peptide Toxins for Cell-Autonomous Modulation of Ion Channels and Receptors In Vivo

The physiologies of cells depend on electrochemical signals carried by ion channels and receptors. Venomous animals produce an enormous variety of peptide toxins with high affinity for specific ion channels and receptors. The mammalian prototoxin lynx1 shares with alpha-bungarotoxin the ability to bind and modulate nicotinic receptors (nAChRs); however, lynx1 is tethered to the membrane via a GPI anchor. We show here that several classes of neurotoxins, including bungarotoxins and cobratoxins, retain their selective antagonistic properties when tethered to the membrane. Targeted elimination of nAChR function in zebrafish can be achieved with tethered alpha-bungarotoxin, silencing synaptic transmission without perturbing synapse formation. These studies harness the pharmacological properties of peptide toxins for use in genetic experiments. When combined with specific methods of cell and temporal expression, the extension of this approach to hundreds of naturally occurring peptide toxins opens a new landscape for cell-autonomous regulation of cellular physiology in vivo.

[1]  M. Wilson,et al.  NMDA receptors, place cells and hippocampal spatial memory , 2004, Nature Reviews Neuroscience.

[2]  A. Sali,et al.  lynx1, an Endogenous Toxin-like Modulator of Nicotinic Acetylcholine Receptors in the Mammalian CNS , 1999, Neuron.

[3]  B. Olivera,et al.  Conus venoms: a rich source of novel ion channel-targeted peptides. , 2004, Physiological reviews.

[4]  H. Peng,et al.  Acetylcholinesterase Clustering at the Neuromuscular Junction Involves Perlecan and Dystroglycan , 1999, The Journal of cell biology.

[5]  Joseph E LeDoux,et al.  NMDA Receptors and L-Type Voltage-Gated Calcium Channels Contribute to Long-Term Potentiation and Different Components of Fear Memory Formation in the Lateral Amygdala , 2002, The Journal of Neuroscience.

[6]  R. Mains,et al.  Inducible Genetic Suppression of Neuronal Excitability , 1999, The Journal of Neuroscience.

[7]  S. Higashijima,et al.  Paralytic Zebrafish Lacking Acetylcholine Receptors Fail to Localize Rapsyn Clusters to the Synapse , 2001, The Journal of Neuroscience.

[8]  D. Lipscombe,et al.  Identification of Functionally Distinct Isoforms of the N-Type Ca2+ Channel in Rat Sympathetic Ganglia and Brain , 1997, Neuron.

[9]  W. Catterall.  Molecular properties of brain sodium channels: an important target for anticonvulsant drugs. , 1999, Advances in neurology.

[10]  D. Yoshikami,et al.  Single-Residue Alteration in α-Conotoxin PnIA Switches Its nAChR Subtype Selectivity† , 1999 .

[11]  J. McIntosh,et al.  Conus peptides targeted to specific nicotinic acetylcholine receptor subtypes. , 1999, Annual review of biochemistry.

[12]  Joseph R. Manns,et al.  Recognition Memory and the Human Hippocampus , 2003, Neuron.

[13]  E. Bennett,et al.  Radiolabeled alpha-bungarotoxin derivatives: kinetic interaction with nicotinic acetylcholine receptors. , 1981, Biochemistry.

[14]  D. Yoshikami,et al.  A New -Conotoxin Which Targets 32 Nicotinic Acetylcholine Receptors (*) , 1996, The Journal of Biological Chemistry.

[15]  G. Bi,et al.  Cell membrane resealing by a vesicular mechanism similar to neurotransmitter release. , 1994, Science.

[16]  B. Rudy,et al.  Evidence for the presence of a novel Kv4‐mediated A‐type K+ channel‐modifying factor , 2001, The Journal of physiology.

[17]  M. Adams,et al.  Neurotoxins: overview of an emerging research technology , 1994, Trends in Neurosciences.

[18]  M. Stocker,et al.  MicroO-conotoxin MrVIA inhibits mammalian sodium channels, but not through site I. , 1996, Journal of neurophysiology.

[19]  F. Hucho,et al.  Snake and snail toxins acting on nicotinic acetylcholine receptors: fundamental aspects and medical applications , 2004, FEBS letters.

[20]  B. Rudy,et al.  Molecular Diversity of K+ Channels , 1999, Annals of the New York Academy of Sciences.

[21]  N. Heintz,et al.  Novel Modulation of Neuronal Nicotinic Acetylcholine Receptors by Association with the Endogenous Prototoxin lynx1 , 2002, Neuron.

[22]  D. Kohda,et al.  Structure-activity relationships of mu-conotoxin GIIIA: structure determination of active and inactive sodium channel blocker peptides by NMR and simulated annealing calculations. , 1994, Biochemistry.

[23]  M. Adams,et al.  CALCIUM CHANNEL DIVERSITY AND NEUROTRANSMITTER RELEASE : THE OMEGA -CONOTOXINS AND OMEGA -AGATOXINS , 1994 .

[24]  Hermann Bujard,et al.  Tetracycline-regulated gene expression in the brain , 2000, Current Opinion in Neurobiology.

[25]  Michael N Nitabach,et al.  Electrical Silencing of Drosophila Pacemaker Neurons Stops the Free-Running Circadian Clock , 2002, Cell.

[26]  M. Stryker,et al.  Ocular dominance in layer IV of the cat's visual cortex and the effects of monocular deprivation. , 1978, The Journal of physiology.

[27]  R. Loring,et al.  Amino acid sequence of toxin F, a snake venom toxin that blocks neuronal nicotinic receptors , 1986, Brain Research.

[28]  R. Tsien.  Calcium channels in excitable cell membranes. , 1983, Annual review of physiology.

[29]  J. Changeux,et al.  An H‐bond between two residues from different loops of the acetylcholine binding site contributes to the activation mechanism of nicotinic receptors , 2003, The EMBO journal.

[30]  D. Yoshikami,et al.  A new alpha-conotoxin which targets alpha3beta2 nicotinic acetylcholine receptors. , 1996, The Journal of biological chemistry.

[31]  P. Drapeau,et al.  Synchronization of an Embryonic Network of Identified Spinal Interneurons Solely by Electrical Coupling , 2001, Neuron.

[32]  G. Davis,et al.  Homeostatic Control of Presynaptic Release Is Triggered by Postsynaptic Membrane Depolarization , 2001, Neuron.

[33]  H. Lester,et al.  Genetic Inactivation of an Inwardly Rectifying Potassium Channel (Kir4.1 Subunit) in Mice: Phenotypic Impact in Retina , 2000, The Journal of Neuroscience.

[34]  D Bertrand,et al.  Only Snake Curaremimetic Toxins with a Fifth Disulfide Bond Have High Affinity for the Neuronal α7 Nicotinic Receptor* , 1997, The Journal of Biological Chemistry.

[35]  Targeting multi-cellular organisms. , 2003, Current opinion in genetics & development.

[36]  G. A. Grant,et al.  Differential roles for disulfide bonds in the structural integrity and biological activity of kappa-Bungarotoxin, a neuronal nicotinic acetylcholine receptor antagonist. , 1998, Biochemistry.

[37]  M. Stryker,et al.  Prenatal tetrodotoxin infusion blocks segregation of retinogeniculate afferents. , 1988, Science.

[38]  S. Higashijima,et al.  The Zebrafish Motility Mutant twitch once Reveals New Roles for Rapsyn in Synaptic Function , 2002, The Journal of Neuroscience.

[39]  J. Changeux,et al.  Knockout and knockin mice to investigate the role of nicotinic receptors in the central nervous system. , 2002, Progress in brain research.

[40]  C. Van Renterghem,et al.  Binding of six chimeric analogs of omega-conotoxin MVIIA and MVIIC to N- and P/Q-type calcium channels. , 2000, Biochemical and biophysical research communications.

[41]  Shiaoching Gong,et al.  A gene expression atlas of the central nervous system based on bacterial artificial chromosomes , 2003, Nature.

[42]  H. Lester,et al.  ROMK1 (Kir1.1) causes apoptosis and chronic silencing of hippocampal neurons. , 2000, Journal of neurophysiology.