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1.
Proc Natl Acad Sci U S A ; 111(17): 6449-54, 2014 Apr 29.
Article in English | MEDLINE | ID: mdl-24733934

ABSTRACT

Previously we defined neuronal subclasses within the mouse peripheral nervous system using an experimental strategy called "constellation pharmacology." Here we demonstrate the broad applicability of constellation pharmacology by extending it to the CNS and specifically to the ventral respiratory column (VRC) of mouse brainstem, a region containing the neuronal network controlling respiratory rhythm. Analysis of dissociated cells from this locus revealed three major cell classes, each encompassing multiple subclasses. We broadly analyzed the combinations (constellations) of receptors and ion channels expressed within VRC cell classes and subclasses. These were strikingly different from the constellations of receptors and ion channels found in subclasses of peripheral neurons from mouse dorsal root ganglia. Within the VRC cell population, a subset of dissociated neurons responded to substance P, putatively corresponding to inspiratory pre-Bötzinger complex (preBötC) neurons. Using constellation pharmacology, we found that these substance P-responsive neurons also responded to histamine, and about half responded to bradykinin. Electrophysiological studies conducted in brainstem slices confirmed that preBötC neurons responsive to substance P exhibited similar responsiveness to bradykinin and histamine. The results demonstrate the predictive utility of constellation pharmacology for defining modulatory inputs into specific neuronal subclasses within central neuronal networks.


Subject(s)
Central Nervous System/cytology , Neurons/physiology , Animals , Bradykinin/pharmacology , Brain Stem/cytology , Brain Stem/drug effects , Brain Stem/physiology , Calcium/metabolism , Cells, Cultured , Cluster Analysis , Female , Ganglia, Spinal/cytology , Ganglia, Spinal/drug effects , Ganglia, Spinal/physiology , Histamine/pharmacology , Imaging, Three-Dimensional , Male , Mice , Mice, Inbred C57BL , Nerve Net/cytology , Nerve Net/drug effects , Nerve Net/physiology , Neurons/drug effects , Receptors, Cholinergic/metabolism , Receptors, Glutamate/metabolism , Respiratory Center/cytology , Substance P/pharmacology
2.
Toxicon ; 81: 67-79, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24508768

ABSTRACT

In order to decode the roles that N-methyl-D-aspartate (NMDA) receptors play in excitatory neurotransmission, synaptic plasticity, and neuropathologies, there is need for ligands that differ in their subtype selectivity. The conantokin family of Conus peptides is the only group of peptidic natural products known to target NMDA receptors. Using a search that was guided by phylogeny, we identified new conantokins from the marine snail Conus bocki that complement the current repertoire of NMDA receptor pharmacology. Channel currents measured in Xenopus oocytes demonstrate conantokins conBk-A, conBk-B, and conBk-C have highest potencies for NR2D containing receptors, in contrast to previously characterized conantokins that preferentially block NR2B containing NMDA receptors. Conantokins are rich in γ-carboxyglutamate, typically 17-34 residues, and adopt helical structure in a calcium-dependent manner. As judged by CD spectroscopy, conBk-C adopts significant helical structure in a calcium ion-dependent manner, while calcium, on its own, appears insufficient to stabilize helical conformations of conBk-A or conBk-B. Molecular dynamics simulations help explain the differences in calcium-stabilized structures. Two-dimensional NMR spectroscopy shows that the 9-residue conBk-B is relatively unstructured but forms a helix in the presence of TFE and calcium ions that is similar to other conantokin structures. These newly discovered conantokins hold promise that further exploration of small peptidic antagonists will lead to a set of pharmacological tools that can be used to characterize the role of NMDA receptors in nervous system function and disease.


Subject(s)
Conotoxins/chemistry , Phylogeny , Receptors, N-Methyl-D-Aspartate/chemistry , Amino Acid Sequence , Animals , Base Sequence , Circular Dichroism , Conus Snail/chemistry , Ligands , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Sequence Data , Mollusk Venoms/chemistry , Patch-Clamp Techniques , Rats , Receptors, N-Methyl-D-Aspartate/antagonists & inhibitors , Synaptic Transmission/drug effects , Xenopus
3.
J Pept Sci ; 16(8): 375-82, 2010 Aug.
Article in English | MEDLINE | ID: mdl-20572027

ABSTRACT

A multidisciplinary strategy for discovery of new Conus venom peptides combines molecular genetics and phylogenetics with peptide chemistry and neuropharmacology. Here we describe application of this approach to the conantokin family of conopeptides targeting NMDA receptors. A new conantokin from Conus rolani, ConRl-A, was identified using molecular phylogeny and subsequently synthesized and functionally characterized. ConRl-A is a 24-residue peptide containing three gamma-carboxyglutamic acid residues with a number of unique sequence features compared to conantokins previously characterized. The HPLC elution of ConRl-A suggested that this peptide exists as two distinct, slowly exchanging conformers. ConRl-A is predominantly helical (estimated helicity of 50%), both in the presence and absence of Ca(++). The order of potency for blocking the four NMDA receptor subtypes by ConRl-A was NR2B > NR2D > NR2A > NR2C. This peptide has a greater discrimination between NR2B and NR2C than any other ligand reported so far. In summary, ConRl-A is a new member of the conantokin family that expands our understanding of structure/function of this group of peptidic ligands targeted to NMDA receptors. Thus, incorporating phylogeny in the discovery of novel ligands for the given family of ion channels or receptors is an efficient means of exploring the megadiverse group of peptides from the genus Conus.


Subject(s)
Conotoxins/metabolism , Conus Snail/metabolism , Peptides/metabolism , Phylogeny , Animals , Circular Dichroism , Conotoxins/genetics , Conus Snail/classification , DNA, Complementary , Electrophysiology , Mollusk Venoms/metabolism , Oocytes , Peptides/genetics , Receptors, N-Methyl-D-Aspartate/antagonists & inhibitors , Xenopus
4.
Curr Opin Drug Discov Devel ; 12(2): 231-9, 2009 Mar.
Article in English | MEDLINE | ID: mdl-19333868

ABSTRACT

The peptides in the venoms of predatory marine snails belonging to the genus Conus ('cone snails') have well-established therapeutic applications for the treatment of pain and epilepsy. This review discusses the neuroprotective and cardioprotective potential of four families of Conus peptides (conopeptides), including omega-conotoxins that target voltage-gated Ca2+ channels, conantokins that target NMDA receptors, mu-conotoxins that target voltage-gated Na+ channels, and kappa- and kappaM-conotoxins that target K+ channels. The diversity of Conus peptides that have already been shown to exhibit neuroprotective/cardioprotective activity suggests that marine snail venoms are a potentially rich source of drug leads with diverse mechanisms.


Subject(s)
Cardiovascular Agents/pharmacology , Conotoxins/pharmacology , Drug Discovery , Neuroprotective Agents/pharmacology , Animals , Calcium Channel Blockers/pharmacology , Cardiovascular Agents/chemistry , Cardiovascular Agents/isolation & purification , Conotoxins/chemistry , Conotoxins/isolation & purification , Excitatory Amino Acid Antagonists/pharmacology , Humans , Neuroprotective Agents/chemistry , Neuroprotective Agents/isolation & purification , Potassium Channel Blockers/pharmacology , Receptors, N-Methyl-D-Aspartate/antagonists & inhibitors , Sodium Channel Blockers/pharmacology , Structure-Activity Relationship , omega-Conotoxins/pharmacology
5.
Biochemistry ; 48(19): 4063-73, 2009 May 19.
Article in English | MEDLINE | ID: mdl-19309162

ABSTRACT

Conantokins are venom peptides from marine cone snails that are NMDA receptor antagonists. Here, we report the characterization of a 24 AA conantokin from Conus brettinghami Coomans , H. E. , Moolenbeek , R. G. and Wils , E. ( 1982 ) Basteria 46 ( 1/4 ), 3 - 67 , conantokin-Br (con-Br), the first conantokin that does not have the conserved glutamate residue at position 2. Molecular modeling studies suggest that con-Br has a helical structure between residues 2-13. In contrast to other characterized conantokins, con-Br has a high potency for NMDA receptors with NR2D subunits. To identify determinants for NR2D potency, we synthesized chimeras of con-Br and conantokin-R (con-R); the latter has a approximately 30-fold lower potency for the NR2D subtype. The characterization of two reciprocal chimeras (con-Br/R and con-R/Br), comprising the first 9-10 N-terminal AAs of each conantokin followed by the corresponding C-terminal AAs of the other conantokin demonstrates that determinants for NR2D selectivity are at the N-terminal region. Additional analogues comprising 1-3 amino acid substitutions from each peptide into the homologous region of the other led to the identification of a key determinant; a Tyr residue in position 5 increases potency for NR2D, while Val at this locus causes a decrease. The systematic definition of key determinants in the conantokin peptides for NMDA receptor subtype selectivity is an essential component in the development of conantokin peptides that are highly selective for each specific NMDA receptor subtype.


Subject(s)
Conotoxins/chemistry , Conus Snail/chemistry , Peptides/chemistry , Receptors, N-Methyl-D-Aspartate/chemistry , Amino Acid Sequence , Amino Acid Substitution , Animals , Computer Simulation , Conotoxins/metabolism , Conotoxins/pharmacology , Electrophysiology , Female , Inhibitory Concentration 50 , Models, Molecular , Molecular Sequence Data , Oocytes/metabolism , Oxidation-Reduction , Patch-Clamp Techniques , Peptides/metabolism , Peptides/pharmacology , Perfusion , Protein Folding , Protein Structure, Secondary , Protein Subunits/metabolism , Protein Subunits/pharmacology , Receptors, N-Methyl-D-Aspartate/antagonists & inhibitors , Receptors, N-Methyl-D-Aspartate/metabolism , Sequence Homology, Amino Acid , Structure-Activity Relationship , Tyrosine/metabolism , Xenopus
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