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Voltage-gated potassium channels (VGKCs) comprise the largest and most complex families of ion channels. Approximately 70 genes encode VGKC alpha subunits, which assemble into functional tetrameric channel complexes. These subunits can also combine to form heteromeric channels, significantly expanding the potential diversity of VGKCs. The functional expression and physiological role of heteromeric K-channels have remained largely unexplored due to the lack of tools to probe their functions. Conotoxins, from predatory cone snails, have high affinity and specificity for heteromeric combinations of K-channels and show great promise for defining their physiological roles. In this work, using conotoxin KM-RIIIJ as a pharmacological probe, we explore the expression and physiological functions of heteromeric K v 1.2 channels using constellation pharmacology platform. We report that heteromers of K v 1.2/1.1 are highly expressed in proprioceptive neurons found in the dorsal root ganglion (DRG). Inhibition of K v 1.2/1.1 heteromers leads to an influx of calcium ions, suggesting that these channels regulate neuronal excitability. We also present evidence that K v 1.2/1.1 heteromers counteract persistent sodium currents, and that inhibiting these channels leads to tonic firing of action potentials. Additionally, KM-RIIIJ impaired proprioception in mice, uncovering a previously unrecognized physiological function of heteromeric K v 1.2/1.1 channels in proprioceptive sensory neurons of the DRG., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Marine tunicates produce defensive amino-acid-derived metabolites, including 2-(3,5-diiodo-4-methoxyphenyl)ethan-1-amine (DIMTA), but their mechanisms of action are rarely known. Using an assay-guided approach, we found that out of the many different sensory cells in the mouse dorsal root ganglion (DRG), DIMTA selectively affected low-threshold cold thermosensors. Whole-cell electrophysiology experiments using DRG cells, channels expressed in Xenopus oocytes, and human cell lines revealed that DIMTA blocks several potassium channels, reducing the magnitude of the afterhyperpolarization and increasing the baseline intracellular calcium concentration [Ca 2+ ] i of low-threshold cold thermosensors. When injected into mice, DIMTA increased the threshold of cold sensation by >3 °C. DIMTA may thus serve as a lead in the further design of compounds that inhibit problems in the cold-sensory system, such as cold allodynia and other neuropathic pain conditions.

Current drug discovery efforts focus on identifying lead compounds acting on a molecular target associated with an established pathological state. Concerted molecular changes that occur in specific cell types during disease progression have generally not been identified. Here, we used constellation pharmacology to investigate rat dorsal root ganglion neurons using two models of peripheral nerve injury: chronic constriction injury (CCI) and spinal nerve ligation (SNL). In these well-established models of neuropathic pain, we show that the onset of chronic pain is accompanied by a dramatic, previously unreported increase in the number of bradykinin-responsive neurons, with larger increases observed after SNL relative to CCI. To define the neurons with altered expression, we charted the temporal course of molecular changes following 1, 3, 6, and 14 d after SNL injury and demonstrated that specific molecular changes have different time courses during the progression to a pain state. In particular, ATP receptors up-regulated on day 1 postinjury, whereas the increase in bradykinin receptors was gradual after day 3 postinjury. We specifically tracked changes in two subsets of neurons: peptidergic and nonpeptidergic nociceptors. Significant increases occurred in ATP responses in nAChR-expressing isolectin B4+ nonpeptidergic neurons 1 d postinjury, whereas peptidergic neurons did not display any significant change. We propose that remodeling of ion channels and receptors occurs in a concerted and cell-specific manner, resulting in the appearance of bradykinin-responsive neuronal subclasses that are relevant to chronic pain., Competing Interests: The authors declare no competing interest.

Somatosensory neurons have historically been classified by a variety of approaches, including structural, anatomical, and genetic markers; electrophysiological properties; pharmacological sensitivities; and more recently, transcriptional profile differentiation. These methodologies, used separately, have yielded inconsistent classification schemes. Here, we describe phenotypic differences in response to pharmacological agents as measured by changes in cytosolic calcium concentration for the rapid classification of neurons in vitro; further analysis with genetic markers, whole-cell recordings, and single-cell transcriptomics validated these findings in a functional context. Using this general approach, which we refer to as tripartite constellation analysis (TCA), we focused on large-diameter dorsal-root ganglion (L-DRG) neurons with myelinated axons. Divergent responses to the K-channel antagonist, κM-conopeptide RIIIJ (RIIIJ), reliably identified six discrete functional cell classes. In two neuronal subclasses (L1 and L2), block with RIIIJ led to an increase in [Ca] i Simultaneous electrophysiology and calcium imaging showed that the RIIIJ-elicited increase in [Ca] i corresponded to different patterns of action potentials (APs), a train of APs in L1 neurons, and sporadic firing in L2 neurons. Genetically labeled mice established that L1 neurons are proprioceptors. The single-cell transcriptomes of L1 and L2 neurons showed that L2 neurons are Aδ-low-threshold mechanoreceptors. RIIIJ effects were replicated by application of the K v 1.1 selective antagonist, Dendrotoxin-K, in several L-DRG subclasses (L1, L2, L3, and L5), suggesting the presence of functional K v 1.1/K v 1.2 heteromeric channels. Using this approach on other neuronal subclasses should ultimately accelerate the comprehensive classification and characterization of individual somatosensory neuronal subclasses within a mixed population., Competing Interests: The authors declare no competing interest.

Transient receptor potential (TRP) cation channels are molecular targets of various natural products. TRPA1, a member of TRP channel family, is specifically activated by natural products such as allyl isothiocyanate (mustard oil), cinnamaldehyde (cinnamon), and allicin (garlic). In this study, we demonstrated that TRPA1 is also a target of trans -anethole in fennel oil (FO) and fennel seed extract. Similar to FO, trans -anethole selectively elicited calcium influx in TRPA1-expressing mouse sensory neurons of the dorsal root and trigeminal ganglia. These FO- and anethole-induced calcium responses were blocked by a selective TRPA1 channel antagonist, HC-030031. Moreover, both FO and trans -anethole induced calcium influx and transmembrane currents in HEK293 cells stably overexpressing human TRPA1 channels, but not in regular HEK293 cells. Mutation of the amino acids S873 and T874 binding site of human TRPA1 significantly attenuated channel activation by trans -anethole, whereas pretreating with glutathione, a nucleophile, did not. Conversely, activation of TRPA1 by the electrophile allyl isothiocyanate was abolished by glutathione, but was ostensibly unaffected by mutation of the ST binding site. Finally, it was found that trans -anethole was capable of desensitizing TRPA1, and unlike allyl isothiocyanate, it failed to induce nocifensive behaviors in mice. We conclude that trans -anethole is a selective, nonelectrophilic, and seemingly less-irritating agonist of TRPA1., (Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.)

The vast complexity of native heteromeric K + channels is largely unexplored. Defining the composition and subunit arrangement of individual subunits in native heteromeric K + channels and establishing their physiological roles is experimentally challenging. Here we systematically explored this "zone of ignorance" in molecular neuroscience. Venom components, such as peptide toxins, appear to have evolved to modulate physiologically relevant targets by discriminating among closely related native ion channel complexes. We provide proof-of-principle for this assertion by demonstrating that κM-conotoxin RIIIJ (κM-RIIIJ) from Conus radiatus precisely targets "asymmetric" K v channels composed of three K v 1.2 subunits and one K v 1.1 or K v 1.6 subunit with 100-fold higher apparent affinity compared with homomeric K v 1.2 channels. Our study shows that dorsal root ganglion (DRG) neurons contain at least two major functional K v 1.2 channel complexes: a heteromer, for which κM-RIIIJ has high affinity, and a putative K v 1.2 homomer, toward which κM-RIIIJ is less potent. This conclusion was reached by ( i ) covalent linkage of members of the mammalian Shaker-related K v 1 family to K v 1.2 and systematic assessment of the potency of κM-RIIIJ block of heteromeric K + channel-mediated currents in heterologous expression systems; ( ii ) molecular dynamics simulations of asymmetric K v 1 channels providing insights into the molecular basis of κM-RIIIJ selectivity and potency toward its targets; and ( iii ) evaluation of calcium responses of a defined population of DRG neurons to κM-RIIIJ. Our study demonstrates that bioactive molecules present in venoms provide essential pharmacological tools that systematically target specific heteromeric K v channel complexes that operate in native tissues., Competing Interests: The authors declare no conflict of interest.

The molecular sensor of innocuous (painless) cold sensation is well-established to be transient receptor potential cation channel, subfamily M, member 8 (TRPM8). However, the role of transient receptor potential cation channel, subfamily A, member 1 (TRPA1) in noxious (painful) cold sensation has been controversial. We find that TRPA1 channels contribute to the noxious cold sensitivity of mouse somatosensory neurons, independent of TRPM8 channels, and that TRPA1-expressing neurons are largely non-overlapping with TRPM8-expressing neurons in mouse dorsal-root ganglia (DRG). However, relatively few TRPA1-expressing neurons (e.g., responsive to allyl isothiocyanate or AITC, a selective TRPA1 agonist) respond overtly to cold temperature in vitro, unlike TRPM8-expressing neurons, which almost all respond to cold. Using somatosensory neurons from TRPM8-/- mice and subtype-selective blockers of TRPM8 and TRPA1 channels, we demonstrate that responses to cold temperatures from TRPA1-expressing neurons are mediated by TRPA1 channels. We also identify two factors that affect the cold-sensitivity of TRPA1-expressing neurons: (1) cold-sensitive AITC-sensitive neurons express relatively more TRPA1 transcripts than cold-insensitive AITC-sensitive neurons and (2) voltage-gated potassium (K V ) channels attenuate the cold-sensitivity of some TRPA1-expressing neurons. The combination of these two factors, combined with the relatively weak agonist-like activity of cold temperature on TRPA1 channels, partially explains why few TRPA1-expressing neurons respond to cold. Blocking K V channels also reveals another subclass of noxious cold-sensitive DRG neurons that do not express TRPM8 or TRPA1 channels. Altogether, the results of this study provide novel insights into the cold-sensitivity of different subclasses of somatosensory neurons., (Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.)

Cone snails in the Virgiconus clade prey on marine worms. Here, we identify six related conotoxins in the O1-superfamily from three species in this clade, Conus virgo, Conus terebra and Conus kintoki. One of these peptides, vi6a, was directly purified from the venom of C. virgo by following its activity using calcium imaging of dissociated mouse dorsal root ganglion (DRG) neurons. The purified peptide was biochemically characterized, synthesized and tested for activity in mice. Hyperactivity was observed upon both intraperitoneal and intracranial injection of the peptide. The effect of the synthetic peptide on DRG neurons was identical to that of the native peptide. Using the vi6a sequence, five other homologs were identified. These peptides define a glycine-rich subgroup of the O1-superfamily from the Virgiconus clade, with biological activity in mice., (Published by Elsevier Ltd.)

A pressing need in neurobiology is the comprehensive identification and characterization of neuronal subclasses within the mammalian nervous system. To this end, we used constellation pharmacology as a method to interrogate the neuronal and glial subclasses of the mouse cerebellum individually and simultaneously. We then evaluated the data obtained from constellation-pharmacology experiments by cluster analysis to classify cells into neuronal and glial subclasses, based on their functional expression of glutamate, acetylcholine, and GABA receptors, among other ion channels. Conantokin peptides were used to identify N-methyl-d-aspartate (NMDA) receptor subtypes, which revealed that neurons of the young mouse cerebellum expressed NR2A and NR2B NMDA receptor subunits. Additional pharmacological tools disclosed differential expression of α-amino-3-hydroxy-5-methyl-4-isoxazloepropionic, nicotinic acetylcholine, and muscarinic acetylcholine receptors in different neuronal and glial subclasses. Certain cell subclasses correlated with known attributes of granule cells, and we combined constellation pharmacology with genetically labeled neurons to identify and characterize Purkinje cells. This study illustrates the utility of applying constellation pharmacology to classify neuronal and glial subclasses in specific anatomical regions of the brain., (Copyright © 2016 the American Physiological Society.)

Prey shifts in carnivorous predators are events that can initiate the accelerated generation of new biodiversity. However, it is seldom possible to reconstruct how the change in prey preference occurred. Here we describe an evolutionary "smoking gun" that illuminates the transition from worm hunting to fish hunting among marine cone snails, resulting in the adaptive radiation of fish-hunting lineages comprising ∼100 piscivorous Conus species. This smoking gun is δ-conotoxin TsVIA, a peptide from the venom of Conus tessulatus that delays inactivation of vertebrate voltage-gated sodium channels. C. tessulatus is a species in a worm-hunting clade, which is phylogenetically closely related to the fish-hunting cone snail specialists. The discovery of a δ-conotoxin that potently acts on vertebrate sodium channels in the venom of a worm-hunting cone snail suggests that a closely related ancestral toxin enabled the transition from worm hunting to fish hunting, as δ-conotoxins are highly conserved among fish hunters and critical to their mechanism of prey capture; this peptide, δ-conotoxin TsVIA, has striking sequence similarity to these δ-conotoxins from piscivorous cone snail venoms. Calcium-imaging studies on dissociated dorsal root ganglion (DRG) neurons revealed the peptide's putative molecular target (voltage-gated sodium channels) and mechanism of action (inhibition of channel inactivation). The results were confirmed by electrophysiology. This work demonstrates how elucidating the specific interactions between toxins and receptors from phylogenetically well-defined lineages can uncover molecular mechanisms that underlie significant evolutionary transitions.

In this study, a proteogenomic annotation strategy was used to identify a novel bioactive peptide from the venom of the predatory marine snail Conus victoriae. The peptide, conorfamide-Vc1 (CNF-Vc1), defines a new gene family. The encoded mature peptide was unusual for conotoxins in that it was cysteine-free and, despite low overall sequence similarity, contained two short motifs common to known neuropeptides/hormones. One of these was the C-terminal RF-amide motif, commonly observed in neuropeptides from a range of organisms, including humans. The mature venom peptide was synthesized and characterized structurally and functionally. The peptide was bioactive upon injection into mice, and calcium imaging of mouse dorsal root ganglion (DRG) cells revealed that the peptide elicits an increase in intracellular calcium levels in a subset of DRG neurons. Unusually for most Conus venom peptides, it also elicited an increase in intracellular calcium levels in a subset of non-neuronal cells., Biological Significance: Our findings illustrate the utility of proteogenomics for the discovery of novel, functionally relevant genes and their products. CNF-Vc1 should be useful for understanding the physiological role of RF-amide peptides in the molluscan and mammalian nervous systems., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Constellation pharmacology is a cell-based high-content phenotypic-screening platform that utilizes subtype-selective pharmacological agents to elucidate the cell-specific combinations (constellations) of key signaling proteins that define specific cell types. Heterogeneous populations of native cells, in which the different individual cell types have been identified and characterized, are the foundation for this screening platform. Constellation pharmacology is useful for screening small molecules or for deconvoluting complex mixtures of biologically active natural products. This platform has been used to purify natural products and discover their molecular mechanisms. In the ongoing development of constellation pharmacology, there is a positive feedback loop between the pharmacological characterization of cell types and screening for new drug candidates. As constellation pharmacology is used to discover compounds with novel targeting-selectivity profiles, those new compounds then further help to elucidate the constellations of specific cell types, thereby increasing the content of this high-content platform.

The toxinology of the crassispirine snails, a major group of venomous marine gastropods within the superfamily Conoidea, is largely unknown. Here we define the first venom peptide superfamily, the P-like crassipeptides, and show that the organization of their gene sequences is similar to conotoxin precursors. We provide evidence that one peptide family within the P-like crassipeptide superfamily includes potassium-channel (K-channel) blockers, the κP-crassipeptides. Three of these peptides were chemically synthesized (cce9a, cce9b and iqi9a). Using conventional electrophysiology, cce9b was shown to be an antagonist of both a human Kv1.1 channel isoform (Shaker subfamily of voltage-gated K channels) and a Drosophila K-channel isoform. We assessed the bioactivity of these peptides in native mammalian dorsal root ganglion neurons in culture. We demonstrate that two of these crassipeptides, cce9a and cce9b, elicited an excitatory phenotype in a subset of small-diameter capsaicin-sensitive mouse DRG neurons that were also affected by κJ-conotoxin PlXIVA (pl14a), a blocker of Kv1.6 channels. Given the vast complexity of heteromeric K-channel isoforms, this study demonstrates that the crassispirine venoms are a potentially rich source for discovering novel peptides that can help to identify and characterize the diversity of K-channel subtypes expressed in native neurons and other cell types., (Published by Elsevier Ltd.)

Comprehensive understanding of nervous systems would require the unambiguous identification of all neuronal cell types, which are distinguished by gene-expression differences, particularly in plasma-membrane receptors and ion channels. Toward this ultimate goal, a recent paper initiated an approach to identify and study divergent neuronal cell types through comparative cellular physiology.

The griseorhodins belong to a family of extensively modified aromatic polyketides that exhibit activities such as inhibition of HIV reverse transcriptase and human telomerase. The vast structural diversity of this group of polyketides is largely introduced by enzymatic oxidations, which can significantly influence the bioactivity profile. Four new compounds, griseorhodins D-F, were isolated from a griseorhodin producer, Streptomyces sp. CN48+, based upon their enhancement of calcium uptake in a mouse dorsal root ganglion primary cell culture assay. Two of these compounds, griseorhodins D1 and D2, were shown to be identical to the major, previously uncharacterized products of a grhM mutant in an earlier griseorhodin biosynthesis study. Their structures enabled the establishment of a more complete hypothesis for the biosynthesis of griseorhodins and related compounds. The other two compounds, griseorhodins E and F, represent new products of post-polyketide synthase tailoring in griseorhodin biosynthesis and showed significant binding activity in a human dopamine active transporter assay.

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.

Change is intrinsic to nervous systems; change is required for learning and conditioning and occurs with disease progression, normal development, and aging. To better understand mammalian nervous systems and effectively treat nervous-system disorders, it is essential to track changes in relevant individual neurons. A critical challenge is to identify and characterize the specific cell types involved and the molecular-level changes that occur in each. Using an experimental strategy called constellation pharmacology, we demonstrate that we can define a specific somatosensory neuronal subclass, cold thermosensors, across different species and track changes in these neurons as a function of development. Cold thermosensors are uniformly responsive to menthol and innocuous cool temperature (17 °C), indicating that they express TRPM8 channels. A subset of cold thermosensors expressed α7 nicotinic acetylcholine receptors (nAChRs) but not other nAChR subtypes. Differences in temperature threshold of cold thermosensors correlated with functional expression of voltage-gated K channels Kv1.1/1.2: Relatively higher expression of KV1.1/1.2 channels resulted in a higher threshold response to cold temperature. Other signaling components varied during development and between species. In cold thermosensors of neonatal mice and rats, ATP receptors were functionally expressed, but the expression disappeared with development. This developmental change occurred earlier in low-threshold than high-threshold cold thermosensors. Most rat cold thermosensors expressed TRPA1 channels, whereas mouse cold thermosensors did not. The broad implications of this study are that it is now feasible to track changes in receptor and ion-channel expression in individual neuronal subclasses as a function of development, learning, disease, or aging.

We investigated the functional expression of nicotinic acetylcholine receptors (nAChRs) in heterogeneous populations of dissociated rat and mouse lumbar dorsal root ganglion (DRG) neurons by calcium imaging. By this experimental approach, it is possible to investigate the functional expression of multiple receptor and ion-channel subtypes across more than 100 neuronal and glial cells simultaneously. Based on nAChR expression, DRG neurons could be divided into four subclasses: (1) neurons that express predominantly α3β4 and α6β4 nAChRs; (2) neurons that express predominantly α7 nAChRs; (3) neurons that express a combination of α3β4/α6β4 and α7 nAChRs; and (4) neurons that do not express nAChRs. In this comparative study, the same four neuronal subclasses were observed in mouse and rat DRG. However, the expression frequency differed between species: substantially more rat DRG neurons were in the first three subclasses than mouse DRG neurons, at all developmental time points tested in our study. Approximately 70-80% of rat DRG neurons expressed functional nAChRs, in contrast to only ~15-30% of mouse DRG neurons. Our study also demonstrated functional coupling between nAChRs, voltage-gated calcium channels, and mitochondrial Ca(2) (+) transport in discrete subsets of DRG neurons. In contrast to the expression of nAChRs in DRG neurons, we demonstrated that a subset of non-neuronal DRG cells expressed muscarinic acetylcholine receptors and not nAChRs. The general approach to comparative cellular neurobiology outlined in this paper has the potential to better integrate molecular and systems neuroscience by uncovering the spectrum of neuronal subclasses present in a given cell population and the functionally integrated signaling components expressed in each subclass.

In the oceans, secondary metabolites often protect otherwise poorly defended invertebrates, such as shell-less mollusks, from predation. The origins of these metabolites are largely unknown, but many of them are thought to be made by symbiotic bacteria. In contrast, mollusks with thick shells and toxic venoms are thought to lack these secondary metabolites because of reduced defensive needs. Here, we show that heavily defended cone snails also occasionally contain abundant secondary metabolites, γ-pyrones known as nocapyrones, which are synthesized by symbiotic bacteria. The bacteria, Nocardiopsis alba CR167, are related to widespread actinomycetes that we propose to be casual symbionts of invertebrates on land and in the sea. The natural roles of nocapyrones are unknown, but they are active in neurological assays, revealing that mollusks with external shells are an overlooked source of secondary metabolite diversity., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Different types of neurons diverge in function because they express their own unique set or constellation of signaling molecules, including receptors and ion channels that work in concert. We describe an approach to identify functionally divergent neurons within a large, heterogeneous neuronal population while simultaneously investigating specific isoforms of signaling molecules expressed in each. In this study we characterized two subclasses of menthol-sensitive neurons from cultures of dissociated mouse dorsal-root ganglia. Although these neurons represent a small fraction of the dorsal-root ganglia neuronal population, we were able to identify them and investigate the cell-specific constellations of ion channels and receptors functionally expressed in each subclass, using a panel of selective pharmacological tools. Differences were found in the functional expression of ATP receptors, TRPA1 channels, voltage-gated calcium-, potassium-, and sodium channels, and responses to physiologically relevant cold temperatures. Furthermore, the cell-specific responses to various stimuli could be altered through pharmacological interventions targeted to the cell-specific constellation of ion channels expressed in each menthol-sensitive subclass. In fact, the normal responses to cold temperature could be reversed in the two neuronal subclasses by the coapplication of the appropriate combination of pharmacological agents. This result suggests that the functionally integrated constellation of signaling molecules in a particular type of cell is a more appropriate target for effective pharmacological intervention than a single signaling molecule. This shift from molecular to cellular targets has important implications for basic research and drug discovery. We refer to this paradigm as "constellation pharmacology."

We describe a functional profiling strategy to identify and characterize subtypes of neurons present in a peripheral ganglion, which should be extendable to neurons in the CNS. In this study, dissociated dorsal-root ganglion neurons from mice were exposed to various pharmacological agents (challenge compounds), while at the same time the individual responses of >100 neurons were simultaneously monitored by calcium imaging. Each challenge compound elicited responses in only a subset of dorsal-root ganglion neurons. Two general types of challenge compounds were used: agonists of receptors (ionotropic and metabotropic) that alter cytoplasmic calcium concentration (receptor-agonist challenges) and compounds that affect voltage-gated ion channels (membrane-potential challenges). Notably, among the latter are K-channel antagonists, which elicited unexpectedly diverse types of calcium responses in different cells (i.e., phenotypes). We used various challenge compounds to identify several putative neuronal subtypes on the basis of their shared and/or divergent functional, phenotypic profiles. Our results indicate that multiple receptor-agonist and membrane-potential challenges may be applied to a neuronal population to identify, characterize, and discriminate among neuronal subtypes. This experimental approach can uncover constellations of plasma membrane macromolecules that are functionally coupled to confer a specific phenotypic profile on each neuronal subtype. This experimental platform has the potential to bridge a gap between systems and molecular neuroscience with a cellular-focused neuropharmacology, ultimately leading to the identification and functional characterization of all neuronal subtypes at a given locus in the nervous system.

An accelerated rate of natural-product discovery is critical for the future of ion channel pharmacology. For the full potential of natural products to be realized, an interdisciplinary initiative is required that combines chemical ecology and ion channel physiology. A prime source of future drug leads targeted to ion channels is the vast assortment of compounds that mediate biotic interactions in the marine environment. Many animals have evolved a chemical strategy to change the behavior of their prey, predators or competitors, which appears to require a large set of ion channel-targeted compounds acting in concert. Some of these compounds (e.g., ziconotide [Prialt(®)]) have already found important biomedical applications. The elucidation of molecular mechanisms mediating biotic interactions should yield a rich stream of potent and selective natural products for the drug pipeline.

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.

The conantokins are a family of Conus venom peptides (17-27AA) that are N-methyl-d-aspartate (NMDA) receptor antagonists. Conantokins lack disulfide bridges (six out of seven previously characterized peptides are linear), but contain multiple residues of gamma-carboxyglutamate. These post-translationally modified amino acids confer the largely helical structure of conantokins by coordinating divalent metal ions. Here, we report that a group of fish-hunting cone snails, Conus purpurascens and Conus ermineus, express a distinctive branch of the conantokin family in their venom ducts. Two novel conantokins, conantokin-P (Con-P) and conantokin-E (Con-E) are 24AA long and contain five gamma-carboxyglutamate residues. These two peptides are characterized by a long disulfide loop (12 amino acids including two Gla residues between the Cys residues). The oxidative folding studies of Con-P revealed that the formation of the disulfide bond proceeded significantly faster in the presence of Ca(++) ions. Circular dichroism suggested that Con-P is less helical than other previously characterized conantokins. Con-P blocks NMDA receptors containing NR2B subunit with submicromolar potency. Furthermore, the subtype-selectivity for different NR2 subunits differs from that of the previously characterized conantokins. Our results suggest that different branches of the phylogenetic tree of cone snails have evolved distinct groups of conantokins, each with its own unique biochemical features.

Formation of the vertebrate neuromuscular junction (NMJ) takes place in a stereotypic pattern in which nerves terminate at select sarcolemmal sites often localized to the central region of the muscle fibers. Several lines of evidence indicate that the muscle fibers may initiate postsynaptic differentiation independent of the ingrowing nerves. For example, nascent acetylcholine receptors (AChRs) are pre-patterned at select regions of the muscle during the initial stage of neuromuscular synaptogenesis. It is not clear how these pre-patterned AChR clusters are assembled, and to what extent they contribute to pre- and post-synaptic differentiation during development. Here, we show that genetic deletion of the AChR gamma-subunit gene in mice leads to an absence of pre-patterned AChR clusters during initial stages of neuromuscular synaptogenesis. The absence of pre-patterned AChR clusters was associated with excessive nerve branching, increased motoneuron survival, as well as aberrant distribution of acetylcholinesterase (AChE) and rapsyn. However, clustering of muscle specific kinase (MuSK) proceeded normally in the gamma-null muscles. AChR clusters emerged at later stages owing to the expression of the AChR epsilon-subunit, but these delayed AChR clusters were broadly distributed and appeared at lower level compared with the wild-type muscles. Interestingly, despite the abnormal pattern, synaptic vesicle proteins were progressively accumulated at individual nerve terminals, and neuromuscular synapses were ultimately established in gamma-null muscles. These results demonstrate that the gamma-subunit is required for the formation of pre-patterned AChR clusters, which in turn play an essential role in determining the subsequent pattern of neuromuscular synaptogenesis.

In an attempt to determine whether the rescue of developing motoneurons (MNS) from programmed cell death (PCD) in the chick embryo following reductions in neuromuscular function involves muscle or neuronal nicotinic acetylcholine receptors (nAChRs), we have employed a novel cone snail toxin alphaA-OIVA that acts selectively to antagonize the embryonic/fetal form of muscle nAChRs. The results demonstrate that alphaA-OIVA is nearly as effective as curare or alpha-bungarotoxin (alpha-BTX) in reducing neuromuscular function and is equally effective in increasing MN survival and intramuscular axon branching. Together with previous reports, we also provide evidence consistent with a transition between the embryonic/fetal form to the adult form of muscle nAChRs in chicken that involves the loss of the gamma subunit in the adult receptor. We conclude that selective inhibition of the embryonic/fetal form of the chicken muscle nAChR is sufficient to rescue MNs from PCD without any involvement of neuronal nAChRs.

Although the neuromuscular nicotinic acetylcholine receptor (nAChR) is one of the most intensively studied ion channels in the nervous system, the differential roles of fetal and adult subtypes of the nAChR under normal and pathological conditions are still incompletely defined. Until recently, no pharmacological tools distinguished between fetal and adult subtypes. Waglerin toxins (from snake venom) and alphaA(S)-conotoxins (from cone-snail venom) have provided such tools. Because these peptides were characterized by different research groups using different methods, we have: 1) more extensively tested their subtype selectivity, and 2) begun to explore how these peptides may be used in concert to elucidate expression patterns and functions of fetal and adult nAChRs. In heterologous expression systems and native tissues, Waglerin-1 and an alphaA(S)-conotoxin analog, alphaA-OIVA[K15N], are high-affinity, highly selective inhibitors of the adult and fetal muscle nAChRs, respectively. We have used the peptides and their fluorescent derivatives to explore the expression and function of the fetal and adult nAChR subtypes. While fluorescent derivatives of these peptides indicated a gradual transition from fetal to adult muscle nAChRs in mice during the first 2 weeks postnatal, we unexpectedly observed a steeper transition in functional expression in the mouse diaphragm muscle using electrophysiology. As a toolkit of pharmacological agents with complementary specificity, alphaA-OIVA[K15N] and Waglerin-1 should have further utility in determining the roles of fetal and adult nAChR subtypes in development, in mature tissues, and under pathological conditions.

Over two decades of research on venom peptides derived from cone snails ("conopeptides or conotoxins") has led to several compounds that have reached human clinical trials, most of them for the treatment of pain. Remarkably, none of the conopeptides in clinical development mediate analgesia through the opioid receptors, underlying the diverse and novel neuropharmacology evolved by Conus snails. These predatory animals produce an estimated approximately 100,000 distinct conotoxins, a vast majority yet to be discovered and characterized. The conopeptides studied to-date in animal models, have exhibited antinociceptive, antiepileptic, neuroprotective or cardioprotective activities. Screening results also suggest applications of conotoxins in cancer, neuromuscular and psychiatric disorders. Additional potentially important applications of conotoxin research are the discovery and validation of new therapeutic targets, also defining novel binding sites on already validated molecular targets. As the structural and functional diversity of conotoxins is being investigated, the Conus venoms continue to surprise with the plethora of neuropharmacological compounds and potential new therapeutics. This review summarizes recent efforts in the discovery of conopeptides, and their preclinical and clinical development.

We report the discovery and characterization of three conantokin peptides from the venom of Conus parius. Each peptide (conantokin-Pr1, -Pr2, and -Pr3) contains 19 amino acids with three gamma-carboxyglutamate (Gla) residues, a post-translationally modified amino acid characteristic of conantokins. The new peptides contain several amino acid residues that differ from previous conantokin consensus sequences. Notably, the new conantokins lack Gla at the 3rd position from the N terminus, where the Gla residue is replaced by either aspartate or by another post-translationally modified residue, 4-trans-hydroxyproline. Conantokin-Pr3 is the first conantokin peptide to have three different post-translational modifications. Conantokins-Pr1 and -Pr2 adopt alpha-helical conformations in the presence of divalent cations (Mg2+ and Ca2+) but are generally unstructured in the absence of divalent cations. Conantokin-Pr3 adopts an alpha-helical conformation even in the absence of divalent cations. Like other conantokins, the new peptides induced sleep in young mice and hyperactivity in older mice upon intracranial injection. Electrophysiological assays confirmed that conantokins-Pr1, -Pr2, and -Pr3 are N-methyl-d-aspartate (NMDA) receptor antagonists, with highest potency for NR2B-containing NMDA receptors. Conantokin-Pr3 demonstrated approximately 10-fold selectivity for NR2B-containing NMDA receptors. However, conantokin-Pr2 showed minimal differences in potency between NR2B and NR2D. Conantokins-Pr1, -Pr2, and -Pr3 all demonstrated high specificity of block for NMDA receptors, when tested against various ligand-gated ion channels. Conus parius conantokins allow for a better definition of structural and functional features of conantokins as ligands targeting NMDA receptors.

Predatory cone snails (genus Conus) produce a rich array of venoms that collectively contain an estimated 100,000 small, disulfide-rich peptides (i.e., conotoxins, or conopeptides). Over the last few decades, the conopeptides have revealed a remarkable diversity of pharmacological function and utility. An evolutionary rationale for the existence of such a large and pharmacologically diverse set of gene products can be premised on the complexity of intra- and interspecies interactions that define the ecology of Conus snails. Insights into these evolutionary trends, moreover, have been exploited with great neuropharmacological success, so that research into the Conus snails effectively recapitulates a new concerted discovery approach, which we discuss here, for developing unique ligands for both laboratory and therapeutic applications. The Conus peptides thus serve as a model system for reaping the pharmacological potential of biodiverse animal lineages.

We have purified a novel paralytic peptide with 32 AA and a single disulfide bond from the venom of Conus parius, a fish-hunting species. The peptide has the following sequence: TYGIYDAKPOFSCAGLRGGCVLPONLROKFKE-NH2, where O is 4-trans-hydroxyproline. The peptide, designated alphaC-conotoxin PrXA (alphaC-PrXA), is the defining member of a new, structurally distinct family of Conus peptides. The peptide is a competitive nAChR antagonist; all previously characterized conotoxins that competitively antagonize nAChRs are structurally and genetically unrelated. (Most belong to the alpha- and alphaA-conotoxin families.) When administered to mice and fish in vivo, alphaC-PrXA caused paralysis and death. In electrophysiological assays, alphaC-PrXA potently antagonized mouse muscle nicotinic acetylcholine receptors (nAChRs), with IC50 values of 1.8 and 3.0 nM for the adult (alpha1beta1 epsilondelta subunits) and fetal (alpha1beta1 gammadelta subunits) muscle nAChR subtypes, respectively. When tested on a variety of ligand-gated and voltage-gated ion channels, alphaC-PrXA proved to be a highly specific inhibitor of the neuromuscular nAChR. The peptide competes with alpha-bungarotoxin for binding at the alpha/delta and alpha/gamma subunit interfaces of the nAChR, with higher affinity for the alpha/delta subunit interface. AlphaC-PrXA is strikingly different from the many conopeptides shown to be nicotinic antagonists; it is most similar in its general biochemical features to the snake toxins known as Waglerins.

The alpha-conotoxin family is comprised of peptides that share the following arrangement of cysteine residues in the primary amino acid sequence: -CC-C-C-, where each dash represents a variable number of amino acids. The number of amino acids between cysteine residues has been used to group the alpha-conotoxins into distinct subfamilies. These subfamilies include the alpha 4/7-, alpha 4/3- and alpha 3/5-conotoxins, so named for the number of amino acids between 2nd/3rd and 3rd/4th cysteine residues, respectively. The alpha 3/5-conotoxins antagonize vertebrate-muscle nicotinic acetylcholine receptors (nAChRs), while the alpha 4/7- and alpha 4/3-conotoxins primarily inhibit vertebrate neuronal nAChRs. To date, these three subfamilies are the most extensively characterized of the alpha-conotoxin family. Here we report the purification and characterization of an unusual alpha 4/4-conotoxin, alpha-conotoxin PIB (alpha-PIB), from the venom of Conus purpurascens, with the following amino-acid sequence: ZSOGCCWNPACVKNRC (Z=pyroglutamate, O=hydroxyproline). This peptide demonstrates high affinity inhibition of vertebrate-muscle nAChRs, and paralytic effects when injected in vivo. Testing of alpha-PIB against other receptors indicated that the inhibitory effect is specific for skeletal muscle nAChRs. alpha-PIB shares the key biochemical and pharmacological characteristics of the alpha-conotoxin family.

We have characterized the defining members of a novel subfamily of excitatory conotoxins, the short kappaA-conotoxins (kappaA(S)-conotoxins). kappaA-conotoxins PIVE and PIVF (kappaA-PIVE and kappaA-PIVF) were purified from Conus purpurascens venom. Both peptides elicited excitatory activity upon injection into fish. kappaA-PIVE was synthesized for further characterization. The excitatory effects of kappaA-PIVE in vivo were dose dependent, causing hyperactivity at low doses and rapid immobilization at high doses, symptomatic of a type of excitotoxic shock. Consistent with these observations, kappaA-PIVE caused repetitive action potentials in frog motor axons in vitro. Similar results have been reported for other structurally distinct conotoxin families; such peptides appear to be required by most fish-hunting cone snails for the rapid immobilization of prey. Unexpected structure-function relationships were revealed between these peptides and two families of homologous conotoxins: the alphaA-conotoxins (muscle nAChR antagonists) and kappaA-conotoxins (excitotoxins), which all share a common arrangement of cysteine residues (CC-C-C-C-C). Biochemically, the kappaA(S)-conotoxins more closely resemble the alphaA(S)-conotoxins than the other kappaA-conotoxin subfamily, the long kappaA-conotoxins (kappaA(L)-conotoxins); however, kappaA(S)- and alphaA(S)-conotoxins produce different physiological effects. In contrast, the kappaA(S)-and kappaA(L)-conotoxins that diverge in several biochemical characteristics are clearly more similar in their physiological effects.

It has been shown previously in a number of systems that after an extended block of activity, synaptic strength is increased. We found that an extended block of synaptic activity at the mouse neuromuscular junction, using a tetrodotoxin cuff in vivo, increased synaptic strength by prolonging the evoked endplate current (EPC) decay. Prolongation of EPC decay was accompanied by only modest prolongation of spontaneous miniature EPC (MEPC) decay. Prolongation of EPC decay was reversed when quantal content was lowered by reducing extracellular calcium. These findings suggested that the cause of EPC prolongation was presynaptic in origin. However, when we acutely inhibited fetal-type acetylcholine receptors (AChRs) using a novel peptide toxin (alphaA-conotoxin OIVA[K15N]), prolongation of both EPC and MEPC decay were reversed. We also blocked synaptic activity in a mutant strain of mice in which persistent muscle activity prevents upregulation of fetal-type AChRs. In these mice, there was no prolongation of EPC decay. We conclude that upregulation of fetal-type AChRs after blocking synaptic activity causes modest prolongation of MEPC decay that is accompanied by much greater prolongation of EPC decay. This might occur if acetylcholine escapes from endplates and binds to extrajunctional fetal-type AChRs only during large, evoked EPCs. Our study is the first to demonstrate a functional role for upregulation of extrajunctional AChRs.

We report the definition and characterization of a conotoxin subfamily, designated the short alphaA-conotoxins (alphaA(S)) and demonstrate that all of these share the unique property of selectively antagonizing the fetal subtype of the mammalian neuromuscular nicotinic acetylcholine receptor (nAChR). We have characterized newly identified alphaA(S)-conotoxins from Conus pergrandis and have conducted a more detailed characterization of alphaA-conotoxins previously reported from additional Conus species. Among the results, the characterization of the short alphaA-conotoxins revealed diverse kinetics of a block of the fetal muscle nAChR, particularly in dissociation rates. The structure-function relationships of native alphaA(S)-conotoxins and some analogues revealed a single amino acid locus (alternatively either His or Pro in native peptides) that is a critical determinant of the dissociation kinetics. The unprecedented binding selectivity for the fetal muscle nAChR, coupled with the kinetic diversity, should make alphaA(S)-conotoxins useful ligands for a diverse set of studies. The rapidly reversible peptides may be most suitable for electrophysiological studies, while the relatively irreversible peptides should be most useful for binding and localization studies.

We report the purification and characterization of a new conotoxin from the venom of Conus radiatus. The peptide, alphaS-conotoxin RVIIIA (alphaS-RVIIIA), is biochemically unique with respect to its amino acid sequence, post-translational modification, and molecular targets. In comparison to other nicotinic antagonists from Conus venoms, alphaS-RVIIIA exhibits an unusually broad targeting specificity for nicotinic acetylcholine receptor (nAChR) subtypes, as assayed by electrophysiology. The toxin is paralytic to mice and fish, consistent with its nearly irreversible block of the neuromuscular nAChR. Similar to other antagonists of certain neuronal nAChRs, the toxin also elicits seizures in mice upon intracranial injection. The only previously characterized conotoxin from the S superfamily, sigma-conotoxin GVIIIA, is a specific competitive antagonist of the 5-HT3 receptor; thus, alphaS-RVIIIA defines a novel family of nicotinic antagonists within the S superfamily. All previously characterized competitive conotoxin nAChR antagonists have been members of the A superfamily of conotoxins. Our working hypothesis is that the particular group of fish-hunting Conus species that includes Conus radiatus uses the alphaS-conotoxin family to target the muscle nAChR and paralyze prey.

We have purified and characterized a novel conotoxin from the venom of Conus obscurus, which has the unique property of selectively and potently inhibiting the fetal form of the mammalian neuromuscular nicotinic acetylcholine receptor (nAChR) (alpha1beta1gammadelta-subunits). Although this conotoxin, alphaA-conotoxin OIVB (alphaA-OIVB), is a high-affinity antagonist (IC50 of 56 nm) of the fetal muscle nAChR, it has >1800-fold lower affinity for the adult muscle nAChR (alpha1beta1epsilondelta-subunits) and virtually no inhibitory activity at a high concentration on various neuronal nAChRs (IC50 > 100 microm in all cases). The peptide (amino acid sequence, CCGVONAACPOCVCNKTCG), with three disulfide bonds, has been chemically synthesized in a biologically active form. Although the neuromuscular nAChRs are perhaps the most extensively characterized of the receptors/ion channels of the nervous system, the precise physiological roles of the fetal form of the muscle nAChR are essentially unknown.alphaA-OIVB is a potentially important tool for delineating the functional roles ofalpha1beta1gammadelta receptors in normal development, as well as in various adult tissues and in pathological states. In addition to its potential as a research tool, alphaA-OIVB may have some direct biomedical applications.

The venoms of cone snails are rich in multiply disulfide-crosslinked peptides, the conotoxins. Conotoxins are grouped into families on the basis of shared cysteine patterns and homologous molecular targets. For example, both the kappaA- and alphaA-conotoxin families share the same Class IV Cys pattern (-CC-C-C-C-C-), but differ in their molecular targets. The kappaA-conotoxins are excitatory toxins that purportedly block potassium channels, while the alphaA-conotoxins are paralytic conotoxins that inhibit nicotinic acetylcholine receptors (nAChRs). In this work, we describe the isolation and characterization of a novel Conus peptide from venom milked from Hawaiian specimens of Conus obscurus. This peptide shares the Class IV Cys pattern but differs from both previously characterized alphaA- and kappaA-conotoxins in the spacing of amino acids between Cys resides. However, the peptide is similar to previously characterized alphaA-conotoxins in its paralytic effects on fish and its antagonist activity on the neuromuscular nAChR. Unexpectedly, the peptide differs in its disulfide bonding from alphaA-conotoxin PIVA. We have named this unique peptide alphaA-conotoxin OIVA, and we consider it the defining member of a subfamily of alphaA-conotoxins that we designate the alphaA(1-3)-conotoxins to identify them by their unique disulfide bonding framework. These results indicate that the alphaA-conotoxin family is both more structurally diverse and broadly distributed than previously believed.