α9-Containing Nicotinic Acetylcholine Receptors Are Required for RgIA-5474 Attenuation of Chemotherapy-Induced Neuropathic Pain.

Nicotinic acetylcholine receptors containing the α9 subunit have been mechanistically implicated in alleviating chemotherapy-induced neuropathic pain. However, the cell types that underlie these effects are currently unknown. RgIA-5474 is a recently developed, synthetic α-conotoxin analog that is a potent antagonist of human α9α10 nAChRs.

αM-Conotoxin MIIIJ Blocks Nicotinic Acetylcholine Receptors at Neuromuscular Junctions of Frog and Fish.

We report the discovery and functional characterization of αM-Conotoxin MIIIJ, a peptide from the venom of the fish-hunting cone snail . Injections of αM-MIIIJ induced paralysis in goldfish () but not mice. Intracellular recording from skeletal muscles of fish () and frog () revealed that αM-MIIIJ inhibited postsynaptic nicotinic acetylcholine receptors (nAChRs) with an IC of ~0.

α-Conotoxins Identify the α3β4* Subtype as the Predominant Nicotinic Acetylcholine Receptor Expressed in Human Adrenal Chromaffin Cells.

Ligands that selectively inhibit human α3β2 and α6β2 nicotinic acetylcholine receptor (nAChRs) and not the closely related α3β4 and α6β4 subtypes are lacking. Current α-conotoxins (α-Ctxs) that discriminate among these nAChR subtypes in rat fail to discriminate among the human receptor homologs. In this study, we describe the development of α-Ctx LvIA(N9R,V10A) that is 3000-fold more potent on oocyte-expressed human α3β2 than α3β4 and 165-fold more potent on human α6/α3β2β3 than α6/α3β4 nAChRs.

Probing the Redox States of Sodium Channel Cysteines at the Binding Site of μO§-Conotoxin GVIIJ.

μO§-Conotoxin GVIIJ is a 35-amino acid peptide that readily blocks six of eight tested NaV1 subunit isoforms of voltage-gated sodium channels. μO§-GVIIJ is unusual in having an S-cysteinylated cysteine (at residue 24). A proposed reaction scheme involves the peptide-channel complex stabilized by a disulfide bond formed via thiol-disulfide exchange between Cys24 of the peptide and a Cys residue at neurotoxin receptor site 8 in the pore module of the channel (specifically, Cys910 of rat NaV1.

Molecular interaction of α-conotoxin RgIA with the rat α9α10 nicotinic acetylcholine receptor.

The α9α10 nicotinic acetylcholine receptor (nAChR) was first identified in the auditory system, where it mediates synaptic transmission between efferent olivocochlear cholinergic fibers and cochlea hair cells. This receptor gained further attention due to its potential role in chronic pain and breast and lung cancers. We previously showed that α-conotoxin (α-CTx) RgIA, one of the few α9α10 selective ligands identified to date, is 300-fold less potent on human versus rat α9α10 nAChR.

Α- and β-subunit composition of voltage-gated sodium channels investigated with μ-conotoxins and the recently discovered μO§-conotoxin GVIIJ.

We investigated the identities of the isoforms of the α (NaV1)- and β (NaVβ)-subunits of voltage-gated sodium channels, including those responsible for action potentials in rodent sciatic nerves. To examine α-subunits, we used seven μ-conotoxins, which target site 1 of the channel. With the use of exogenously expressed channels, we show that two of the μ-conotoxins, μ-BuIIIB and μ-SxIIIA, are 50-fold more potent in blocking NaV1.

A disulfide tether stabilizes the block of sodium channels by the conotoxin μO§-GVIIJ.

A cone snail venom peptide, μO§-conotoxin GVIIJ from Conus geographus, has a unique posttranslational modification, S-cysteinylated cysteine, which makes possible formation of a covalent tether of peptide to its target Na channels at a distinct ligand-binding site. μO§-conotoxin GVIIJ is a 35-aa peptide, with 7 cysteine residues; six of the cysteines form 3 disulfide cross-links, and one (Cys24) is S-cysteinylated. Due to limited availability of native GVIIJ, we primarily used a synthetic analog whose Cys24 was S-glutathionylated (abbreviated GVIIJSSG).

Positional scanning mutagenesis of α-conotoxin PeIA identifies critical residues that confer potency and selectivity for α6/α3β2β3 and α3β2 nicotinic acetylcholine receptors.

The nicotinic acetylcholine receptor (nAChR) subtype α6β2* (the asterisk denotes the possible presence of additional subunits) has been identified as an important molecular target for the pharmacotherapy of Parkinson disease and nicotine dependence. The α6 subunit is closely related to the α3 subunit, and this presents a problem in designing ligands that discriminate between α6β2* and α3β2* nAChRs. We used positional scanning mutagenesis of α-conotoxin PeIA, which targets both α6β2* and α3β2*, in combination with mutagenesis of the α6 and α3 subunits, to gain molecular insights into the interaction of PeIA with heterologously expressed α6/α3β2β3 and α3β2 receptors.

Co-expression of Na(V)β subunits alters the kinetics of inhibition of voltage-gated sodium channels by pore-blocking μ-conotoxins.

Background And Purpose: Voltage-gated sodium channels (VGSCs) are assembled from two classes of subunits, a pore-bearing α-subunit (NaV 1) and one or two accessory β-subunits (NaV βs). Neurons in mammals can express one or more of seven isoforms of NaV 1 and one or more of four isoforms of NaV β. The peptide μ-conotoxins, like the guanidinium alkaloids tetrodotoxin (TTX) and saxitoxin (STX), inhibit VGSCs by blocking the pore in NaV 1.

Molecular basis for the differential sensitivity of rat and human α9α10 nAChRs to α-conotoxin RgIA.

The α9α10 nicotinic acetylcholine receptor (nAChR) may be a potential target in pathophysiology of the auditory system, chronic pain, and breast and lung cancers. Alpha-conotoxins, from the predatory marine snail Conus, are potent nicotinic antagonists, some of which are selective for the α9α10 nAChR. Here, we report a two order of magnitude species difference in the potency of α-conotoxin RgIA for the rat versus human α9α10 nAChR.

μ-Conotoxins that differentially block sodium channels NaV1.1 through 1.8 identify those responsible for action potentials in sciatic nerve.

Voltage-gated sodium channels (VGSCs) are important for action potentials. There are seven major isoforms of the pore-forming and gate-bearing α-subunit (Na(V)1) of VGSCs in mammalian neurons, and a given neuron can express more than one isoform. Five of the neuronal isoforms, Na(V)1.

Navβ subunits modulate the inhibition of Nav1.8 by the analgesic gating modifier μO-conotoxin MrVIB.

Voltage-gated sodium channels (VGSCs) consist of a pore-forming α-subunit and regulatory β-subunits. Several families of neuroactive peptides of Conus snails target VGSCs, including μO-conotoxins and μ-conotoxins. Unlike μ-conotoxins and the guanidinium alkaloid saxitoxin (STX), which are pore blockers, μO-conotoxins MrVIA and MrVIB inhibit VGSCs by modifying channel gating.

α-Conotoxin BuIA[T5A;P6O]: a novel ligand that discriminates between α6ß4 and α6ß2 nicotinic acetylcholine receptors and blocks nicotine-stimulated norepinephrine release.

α6* (asterisk indicates the presence of additional subunits) nicotinic acetylcholine receptors (nAChRs) are broadly implicated in catecholamine-dependent disorders that involve attention, motor movement, and nicotine self-administration. Different molecular forms of α6 nAChRs mediate catecholamine release, but receptor differentiation is greatly hampered by a paucity of subtype selective ligands. α-Conotoxins are nAChR-targeted peptides used by Conus species to incapacitate prey.

Alpha-conotoxins as pharmacological probes of nicotinic acetylcholine receptors.

Cysteine-rich peptides from the venom of cone snails (Conus) target a wide variety of different ion channels. One family of conopeptides, the alpha-conotoxins, specifically target different isoforms of nicotinic acetylcholine receptors (nAChRs) found both in the neuromuscular junction and central nervous system. This family is further divided into subfamilies based on the number of amino acids between cysteine residues.

Synergistic and antagonistic interactions between tetrodotoxin and mu-conotoxin in blocking voltage-gated sodium channels.

Tetrodotoxin (TTX) is the quintessential ligand of voltage-gated sodium channels (NaVs). Like TTX, mu-conotoxin peptides are pore blockers, and both toxins have helped to define the properties of neurotoxin receptor Site 1 of NaVs. Here, we report unexpected results showing that the recently discovered mu-conotoxin KIIIA and TTX can simultaneously bind to Site 1 and act in concert.

Pruning nature: Biodiversity-derived discovery of novel sodium channel blocking conotoxins from Conus bullatus.

Described herein is a general approach to identify novel compounds using the biodiversity of a megadiverse group of animals; specifically, the phylogenetic lineage of the venomous gastropods that belong to the genus Conus ("cone snails"). Cone snail biodiversity was exploited to identify three new mu-conotoxins, BuIIIA, BuIIIB and BuIIIC, encoded by the fish-hunting species Conus bullatus. BuIIIA, BuIIIB and BuIIIC are strikingly divergent in their amino acid composition compared to previous mu-conotoxins known to target the voltage-gated Na channel skeletal muscle subtype Na(v)1.

Structure, dynamics, and selectivity of the sodium channel blocker mu-conotoxin SIIIA.

mu-SIIIA, a novel mu-conotoxin from Conus striatus, appeared to be a selective blocker of tetrodotoxin-resistant sodium channels in frog preparations. It also exhibited potent analgesic activity in mice, although its selectivity profile against mammalian sodium channels remains unknown. We have determined the structure of mu-SIIIA in aqueous solution and characterized its backbone dynamics by NMR and its functional properties electrophysiologically.

Specificity, affinity and efficacy of iota-conotoxin RXIA, an agonist of voltage-gated sodium channels Na(V)1.2, 1.6 and 1.7.

The excitotoxic conopeptide iota-RXIA induces repetitive action potentials in frog motor axons and seizures upon intracranial injection into mice. We recently discovered that iota-RXIA shifts the voltage-dependence of activation of voltage-gated sodium channel Na(V)1.6 to a more hyperpolarized level.

Amino acid residues that confer high selectivity of the alpha6 nicotinic acetylcholine receptor subunit to alpha-conotoxin MII[S4A,E11A,L15A].

Nicotinic acetylcholine receptors (nAChRs) containing alpha3 and beta2 subunits are found in autonomic ganglia and mediate ganglionic transmission. The closely related alpha6 nAChR subtype is found in the central nervous system where changes in its level of expression are observed in Parkinson's disease. To obtain a ligand that discriminates between these two receptors, we designed and synthesized a novel analog ofalpha-conotoxin MII, MII[S4A,E11A,L15A], and tested it on nAChRs expressed in Xenopus oocytes.

Structure/function characterization of micro-conotoxin KIIIA, an analgesic, nearly irreversible blocker of mammalian neuronal sodium channels.

Peptide neurotoxins from cone snails continue to supply compounds with therapeutic potential. Although several analgesic conotoxins have already reached human clinical trials, a continuing need exists for the discovery and development of novel non-opioid analgesics, such as subtype-selective sodium channel blockers. Micro-conotoxin KIIIA is representative of micro-conopeptides previously characterized as inhibitors of tetrodotoxin (TTX)-resistant sodium channels in amphibian dorsal root ganglion neurons.

Characterization of nicotinic acetylcholine receptors that modulate nicotine-evoked [3H]norepinephrine release from mouse hippocampal synaptosomes.

Nicotine's modulation of hippocampal noradrenergic neurotransmission may contribute to its mnemonic properties, but the nicotinic acetylcholine receptor (nAChR) subtypes that modulate terminal release of norepinephrine are unknown. In the present study, we used a number of subtype-selective alpha-conotoxins in combination with nicotinic receptor subunit-deficient mice to characterize nAChRs that modulate [3H]nore-pinephrine release from synaptosomes. The results indicate that at least two populations of nAChRs contribute to this release: a novel alpha6(alpha4)beta2beta3beta4 subtype and an alpha6(alpha4)beta2beta3 subtype.

Alpha-conotoxin BuIA, a novel peptide from Conus bullatus, distinguishes among neuronal nicotinic acetylcholine receptors.

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels. Alpha subunits, together with beta 2 and/or beta 4 subunits, form ligand-binding sites at alpha/beta subunit interfaces. Predatory marine snails of the genus Conus are a rich source of nAChR-targeted peptides.

Effect of novel alpha-conotoxins on nicotine-stimulated [3H]dopamine release from rat striatal synaptosomes.

Nicotine's action on the midbrain dopaminergic neurons is mediated by nicotinic acetylcholine receptors (nAChRs) that are present on the cell bodies and the terminals of these neurons. Previously, it was suggested that one of the nAChR subtypes located on striatal dopaminergic terminals may be an alpha3beta2 subtype, based on partial inhibition of nicotine-stimulated [(3)H]dopamine release by alpha-conotoxin MII, a potent inhibitor of heterologously expressed alpha3beta2 nAChRs. More recent studies indicated that alpha-conotoxin MII also potently blocks alpha6-containing nAChRs.

Analogs of alpha-conotoxin MII are selective for alpha6-containing nicotinic acetylcholine receptors.

Neuronal nicotinic acetylcholine receptors (nAChRs) both mediate direct cholinergic synaptic transmission and modulate synaptic transmission by other neurotransmitters. Novel ligands are needed as probes to discriminate among structurally related nAChR subtypes. Alpha-conotoxin MII, a selective ligand that discriminates among a variety of nAChR subtypes, fails to discriminate well between some subtypes containing the closely related alpha3 and alpha6 subunits.

Expression of neuronal nicotinic acetylcholine receptor subunit mRNAs within midbrain dopamine neurons.

Many behavioral effects of nicotine result from activation of nigrostriatal and mesolimbic dopaminergic systems. Nicotine regulates dopamine release not only by stimulation of nicotinic acetylcholine receptors (nAChRs) on dopamine cell bodies within the substantia nigra and ventral tegmental area (SN/VTA), but also on presynaptic nAChRs located on striatal terminals. The nAChR subtype(s) present on both cell bodies and terminals is still a matter of controversy.