Sean Christensen

α-Conotoxin BuIA[T5A;P6O]: a novel ligand that discriminates between α6β4 and α6β2 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. We hypothesized that distinct conotoxin-binding kinetics could be exploited to develop a series of selective probes to enable study of native receptor subtypes. Proline6 of α-conotoxin BuIA was found to be critical for nAChR selectivity; substitution of proline6 with 4-hydroyxproline increased the IC(50) by 2800-fold at α6/α3β2β3 but only by 6-fold at α6/α3β4 nAChRs (to 1300 and 12 nM, respectively). We used conotoxin probes together with subunit-null mice to interrogate nAChR subtypes that modulate hippocampal norepinephrine release. Release was abolished in α6-null mutant mice. α-Conotoxin BuIA[T5A;P6O] partially blocked norepinephrine release in wild-type controls but failed to block release in β4(-/-) mice. In contrast, BuIA[T5A;P6O] failed to block dopamine release in the wild-type striatum known to contain α6β2* nAChRs. BuIA[T5A;P6O] is a novel ligand for distinguishing between closely related α6* nAChRs; α6β4* nAChRs modulate norepinephrine release in hippocampus but not dopamine release in striatum.

Evolution of Conus peptide toxins: Analysis of Conus californicus Reeve, 1844

Conus species are characterized by their hyperdiverse toxins, encoded by a few gene superfamilies. Our phylogenies of the genus, based on mitochondrial genes, confirm previous results that C. californicus is highly divergent from all other species. Genetic and biochemical analysis of their venom peptides comprise the fifteen most abundant conopeptides and over 50 mature cDNA transcripts from the venom duct. Although C. californicus venom retains many of the general properties of other Conus species, they share only half of the toxin gene superfamilies found in other Conus species. Thus, in these two lineages, approximately half of the rapidly diversifying gene superfamilies originated after an early Tertiary split. Such results demonstrate that, unlike endogenously acting gene families, these genes are likely to be significantly more restricted in their phylogenetic distribution. In concordance with the evolutionary distance of C. californicus from other species, there are aspects of prey-capture behavior and prey preferences of this species that diverges significantly from all other Conus.

α-Conotoxin OmIA Is a Potent Ligand for the Acetylcholine-binding Protein as Well as α3β2 and α7 Nicotinic Acetylcholine Receptors

The molluskan acetylcholine-binding protein (AChBP) is a homolog of the extracellular binding domain of the pentameric ligand-gated ion channel family. AChBP most closely resembles the α-subunit of nicotinic acetylcholine receptors and in particular the homomeric α7 nicotinic receptor. We report the isolation and characterization of an α-conotoxin that has the highest known affinity for the Lymnaea AChBP and also potently blocks the α7 nAChR subtype when expressed in Xenopus oocytes. Remarkably, the peptide also has high affinity for the α3β2 nAChR indicating that α-conotoxin OmIA in combination with the AChBP may serve as a model system for understanding the binding determinants of α3β2 nAChRs. α-Conotoxin OmIA was purified from the venom of Conus omaria. It is a 17-amino-acid, two-disulfide bridge peptide. The ligand is the first α-conotoxin with higher affinity for the closely related receptor subtypes, α3β2 versus α6β2, and selectively blocks these two subtypes when compared with α2β2, α4β2, and α1β1δϵ nAChRs.

Atypical α-Conotoxin LtIA from Conus litteratus Targets a Novel Microsite of the α3β2 Nicotinic Receptor

Different nicotinic acetylcholine receptor (nAChR) subtypes are implicated in learning, pain sensation, and disease states, including Parkinson disease and nicotine addiction. α-Conotoxins are among the most selective nAChR ligands. Mechanistic insights into the structure, function, and receptor interaction of α-conotoxins may serve as a platform for development of new therapies. Previously characterized α-conotoxins have a highly conserved Ser-Xaa-Pro motif that is crucial for potent nAChR interaction. This study characterized the novel α-conotoxin LtIA, which lacks this highly conserved motif but potently blocked α3β2 nAChRs with a 9.8 nm IC50 value. The off-rate of LtIA was rapid relative to Ser-Xaa-Pro-containing α-conotoxin MII. Nevertheless, pre-block of α3β2 nAChRs with LtIA prevented the slowly reversible block associated with MII, suggesting overlap in their binding sites. nAChR β subunit ligand-binding interface mutations were used to examine the >1000-fold selectivity difference of LtIA for α3β2 versus α3β4 nAChRs. Unlike MII, LtIA had a >900-fold increased IC50 value on α3β2(F119Q) versus wild type nAChRs, whereas T59K and V111I β2 mutants had little effect. Molecular docking simulations suggested that LtIA had a surprisingly shallow binding site on the α3β2 nAChR that includes β2 Lys-79. The K79A mutant disrupted LtIA binding but was without effect on an LtIA analog where the Ser-Xaa-Pro motif is present, consistent with distinct binding modes.

A novel inhibitor of α9α10 nicotinic acetylcholine receptors from Conus vexillum delineates a new conotoxin superfamily.

Conotoxins (CTxs) selectively target a range of ion channels and receptors, making them widely used tools for probing nervous system function. Conotoxins have been previously grouped into superfamilies according to signal sequence and into families based on their cysteine framework and biological target. Here we describe the cloning and characterization of a new conotoxin, from Conus vexillum, named αB-conotoxin VxXXIVA. The peptide does not belong to any previously described conotoxin superfamily and its arrangement of Cys residues is unique among conopeptides. Moreover, in contrast to previously characterized conopeptide toxins, which are expressed initially as prepropeptide precursors with a signal sequence, a ‘‘pro’’ region, and the toxin-encoding region, the precursor sequence of αB-VxXXIVA lacks a ‘‘pro’’ region. The predicted 40-residue mature peptide, which contains four Cys, was synthesized in each of the three possible disulfide arrangements. Investigation of the mechanism of action of αB-VxXXIVA revealed that the peptide is a nicotinic acetylcholine receptor (nAChR) antagonist with greatest potency against the α9α10 subtype. 1H nuclear magnetic resonance (NMR) spectra indicated that all three αB-VxXXIVA isomers were poorly structured in aqueous solution. This was consistent with circular dichroism (CD) results which showed that the peptides were unstructured in buffer, but adopted partially helical conformations in aqueous trifluoroethanol (TFE) solution. The α9α10 nAChR is an important target for the development of analgesics and cancer chemotherapeutics, and αB-VxXXIVA represents a novel ligand with which to probe the structure and function of this protein.

The atypical alpha-conotoxin LtIA from Conus litteratus targets a novel microsite of the alpha3beta2 nicotinic receptor

Different nicotinic acetylcholine receptor (nAChR) subtypes are implicated in learning, pain sensation, and disease states, including Parkinson disease and nicotine addiction. α-Conotoxins are among the most selective nAChR ligands. Mechanistic insights into the structure, function, and receptor interaction of α-conotoxins may serve as a platform for development of new therapies. Previously characterized α-conotoxins have a highly conserved Ser-Xaa-Pro motif that is crucial for potent nAChR interaction. This study characterized the novel α-conotoxin LtIA, which lacks this highly conserved motif but potently blocked α3β2 nAChRs with a 9.8 nm IC50 value. The off-rate of LtIA was rapid relative to Ser-Xaa-Pro-containing α-conotoxin MII. Nevertheless, pre-block of α3β2 nAChRs with LtIA prevented the slowly reversible block associated with MII, suggesting overlap in their binding sites. nAChR β subunit ligand-binding interface mutations were used to examine the >1000-fold selectivity difference of LtIA for α3β2 versus α3β4 nAChRs. Unlike MII, LtIA had a >900-fold increased IC50 value on α3β2(F119Q) versus wild type nAChRs, whereas T59K and V111I β2 mutants had little effect. Molecular docking simulations suggested that LtIA had a surprisingly shallow binding site on the α3β2 nAChR that includes β2 Lys-79. The K79A mutant disrupted LtIA binding but was without effect on an LtIA analog where the Ser-Xaa-Pro motif is present, consistent with distinct binding modes.

Inhibition of α9α10 nicotinic acetylcholine receptors prevents chemotherapy-induced neuropathic pain

Significance This study addresses the need to phase out opioids as the major analgesic drugs for moderate to severe chronic pain. We establish that a highly selective and potent inhibitor of the α9α10 nicotinic acetylcholine receptor (nAChR) subtype prevents the expression of chemotherapy-induced neuropathic pain. Thus, selective antagonists of the α9α10 nAChR are potential leads for nonopioid analgesic drug development. The effects of inhibitors of the α9α10 receptor, together with genetic studies, suggest a key role for the α9α10 nAChR subtype in an intercellular signaling network that can be activated by diverse insults (e.g., chemotherapy, nerve injury, and diabetes). Opioids are first-line drugs for moderate to severe acute pain and cancer pain. However, these medications are associated with severe side effects, and whether they are efficacious in treatment of chronic nonmalignant pain remains controversial. Medications that act through alternative molecular mechanisms are critically needed. Antagonists of α9α10 nicotinic acetylcholine receptors (nAChRs) have been proposed as an important nonopioid mechanism based on studies demonstrating prevention of neuropathology after trauma-induced nerve injury. However, the key α9α10 ligands characterized to date are at least two orders of magnitude less potent on human vs. rodent nAChRs, limiting their translational application. Furthermore, an alternative proposal that these ligands achieve their beneficial effects by acting as agonists of GABAB receptors has caused confusion over whether blockade of α9α10 nAChRs is the fundamental underlying mechanism. To address these issues definitively, we developed RgIA4, a peptide that exhibits high potency for both human and rodent α9α10 nAChRs, and was at least 1,000-fold more selective for α9α10 nAChRs vs. all other molecular targets tested, including opioid and GABAB receptors. A daily s.c. dose of RgIA4 prevented chemotherapy-induced neuropathic pain in rats. In wild-type mice, oxaliplatin treatment produced cold allodynia that could be prevented by RgIA4. Additionally, in α9 KO mice, chemotherapy-induced development of cold allodynia was attenuated and the milder, temporary cold allodynia was not relieved by RgIA4. These findings establish blockade of α9-containing nAChRs as the basis for the efficacy of RgIA4, and that α9-containing nAChRs are a critical target for prevention of chronic cancer chemotherapy-induced neuropathic pain.

Synthesis and Characterization of 125I-α-Conotoxin ArIB[V11L;V16A], a Selective α7 Nicotinic Acetylcholine Receptor Antagonist

The α7 nicotinic acetylcholine receptors (nAChRs) are widely expressed both in the central nervous system (CNS) and periphery. In the CNS, 125I-α-bungarotoxin is commonly used to identify α7 nAChRs specifically. However, α-bungarotoxin also interacts potently with α1* and α9α10 nAChRs, two receptor subtypes in peripheral tissues that are colocalized with the α7 subtype. [3H]Methyllycaconitine is also frequently used as an α7-selective antagonist, but it has significant affinity for α6* and α9α10 nAChR subtypes. In this study, we have developed a highly α7-selective α-conotoxin radioligand by iodination of a naturally occurring histidine. Both mono- and diiodo derivatives were generated and purified (specific activities were 2200 and 4400 Ci mmol-1, respectively). The properties of the mono- and diiodo derivatives were very similar to each other, but the diiodo was less stable. For monoidodo peptide, saturation binding to mouse hippocampal membranes demonstrated a Kd value of 1.15 ± 0.13 nM, similar to that of 125I-α-bungarotoxin in the same preparations (0.52 ± 0.16 nM). Association and dissociation kinetics were relatively rapid (kobs for association at 1 nM was 0.027 ± 0.007 min-1; koff = 0.020 ± 0.001 min-1). Selectivity was confirmed with autoradiography using α7-null mutant tissue: specific binding was abolished in all regions of α7-/- brains, whereas wild-type mice expressed high levels of labeling and low nonspecific binding. 125I-α-conotoxin ArIB[V11L; V16A] should prove useful where α7 nAChRs are coexpressed with other subtypes that are also labeled by existing ligands. Furthermore, true equilibrium binding experiments could be performed on α7 nAChRs, something that is impossible with 125I-α-bungarotoxin.

Cloning, synthesis, and characterization of αO-conotoxin GeXIVA, a potent α9α10 nicotinic acetylcholine receptor antagonist

Significance The α9α10 nicotinic AChR (nAChR) subtype is a recently identified target for the development of breast cancer chemotherapeutics and analgesics, particularly to treat neuropathic pain. Structure/function analyses of antagonists of this subtype are therefore essential for the development of specific therapeutic compounds. The Conus genus is a rich source of pharmacologically active peptides, and we report here that the αO-conotoxin GeXIVA is a potent and selective antagonist of the α9α10 nAChR subtype. GeXIVA displays unique structural properties among other Conus peptides and represents a previously unidentified template for molecules active against neuropathic pain. We identified a previously unidentified conotoxin gene from Conus generalis whose precursor signal sequence has high similarity to the O1-gene conotoxin superfamily. The predicted mature peptide, αO-conotoxin GeXIVA (GeXIVA), has four Cys residues, and its three disulfide isomers were synthesized. Previously pharmacologically characterized O1-superfamily peptides, exemplified by the US Food and Drug Administration-approved pain medication, ziconotide, contain six Cys residues and are calcium, sodium, or potassium channel antagonists. However, GeXIVA did not inhibit calcium channels but antagonized nicotinic AChRs (nAChRs), most potently on the α9α10 nAChR subtype (IC50 = 4.6 nM). Toxin blockade was voltage-dependent, and kinetic analysis of toxin dissociation indicated that the binding site of GeXIVA does not overlap with the binding site of the competitive antagonist α-conotoxin RgIA. Surprisingly, the most active disulfide isomer of GeXIVA is the bead isomer, comprising, according to NMR analysis, two well-resolved but uncoupled disulfide-restrained loops. The ribbon isomer is almost as potent but has a more rigid structure built around a short 310-helix. In contrast to most α-conotoxins, the globular isomer is the least potent and has a flexible, multiconformational nature. GeXIVA reduced mechanical hyperalgesia in the rat chronic constriction injury model of neuropathic pain but had no effect on motor performance, warranting its further investigation as a possible therapeutic agent.

Characterization of a novel α-conotoxin from conus textile that selectively targets α6/α3β2β3 nicotinic acetylcholine receptors.

Background: Cone snails are a rich source of α-conotoxins that target nicotinic acetylcholine receptors (nAChR). Results: A new α-conotoxin TxIB potently blocked α6/α3β2β3 nAChRs with high selectivity. Conclusion: TxIB is an effective inhibitor of α6/α3β2β3 nAChRs. Its structure was determined by NMR. Significance: TxIB is a new, uniquely selective ligand for querying the structure and function of nAChRs and designing therapeutic drugs. α6β2 Nicotinic acetylcholine receptors (nAChRs) expressed by dopaminergic neurons in the CNS are potential therapeutic targets for the treatment of several neuropsychiatric diseases, including nicotine addiction and Parkinson disease. However, recent studies indicate that the α6 subunit can also associate with the β4 subunit to form α6β4 nAChRs that are difficult to pharmacologically distinguish from α6β2, α3β4, and α3β2 subtypes. The current study characterized a novel 16-amino acid α-conotoxin (α-CTx) TxIB from Conus textile whose sequence is GCCSDPPCRNKHPDLC-amide as deduced from gene cloning. The peptide and an analog with an additional C-terminal glycine were chemically synthesized and tested on rat nAChRs heterologously expressed in Xenopus laevis oocytes. α-CTx TxIB blocked α6/α3β2β3 nAChR with an IC50 of 28 nm. In contrast, the peptide showed little or no block of other tested subtypes at concentrations up to 10 μm. The three-dimensional solution structure of α-CTx TxIB was determined using NMR spectroscopy. α-CTx TxIB represents a uniquely selective ligand for probing the structure and function of α6β2 nAChRs.