Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2018 Dec 1.
Published in final edited form as: Neuropharmacology. 2017 Apr 14;127:243–252. doi: 10.1016/j.neuropharm.2017.04.015

αO-Conotoxin GeXIVA Disulfide Bond Isomers Exhibit Differential Sensitivity for Various Nicotinic Acetylcholine Receptors but Retain Potency and Selectivity for the Human α9α10 Subtype

Dongting Zhangsun a, Xiaopeng Zhu a, Quentin Kaas b, Yong Wu a, David J Craik b, J Michael McIntosh c,d, Sulan Luo a,*
PMCID: PMC6029978  NIHMSID: NIHMS966034  PMID: 28416445

Abstract

Nicotinic acetylcholine receptor (nAChR) subtypes exhibit distinct neuropharmacological properties that are involved in a range of neuropathological conditions, including pain, addiction, epilepsy, autism, schizophrenia, Tourette’s syndrome, Alzheimer’s and Parkinson’s diseases, as well as many types of cancer. The α9α10 nAChR is a potential target in chronic pain, wound healing, the pathophysiology of the auditory system, and breast and lung cancers. αO-conotoxin GeXIVA is a potent antagonist of rat α9α10 nAChRs, with the ‘bead’ disulfide bond isomer displaying the lowest IC50 of the three possible isomers. In the rat chronic constriction injury model of neuropathic pain, this isomer reduced mechanical hyperalgesia without affecting motor performance. Here, we report the effects of the three disulfide bond isomers of GeXIVA on human α9α10 nAChRs, other human nAChR subtypes, various rat nAChR subtypes, and 10 rat α9α10 nAChR mutants. The three isomers displayed only ~5-fold difference in potency on the human vs rat α9α10 receptors and had similar affinities at wild-type rat α9α10 nAChRs and all 10 α9α10 receptor mutants. From these findings, the binding site and mechanism of action of GeXIVA on rat and human α9α10 nAChR was deduced to be different from that of other conotoxins targeting this nAChR subtype. GeXIVA is therefore a unique ligand that might prove useful for further probing of binding sites on the α9α10 nAChR.

Keywords: αO-conotoxin GeXIVA disulfide bond Isomers, human and rat nAChRs, α9α10 nAChR subtype and its mutants, Differential sensitivity, Mechanism of action

1. Introduction

For centuries, the shells of Conus snails have been coveted and collected owing to their beauty, but with this beauty comes danger; cone snails are venomous, immobilizing their prey using a complex concoction of toxins. Each concoction contains a unique complement of a thousand or more diverse components (Davis et al., 2009; Lavergne et al., 2015). Given the existence of more than 800 species of cone snails (Puillandre et al., 2015; Uribe et al., 2017) (REF), the library of toxins is immense. A number of cases of human fatalities from cone snail envenomation have occurred, and reports of “painless death” sparked interest in the analgesic properties of the venom (Livett et al., 2006). Many of the venom compounds, which are small disulfide-rich peptides called conotoxins, have been developed into subtype-selective probes for receptors and ion channels. The sequences of conotoxins are compiled in an online database, ConoServer (Kaas et al., 2012), and their classification schemes and pharmacological activities have been extensively reviewed (Halai and Craik, 2009).

Species differences in target receptors have played a critical role in the development of conotoxins as potential therapeutic agents. One venom component, ω-conotoxin MVIIA, paralyzes fish by targeting calcium channels at the neuromuscular junction. Although calcium channels at the human neuromuscular junction are resistant to ω-conotoxin MVIIA, N-type calcium channels in the dorsal horn of the human spinal cord are highly sensitive to the same toxin. This discovery ultimately led to FDA approval of a chemically synthesized form of ω-conotoxin MVIIA (ziconotide) for use in intractable pain. Another conotoxin, Vc1.1, a potent antagonist of rat α9α10 (rα9α10) nicotinic acetylcholine receptors (nAChRs) and an analgesic in rat models of neuropathic pain (Satkunanathan et al., 2005; Vincler et al., 2006) reached phase 2 human clinical trials. However, the discovery that its potency at human α9α10 nAChRs in vitro was significantly less than in rat, prompted discontinuation of its clinical development.

A structurally novel 28-amino acid peptide, αO-conotoxin (αO-CTx) GeXIVA, which potently blocks nAChRs, was recently discovered in the transcriptome of Conus generalis (Luo et al., 2015). nAChRs are ligand-gated ion channels composed of five subunits, which are either all α subunits or a combination of α and non-α subunits. In mammals, there are nine different α subunits: α1–α7, α9, α10, four β subunits and γ, δ and ε subunits. GeXIVA displays high potency for α9α10 nAChRs. GeXIVA has four cysteine (Cys) residues in its primary sequence, which can be oxidized to form three possible disulfide isomers, referred to as the globular, ribbon and bead isomers. For convenience we hereafter denote them as GeXIVA[1,3], GeXIVA[1,4], and GeXIVA[1,2], respectively, with the numerals in square brackets referring to the first disulfide connectively in each of the isomers (with the 2nd disulfide connectivity following by default). Remarkably, each of these isomers shows antagonist activity at rat α9α10 nAChRs (Luo et al., 2015). Here we investigate the subtype-selectivity of each of the isomers on a range of rat nAChRs and at some human nAChR subtypes. We also probe the potential binding site of the peptide using 10 rat α9α10 receptor mutants.

2. Materials and Methods

2.1. Peptide synthesis

The three isomers of αO-CTx GeXIVA were synthesized as previously described, whereby we used directed synthesis in a two-step oxidation procedure to enforce the disulfide arrangement (Luo et al., 2015). Briefly, the linear peptide was assembled using solid-phase methodology on an ABI 433A peptide synthesizer using FastMoc (N-(9-fluorenyl) methoxycarbonyl) chemistry and standard side-chain protection, except for Cys residues. The Cys residues of the three isomers were protected in pairs with either S-trityl on Cys20 and Cys27 (designated GeXIVA[1,2]), Cys9 and Cys27 (designated GeXIVA[1,3]), Cys9 and Cys20 (designated GeXIVA[1,4]), or S-acetamidomethyl on Cys2 and Cys9, Cys2 and Cys20, Cys2 and Cys27. The peptides were released from the solid support by treatment with reagent K (trifluoroacetic acid/water/ethanedithiol/phenol/thioanisole; 90:5:2.5:7.5:5, v/v/v/v/v). The released peptides were precipitated, washed three times with cold ether, and subjected to a two-step oxidation protocol to fold them selectively (Dowell et al., 2003; Walker et al., 1999). The oxidised peptides were purified using high-performance liquid chromatography on a reversed-phase C18 Vydac column using a linear gradient of 10–50% B60 in 40 min. Solvent B (60% ACN, 40 % H20, 0.092% TFA); Solvent A (0.1% trifluoroacetic acid in H2O). Peptide concentration was measured using a spectrophotometer at a wavelength of 280 nm, and calculated using the Beer-Lambert equation. Matrix-assisted laser desorption ionization (MALDI) time-of-flight mass spectrometry and NMR spectroscopy were used to confirm the identity of the three disulfide isomers, as described in our earlier study (Luo et al., 2015).

2.2. nAChR clones and cRNA preparation

Clones of rat α2–α7 and β2–β4, as well as mouse muscle α1β1δε cDNAs, were kindly provided by S. Heinemann (Salk Institute, San Diego, CA). Clones for rat α9 and α10 were kindly provided by A. B. Elgoyhen (INGEBI, Buenos Aires, Argentina). Clones of β2 and β3 subunits in the high-expressing pGEMHE vector were kindly provided by C.W. Luetje (University of Miami, Miami, FL). Clones of human α1β1δε were from Steven Sine (Mayo Clinic, Rochester, Minn). The human α6 clone was from Jon Lindstrom (Perelman School of Medicine, Philadelphia, PA). The other human subunits were from James Garrett (Formerly of Cognetix Inc., Salt Lake City, Utah). Clones for the human α9 and α10 subunits were kindly provided by Lawrence Lustig (Johns Hopkins University, Baltimore, MD). The human subunits were subcloned into the pSGEM vector for Xenopus oocyte expression. Capped cRNA for the various subunits were made using the mMessage mMachine in vitro transcription kit following linearization of the plasmid. The cRNA was purified using the Qiagen RNeasy kit. The concentration of cRNA was determined by absorbance at 260 nm.

2.3. Construction of rat α9α10 nAChR mutants

Rat α9 or α10 nAChR subunit clones were mutated using the QuikChange system (Stratagene) according to amino acid sequence alignment of N-terminal-binding regions of rat and human α9 and α10 nAChR subunits. In mutated rα9A24Kα10 nAChRs, the codon for Ala24 of the rat α9 subunit was replaced by a codon for Lys, and in rα9α10E61S nAChRs, the codon for Glu of the rat α10 subunit was replaced by a codon for Ser, and so on, as described previously (Azam and McIntosh, 2012). Briefly, primers containing the desired point mutation flanked by at least 15 bases on either side were synthesized. Using the non-strand displacing action of Pfu Turbo DNA polymerase, the mutagenic primers were extended and incorporated by PCR. The methylated, non-mutated parental cDNA was digested with DpnI. The mutated DNA was transformed into DH10B or DH5α competent cells, isolated using the Qiaprep mini prep kit (Qiagen, Valencia, CA, USA), and sequenced to ascertain the incorporation of the desired mutation.

2.4. cRNA injection and expression

Xenopus oocytes were used to heterologously express cloned nAChR subtypes. Oocyte preparation, and expression of nAChR subunits in Xenopus oocytes were performed as described previously (McIntosh et al., 2005). For rat α9 subunit mutants, cRNA of mutant α9 subunit were mixed at a 1:1 ratio with wild-type α10 for a final concentration of at least 500 ng/µL for each subunit cRNA. For rat α10 subunit mutants, cRNA of mutant α10 subunit were mixed at a 1:1 ratio with wild-type α9. Fifty to 150 nL of this mixture was injected into each Xenopus oocyte using a Drummond microdispenser (Drummond Scientific, Broomall, PA), as described previously (Cartier et al., 1996), which were incubated at 17°C. Oocytes were injected within a day of harvesting and recordings were made 2–5 days post-injection.

2.5. Electrophysiology

Oocytes were voltage-clamped and exposed to acetylcholine (ACh) and peptide, as described previously (Cartier et al., 1996). The oocyte recording chamber was fabricated from Sylgard and was 30 µL in volume. All recordings were done at room temperature (~22°C). Briefly, oocytes were gravity-perfused with ND-96 buffer containing 1 µM atropine and 0.1 mg/ml BSA at a rate of ~2 mL/min. The ND-96 buffer, with a pH of 7.1–7.5, consisted of 96.0 mM NaCl, 2.0 mM KCl, 1.8 mM CaCl2, 1.0 mM MgCl2 and 5 mM HEPES. In the case of rat and human α9α10, α7, and mouse muscle α1β1δε subtypes, to avoid antagonism of α7-like receptors atropine was omitted from the buffer (Cartier et al., 1996). ACh-gated currents were obtained using a two-electrode voltage-clamp amplifier (model OC-725B, Warner Instruments, Hamden, CT). The membrane potential was clamped at –70 mV for all nAChR subtypes and mutants. The perfused oocyte was then subjected to a 1-sec pulse of 100 µM ACh once a minute repeatedly. In the case of rat and human α9α10 and mouse/human muscle α1β1δε subtypes a 1-sec pulse of 10 µM ACh was used, and for the rat and human α7 subtype a 1-sec pulse of 200 µM ACh was used. To measure conotoxin antagonism, peptide was bath-applied for 5 min before exposure to ACh. All ACh pulses contained no toxin.

2.6. Statistical Analysis

The IC50 values for the oocyte-expressing receptors are reported with 95% confidence intervals. The average of five control responses just preceding a test response was used to normalize the test response to obtain “percent response.” IC50 values were determined by nonlinear regression analysis using Prism (GraphPad Software, Inc., San Diego, CA, USA). All statistical analyses were performed with Prism. Each data point of a concentration-response curve represents the average ± S.E.M. of at least three oocytes.

2.7. Molecular modelling

The molecular models of the two binding modes of ribbon GeXIVA on the α9α10 nAChR that we proposed previously (Luo et al., 2015) were mutated using Modeller9v13 (Sali and Blundell, 1993) for use in the current study. The mutated models were minimised and submitted to 5 ns molecular dynamics simulations using Gromacs 2016 (Abraham et al., 2015) and the Amber99sb-ILDN force field (Lindorff-Larsen et al., 2010). Buried surface areas were computed using the Gromacs package.

3. Results

3.1. GeXIVA isomers are selective for rα9α10 nAChRs

The three disulfide isomers of GeXIVA were tested for their potency against the rat α9α10 (rα9α10) nAChR, as described previously (Luo et al., 2015). We further investigated the effect of the three isomers on other subunit combinations of neuronal and muscle nAChRs heterologously expressed in Xenopus oocytes (Table 1, Fig. 1). Concentration-response analysis indicated that, unlike most α-conotoxins, all three isomers displayed potency against a broad spectrum of rat nAChR subtypes. The three isomers most potently blocked the rα9α10 nAChRs. Generally, the three isomers displayed the lowest potency for subtypes containing the β4 nAChR subunit, and had little effect on rα2β4, rα3β4 and rα4β4 nAChRs, with IC50s > 869 nM.

Table 1.

IC50 and Hill slope values for block of rat nAChR subtypes by αO-CTx GeXIVA[1,2], [1,3] & [1,4].

Subtypes GeXIVA[1,2]b GeXIVA[1,3] GeXIVA[1,4]
IC50 (nM)a Hill slopea IC50 (nM)a Hill slopea IC50 (nM)a Hill slopea
rα9α10 4.61 (3.18–6.65) 0.56 (0.44–0.69) 22.7(11.8–43.5)b 0.78(0.29–1.26)b 7.0(3.6–13.4)b 0.79(0.23–1.36)b
rα7 432(290–644) 1.10(0.72–1.48) 4990(4264–5840) 1.72(1.25–2.18) 1768(1327–2354) 1.00(0.70–1.29)
Mouse α1β1δε 394(311–498) 1.71(0.98–2.43) 683(543–858) 1.47(0.91–2.02) 484(399–587) 1.64(1.15–2.14)
rα6/α3β2β3 449(355–569) 0.69(0.59 –0.79) 369(191–710) 0.69(0.41–0.98) 555(246–1252) 0.64(0.34–0.94)
rα3β2 480(362–638) 0.91(0.67–1.14) 613(432–869) 1.48(0.73–2.23) 489(308–777) 1.06(0.63–1.49)
rα2β2 499(379–656) 1.23(0.91–1.55) 348(237–511) 1.19(0.80–1.57) 122(89.7–166) 0.95(0.70–1.20)
rα4β2 1350(960–1897) 0.99(0.70–1.23) 1261(532–2986) 0.63(0.27–0.99) 215(132–352) 0.82(0.53–1.11)
rα2β4 5523(4600–6632) 1.03(0.80–1.26) 1283(1079–1524) 1.16(0.92–1.39) 1082(911–1285) 1.33(1.01–1.65)
rα4β4 2512(1630–3870) 0.79(0.51–1.08) 1100(795–1521) 1.33(0.72–1.94) 869(675–1118) 1.18(0.81–1.55)
rα6/α3β4 832(718–965) 1.34(0.95–1.74) 497(400–616) 1.75(1.29–2.21) 634(474–821) 1.39(0.84–1.94)
rα3β4 5409(3536–8274) 1.13 (0.53–1.74) 2003(1042–3850) 0.88(0.31–1.45) 3396(2394–4817) 1.06(0.62–1.49)
Open in a new tab
a

Numbers in parentheses are 95% confidence intervals;

Fig. 1.

Fig. 1

Open in a new tab

Percentage inhibition of the mouse muscle type α1β1δε nAChR and rat α7, α2β2, α2β4, α3β2, α3β4, α4β2, α4β4, α6/α3β2β3 and α6/α3β4 nAChR subtypes by various concentrations of the three αO-GeXIVA isomers. Values are mean ± SEM from 3–6 separate oocytes.. IC50 values corresponding to each curve are provided in Table 1.

Despite their potency, the three isomers exhibited differential sensitivity to rat nAChR subtypes. All three isomers were highly selective for rα9α10 nAChR, with IC50s ranging from 4.6–22.7 nM. All three isomers were less potent on the rat α7 nAChR subtype. Of the isomers, GeXIVA[1,2] was most potent at the rα7 nAChR subtype, with an IC50 of 432 nM, which is 10-fold more potent than GeXIVA[1,3] (IC50: 4990 nM), and 4.1-fold more than GeXIVA[1,4] (IC50: 1768 nM). Unlike the rα9α10 and rα7 subtypes, the three isomers blocked the mouse muscle nAChR subtype (mα1β1δε) and the rα6/α3β2β3 subtype with similar potency: IC50s at these subtypes ranged from 394 to 683 nM and 369 to 555 nM, respectively (Table 1, Fig. 1). The three isomers had similar affinities for the rα3β2 subtype, with IC50s ranging from 480 to 613 nM (GeXIVA[1,2] and GeXIVA[1,4] both displayed a similar IC50 of 480 nM and 489 nM) (Table 1, Fig. 1). From these findings, we conclude that there are no differences between the three isomers in terms of potency and selectivity for the mα1β1δε, rα6/α3β2β3, and rα3β2 subtypes, but overall, their potency is far less at these receptors than at the rat and human α9α10 nAChRs. Thus, GeXIVA[1,3] and GeXIVA[1,4] are slightly less selective for rα9α10 nAChR than GeXIVA[1,2], but are 15-fold more selective for rα9α10 nAChR than the other aforementioned subtypes.

The order of potency of the isomers at the rat α9α10 subtype differed to that observed at the rα2β2, rα4β2, rα2β4, rα4β4, rα6/α3β4 and rα3β4 subtypes (Table 3, Fig. 3). GeXIVA[1,4] displayed the greatest potency at rα2β2 and rα4β2, with IC50s of 122 nM at rα2β2, and an IC50 of 215 nM at rα4β2. The IC50s of GeXIVA[1,2] and GeXIVA[1,3] at rα2β2 were 499 nM and 348 nM, respectively, and 1350 nM and 1261 nM, respectively, at rα4β2. GeXIVA[1,4] and GeXIVA[1,3] showed similar activities at rα2β4, rα4β4 and rα6/α3β4. For these six subtypes, GeXIVA[1,2] was the least active of the three isomers, which is interesting given that it had the highest selectivity and was at least 85-fold more potent at rα9α10 than the other subtypes.

Table 3.

IC50 (nM)a values for block of the rat α9α10 nAChR mutants by each isomer of αO-CTx GeXIVA.

Subtype/mutant GeXIVA[1,2] GeXIVA[1,3] GeXIVA[1,4]
rα9α10 4.61 (3.18–6.65)a 22.7 (11.8–43.5)a 7.0 (3.6–13.4)a
hα9α10 20.3(12.4–33.2)a 116(65.4–204) 47.3(29.7–75.3)
rα9S6Nα10 19.8 (13.7–28.6) 119 (79.8–178) 56.8 (32.4–99.6)
rα9S14Nα10 52.5 (45.3–60.8) 123 (65.2–231) 59.7 (51.0–69.9)
rα9A24Kα10 51.6 (42.0–63.5) 170 (82.5–350) 96.0 (66.1–139)
rα9T56Iα10 19.6(14.2–26.9) 80.6 (64.4–101) 29.5 (20.1–43.2)
rα9R71Gα10 59.3 (44.2–79.6) 232 (160–337) 61.6 (47.4–79.9)
rα9S117Aα10 32.1 (21.6–47.8) 182 (87.5–379) 46.7 (7.63–286)
rα9S136Nα10 45.7 (41.3–50.6) 106 (75.5–149) 33.7 (28.4–40.0)
rα9E192Qα10 49.6 (35.1–70.3) 143 (91.7–224) 63.2 (43.7–91.5)
rα9α10E61S 25.1 (17.7–35.4) 67.9 (52.2–88.2) 23.9 (16.7–34.1)
rα9α10D121L 27.6 (18.3–41.7) 122 (67.4–220) 45.1 (22.8–89.4)
Open in a new tab
a

IC50 (nM), Numbers in parentheses are 95% confidence intervals.

Fig. 3.

Fig. 3

Open in a new tab

Concentration-response of the three αO-GeXIVA isomers on human α9α10, α7, α1β1δε and α6/α3β2β3 nAChR subtypes. Values are mean ± SEM from 5–9 separate oocytes. IC50 values corresponding to each curve are provided in Table 2.

3.2. GeXIVA isomers retain potency for hα9α10 nAChRs

The three GeXIVA isomers were tested for their ability to antagonize the response elicited by ACh on the human α9α10 (hα9α10) nAChR, and four closely related human subtypes (Table 2). The three isomers of GeXIVA block human α9α10 nAChRs differently. Fig. 2 shows representative responses of hα9α10 nAChRs to ACh in the presence and absence of the three GeXIVA isomers. The block of hα9α10 nAChR induced by both GeXIVA[1,2] and GeXIVA[1,3] was rapidly reversible (Fig. 2A–B), whereas that induced by GeXIVA[1,4] was slower to reverse (Fig. 2C). The most potent isomer was found to be GeXIVA[1,2], which blocked hα9α10 nAChRs almost completely at 100 nM (Fig. 2A). GeXIVA[1,3] was the least potent with a 57% response at 100 nM (Fig. 2B). GeXIVA[1,4] fell in the middle, with a 33% response at 100 nM (Fig. 2C). Concentration–response experiments were conducted (Table 2, Fig. 3), and the IC50 of the most potent isomer, GeXIVA[1,2], was observed to be 20.3 (12.4–33.2) nM and the IC50 of the least potent isomer, GeXIVA[1,3], was 116 (65.4–204) nM. The IC50 of GeXIVA[1,4] fell in-between, at 47.3 (29.7–75.3) nM – 2.3-fold less potent than GeXIVA[1,2] and 5.7-fold more potent than GeXIVA[1,3].

Table 2.

IC50 and Hill slope values for block of human nAChR subtypes by αO-CTx GeXIVA[1,2], [1,3] & [1,4].

Subtypes αO-GeXIVA[1,2] αO-GeXIVA[1,3] αO-GeXIVA[1,4]
IC50 (nM)a Hill slopea IC50 (nM)a Hill slopea IC50 (nM)a Hill slopea
hα9α10 20.3(12.4–33.2)a 0.91(0.49–1.32)a 116(65–204) 0.73(0.45–1.01) 47.3(29.7–75.3) 0.67(0.46–0.88)
hα7 559(429–728) 1.17(0.85–1.49) 3584(2476–5186) 0.96(0.60–1.32) 889(739–1068) 1.96(1.05–2.87)
hα1β1δε 497(391–633) 1.65(1.02–2.27) 485(398–592) 1.62(1.11–2.13) 365(279–477) 1.80(0.88–2.72)
hα6/α3β2β3 508(345–747) 0.95(0.65–1.24) 149(112–200) 1.00(0.69–1.32) 199(134–295) 1.11(0.68–1.54)
Open in a new tab
a

Numbers in parentheses are 95% confidence intervals

Fig. 2.

Fig. 2

Open in a new tab

The three αO-GeXIVA isomers block human α9α10 nAChR differently at 100 nM concentration. (A) Bead αO-GeXIVA[1,2], (B) globular αO-GeXIVA[1,3], (C) ribbon αO-GeXIVA[1,4]. Oocytes expressing human α9α10 nAChR were voltage clamped at – 70 mV and subjected to a 1 second pulse of 10 µM ACh every minute as described in the Materials and Methods. In panel A, B and C, the initial peaks are control responses, following which the oocyte was exposed to the peptide for 5 min. ND96 perfusion and ACh pulses were then resumed to monitor recovery from toxin block. The arrow denotes the current response trace on α9α10 nAChRs in the presence of 100 nM peptide. The bead isomer αO-GeXIVA[1,2] was most potent as it nearly completely blocked human α9α10 nAChR at 100 nM, whereas the globular αO-GeXIVA[1,3] was the least potent with only 43% block; the ribbon αO-GeXIVA[1,4] had an intermediate activity with 67% block at 100 nM and its recovery was slower than the other two isomers.

3.3. GeXIVA isomers are selective for hα9α10 nAChR

The concentration–response for the three isomers was assessed against each of the other closely related human nAChR subtypes (see Table 2, Fig. 3). The most potent isomer at hα7 nAChR was GeXIVA[1,2], with an IC50 of 559 nM, and the least potent was GeXIVA[1,3] with an IC50 of 3584 nM. GeXIVA[1,4] again fell in-between with an IC50 of 889 nM – 1.6-fold less potent than GeXIVA[1,2] and 4.0-fold more potent than GeXIVA[1,3]. The order of potency of the isomers ([1,2] > [1,4] > [1,3]) was again the same for both the hα7 and hα9α10 nAChRs. Unlike the hα9α10 and hα7 subtypes, the three isomers were equally potent against human muscle nAChR subtype (hα1β1δε) with IC50s of 497 nM ([1,2]), 485 nM ([1,3]), and 365 nM ([1,4]), < 1.4-fold different. GeXIVA[1,3] and GeXIVA[1,4] showed equal potency for hα6/α3β2β3, with IC50s of 149 nM and 199 nM, respectively. GeXIVA[1,2] was 2.6-fold less potent than GeXIVA[1,4], with an IC50 of 508 nM. On the other hand, GeXIVA[1,2] was 25-fold more potent at the hα9α10 nAChR than at hα7, hα1β1δε and hα6/α3β2β3. GeXIVA[1,3] showed similar activities on hα9α10 and hα6/α3β2β3, with closeIC50s; however, it was only weakly potent on hα7 – 31-fold less potent than hα9α10. GeXIVA[1,4] was around 4–19-fold more potent at the hα9α10 nAChR than hα7, hα1β1δε and hα6/α3β2β3. All three isomers of GeXIVA retained selectivity for hα9α10 but not the other nAChR subtypes.

3.4. Effect of the isomers of GeXIVA on rα9α10 nAChR mutants

Mutations within the N-terminal ligand-binding domain of the α9 and α10 subunits of the rat nAChR were used to probe the binding site of GeXIVA. The activity of 10 nAChR α9 and α10 mutants (rα9T56Iα10, rα9S6Nα10, rα9α10E61S, rα9α10D121L, rα9S117Aα10, rα9S14Nα10, rα9S136Nα10, rα9A24Kα10, rα9R71Gα10 and rα9E192Qα10) were measured for various concentrations of the three isomers (Table 3, Fig. 4). All three isomers potently blocked the 10 rα9α10 nAChR mutants, and only small differences in affinity were observed; IC50s were similar to those seen for hα9α10 and although the isomers were less potent than at wild-type rα9α10 nAChRs, they were still within a 10-fold difference. The most potent isomer, GeXIVA[1,2], blocked the 10 rα9α10 mutants with IC50 values ranging from 19.5 to 59.3 nM. IC50s for GeXIVA[1,4] ranged from 24 to 96 nM, and from 68–232 nM for GeXIVA[1,3]. The order of potency of the isomers, in terms of the rα9α10 mutants was [1,2] ≥ [1,4] > [1,3], identical to the trend for inhibition of the rat and human wild-type receptors.

Fig. 4.

Fig. 4

Open in a new tab

Concentration-response curves of the three αO-GeXIVA isomers for inhibition of rat α9α10 nAChR mutants. Values are mean ± SEM from 3–5 separate oocytes. IC50 values corresponding to each curve are provided in Table 3.

Most of the mutations involved residues of the rat receptor being substituted by those of the human receptor, and the absence of measurable effects of these mutations is consistent with the very similar activity of the peptides whatever the origin of the receptor. Fig. 5 shows the location of the mutated positions on the ligand-binding domain of α9α10 nAChR, highlighting that the selected positions cover a large area of the receptor. Because these positions are widespread and because the mutations caused similar changes of activity, the impacts of the mutations on activity are not highly definitive. The positions that were mutated might therefore either not be part of the interface with the peptide ligands, or might be at the peptide binding interface but with the particular amino acids chosen for mutation not measurably impacting the binding affinity.

Fig. 5.

Fig. 5

Open in a new tab

Spatial location of the positions that have been mutated in the α9 and α10 subunits relative to the two proposed binding modes of GeXIVA on the α9α10 nAChR. The ligand binding domain of the α9 and α10 subunits chosen for illustration are represented using a cartoon representation in green and grey, respectively. The positions that have been mutated in these subunits are represented using spheres. The positions the substitution of which led to ~10-fold or ~5-fold decrease binding of GeXIVA bead isomer are in red and blue, respectively. The transmembrane domain is illustrated as rectangles. All α9 subunits are in green and α10 subunits in grey. The two binding modes of GeXIVA that have been proposed previously are located in the pore and in the orthosteric binding α9(+)α10)(−). Ribbon GeXIVA is colored in orange.

Fig. 5 also displays the location of the two potential binding modes that were proposed previously for GeXIVA[1,4] (Luo et al., 2015). Only the two mutations rα9S6N and rα10D121L interrogate the proposed models, but mutations of these residues in the models lead to no change of hydrogen bonding and to a smaller than 4 Å2 difference in contact surface area. The buried surface areas were 2720 Å2 and 1700 Å2 for the binding mode in the agonist binding site and the site at the outskirts of the pore, respectively.

The mutations therefore cannot help prove or disprove the previously proposed models. Mutated positions rα9T56 and rα9S117 are located in the orthosteric binding site α10(+)α9(−), which is distinct from the previously proposed binding mode of GeXIVA[1,4] in the α9(+)α10(−) orthosteric site or in the pore. It was previously suggested that GeXIVA [1,2] did not compete for binding with conotoxin RgIA (Luo et al., 2015), which binds to the α10(+)α9(−) binding site (Azam et al., 2015). The mutation rα9T56I was shown to impact the activity of RgIA (Azam et al., 2015), but the same mutation here had no measurable effect, further supporting the idea that the GeXIVA isomers do not bind to the α10(+)α9(−) binding site.

4. Discussion

Both the bead and ribbon isomers of αO-CTx GeXIVA have previously been shown to significantly reduce mechanical hyperalgesia in the rat chronic constriction injury model of neuropathic pain without affecting motor performance (Li et al., 2016; Luo et al., 2015). In those studies, the analgesic effects of GeXIVA[1,2] and GeXIVA[1,4] occurred at low doses (≤ 2 nmol) without tolerance issues. Although, in both cases, a 2 nmol intramuscular dose produced effects greater than or equal to that observed with 500 nmol of the opioid analgesic morphine (Li et al., 2016; Luo et al., 2015), Li and colleagues observed the analgesic effects of GeXIVA[1,2] to be slightly better than GeXIVA[1,4] (Li et al., 2016). Furthermore, they reported that the pain relieving effect of GeXIVA[1,2] remained for up to 2 weeks after cessation of drug administration. Like GeXIVA, the α-CTxs RgIA and Vc1.1 are known to be potent antagonists of rα9α10 nAChRs, and their potential as human therapeutics is being investigated (Lewis et al., 2012; Livett et al., 2006; McIntosh et al., 2009; Vincler et al., 2006). When tested in rat models of neuropathic pain, both Vc1.1 and RgIA induced analgesia (Azam and McIntosh, 2012). However, when human and rat nAChRs were compared, RgIA was found to be over 300-fold less potent at the hα9α10 nAChR (Table 4) (Azam and McIntosh, 2012) whereas Vc1.1 was found to be around 400-fold less potent at the hα9α10 nAChR compared to the rat receptor (Table 4) (Luo et al., 2015). A molecular basis for the differential sensitivity of rat and human α9α10 nAChRs to RgIA was investigated (Azam and McIntosh, 2012), and it was found that the primary determinant of this disparity is a single amino acid – a Thr56 (rat) to Ile56 (human). The current study aimed to further explore differences between rat and human receptors.

Table 4.

Comparison of the IC50 values of αO- GeXIVA isomers, α- RgIA, α- Vc1.1, and αD-GeXXA at rat and human α9α10 nAChRs and at the rα9T56Iα10 mutant.

Peptide rα9α10
[IC50 (nM)]		 hα9α10
[IC50 (nM)]		 Ratioa
(human /rat)		 rα9T56Iα10
[IC50 (nM)]		 Ratiob
(rα9T56Iα10/
rα9α10)
αO-CTx GeXIVA[1,2] 4.61 20 4.3 19.5 4.2
αO-CTx GeXIVA[1,3] 22.7 116 5.1 80.6 3.6
αO-CTx GeXIVA[1,4] 7 47 6.7 29.5 4.2
α-CTx RgIA[10] 1.49 494 332 2560 1718
α-CTx Vc1.1 19c 7500 395 NA NA
αD-CTx GeXXA 1.2 28 23.3 NA NA
Open in a new tab
a

human α9α10 nAChR subtype IC50/ rat α9α10 IC50.

b

rat α9T56Iα10 nAChR mutant IC50/rat α9α10 IC50.

NA: not available.

Vc1.1 reached phase 2A human clinical trials but trials were halted with the discovery that Vc1.1 had lower affinity for the hα9α10 nAChR compared to the rα9α10 nAChR (http://www.evaluategroup.com/Universal/View.aspx?type=Story&id=135083, Lewis et al., 2012) and in the absence of a clear efficacy signal. Subsequent reports indicated that Vc1.1 has potent GABAB agonist activity (Berecki et al., 2014; Callaghan and Adams, 2010; Callaghan et al., 2008; Castro et al., 2016; Cuny et al., 2012; Huynh et al., 2015; Klimis et al., 2011; Nevin et al., 2007; van Lierop et al., 2013). These findings have prompted an ongoing debate as to whether the analgesic mechanism of action of the α-conotoxins is via α9α10 nAChRs and/ or GABAB receptors (Adams and Berecki, 2013; Mohammadi and Christie, 2014; Mohammadi and Christie, 2015; Wright et al., 2015). The pharmacological profile of GeXIVA coupled with its demonstrated analgesia in rat models of neuropathic pain has prompted further interest in α9α10 nAChRs and GeXIVA as a potential therapeutic agent (Luo et al., 2015, Li et al., 2016).

The present study supports a potential application of GeXIVA isomers in the treatment of neuropathic pain. All three isomers of GeXIVA have similar activities at the human and rat α9α10 nAChRs, with smaller than 5-fold differences (Table 4). They have mid- to low- nanomolar IC50 values (19.6–80.6 nM) for the rat α9α10 mutant rα9T56Iα10, which are similar to the values for the wild-type rat and human α9α10 receptors. In contrast, the IC50 of α-RgIA for rα9T56Iα10 is 2560 nM – 1700-fold less than for rα9α10. The potency of αD-CTx GeXXA (Xu et al., 2015) is 23.3-fold less for the human α9α10 receptor compared to the rat α9α10 receptor (Table 4). Thus, among all of the aforementioned peptides, GeXIVA[1,2] has the greatest affinity for hα9α10 nAChRs (IC50: 20 nM). This finding is consistent with the binding site of GeXIVA on rat and human α9α10 receptors being novel and distinct from that of α-RgIA, α-Vc1.1 and αD-GeXXA.

α-Conotoxins are competitive blockers of nAChRs, with the blocking site of α9α10 nAChRs located at the α10/α9 subunit interface. A species difference renders these conotoxins orders of magnitude less potent on the human vs. rat nAChR (Azam and McIntosh, 2012; Azam et al., 2015; Yu et al., 2013). In the present study, rat α9α10 nAChR mutants were used to compare the binding properties of the three isomers of GeXIVA (Fig. 4, Table 3) to those established for other conotoxins. All of the mutated rα9α10 receptors were found to have a similar affinity for the GeXIVA isomers as the wild-type rat and human α9α10 nAChRs, with a difference of < 14-fold for rat and <3-fold for human. This finding confirms that GeXIVA binds different regions of α9α10 than those identified in α-CTx RgIA and Vc1.1 and αD-CTx ligand-binding studies. Consistent with this, a previous study indicated that block by GeXVIA[1,2] was strongly voltage-dependent, suggesting that the toxin binding site might be allosterically coupled to a voltage-sensitive domain of the nAChR (Luo et al., 2015).

α-Conotoxins with a CC-C-C cysteine framework typically comprise 12–20-amino acids and display a globular disulfide connectivity linking Cys 1 to 3, and 2 to 4. The alternative connectivities – ribbon (Cys1–4, 2–3) and bead (Cys1–2, 3–4) – are generally associated with lower potency. As an example, the native globular isomer of α-conotoxin GI is active at mammalian muscle nAChRs with full biological activity; however, the bead isomer is 10-fold less potent and the ribbon isomer has little or no biological activity (Gehrmann et al., 1998). The ribbon α-GI isomer more readily rearranges to the native globular isomer than the bead isomer, according to stability studies in various assay buffers (Armishaw et al., 2010). In one case, conotoxin α-AuIB, an alternative isomer was shown to be more active at the α3β4 receptor if the receptor contains three α3 and two β4 subunits, but not if the pentamer is formed by assembly of two α3 and three β4 subunits (Grishin et al., 2010). The ribbon disulfide isomer α-AuIB has a less well-defined structure than the globular form (Dutton et al., 2002), and the unexpected activity of the ribbon isomer α-AuIB may be ascribed to the structural flexibility of the molecule, which may allow for a more complementary fit in the binding site.

A disulfide bond between adjacent cysteines is energetically unfavorable, as is the case for the αB-CTx VxXXIVA which has a C-CC-C framework. In the case of GeXIVA, the bead isomer with the 1–2, 3–4 connectivity (GeXIVA[1,2]) blocks rat α9α10 nAChRs, and is 5~6 fold more active than the 1–3, 2–4 (GeXIVA[1,3]) form. VxXXIVA is atypical among disulfide-bridged conotoxins because it has disordered structures in aqueous solution over a range of temperatures and pHs (Luo et al., 2013). In the current study, all three isomers of the GeXIVA (C-C-C-C framework), blocked rat and human α9α10 nAChRs, with the order of potency being bead (Cys1–2, 3–4, GeXIVA [1,2]) ≥ ribbon (Cys1–4, 2–3, GeXIVA [1,4]) > globular (Cys1–3, 2–4, GeXIVA [1,3]) (Table 5). In addition, the three isomers of GeXIVA exhibited differential sensitivity to various nAChRs (Table 1–4). The order of the isomers remained the same for rat and human α9α10 and rα7 subtypes as GeXIVA, which was also similar ([1,2] ≈ [1,3] ≈ [1,4]) for the hα1β1δε, mouse α1β1δε, rα6/α3β2β3 and rα3β2 subtypes. For the hα6/α3β2β3, rα2β4, rα4β4 and rα6/α3β4 subtypes, the order of potency of the isomers differed, with [1,3] ≈ [1,4] ≫ [1,2]; that is to say, the bead isomer GeXIVA[1,2] was the least potent, and the globular and ribbon isomers, GeXIVA[1,3] and [1,4], respectively, had similar potency. The different sensitivities to different nAChRs subtypes may be attributed to the three-dimensional structures of the isomers and corresponding receptors and their interactions. Although both the αO-CTx GeXIVA and α-conotoxins more generally contain four Cys residues, their frameworks are very different. αO-CTx GeXIVA has a ‘four separate’ Cys pattern (C-C-C-C), whereas α-conotoxins and αB-VxXXIVA have a ‘two adjacent and two separate’ Cys pattern (CC-C-C: α-CTx; C-CC-C: αB-CTx) (Table 5). The difference in the functions of the disulfide bond isomers is reflected in the differences between their sequences, Cys arrangements, and three-dimensional structures.

Table 5.

Comparison of the activity of the disulfide isomers of a selection of conotoxins with four cysteines and belonging to different gene superfamilies.

Name Gene
super
family		 Sequence Length
in amino
acids		 nAChR
subtype		 Isomer IC50 nM (activity) Potency ranking
of isomers		 Reference
B[1,2]a G[1,3]b R[1,4]c
αO-GeXIVA O T Inline graphicRSSGRY Inline graphicRSPYDRRRRY Inline graphicRRITDA Inline graphicV 28 rα9α10d 4.6 23 7 [1,2]>[1,4]>[1,3] This work
hα9α10 20 116 47
αB-VxXXIVA B VR Inline graphicLEKSGAQPNKLFRPP Inline graphicQKGPSFARHSR Inline graphicVYYTQSRE 40 rα9α10 1200 3900 >30000 [1,2]>[1,3]>[1,4] (Luo et al., 2013)
α-AuIB A G Inline graphicSYPP Inline graphicFATNPD Inline graphic# 15 rα3β4 (1:10)e n.d. 3000 NA [1,3] >[1,4] (Grishin et al., 2010)
rα3β4 (10:1) n.d. 1100 860 [1,4] >[1,3] (Grishin et al., 2010)
Open in a new tab
#

= C-terminus amidation,

a

B[1,2] or [1,2] is the “bead” isomer with a disulfide connectivity Cys1–Cys2, Cys3–Cys4 (C1–2, C3–4);

b

G[1,3] or [1,3] is the “globular” isomer with a disulfide connectivity Cys1–Cys3, Cys2–Cys4 (C1–3, C2–4);

c

R[1,4] or [1,4] is the “ribbon” isomer with a disulfide connectivity Cys1–Cys4, Cys2–Cys3 (C1–4, C2–3).

d

Data from Luo et al., 2015(Luo et al, 2015). E ratio of quantity of α3 and β4 mRNA injected in oocytes.

E

ratio of quantity of α3 and β4 mRNA injected in oocytes.

Abbreviations: rα9α10 and rα3β4 means rat α9α10 and α3β4; hα9α10 means human α9α10; n.d. is “not determined”; and NA is “not active”.

The α9α10 nAChR is an important target for the development of analgesics and cancer chemotherapeutics (Chen et al., 2011; Chikova et al., 2012; Klimis et al., 2011; Satkunanathan et al., 2005; Wu et al., 2011). The design and development of α*-conotoxin analogs with improved potency at human α9α10 nAChRs and that maintain selectivity over other nAChR subtypes, may especially assist in the treatment of neuropathic pain (Halai et al., 2009). The three isomers of GeXIVA have a unique selectivity among conotoxins as they potently inhibit both rat and human α9α10 nAChRs, potentially allowing a better translation of effects from animal models to the clinic.

Highlights.

  • αO-conotoxin GeXIVA is a potent antagonist of rat α9α10 nAChRs.

  • Three disulfide bond isomers of GeXIVA have differential sensitivity for various nAChRs.

  • The effects of the three isomers on 10 rat α9α10 nAChR mutants are similar.

  • The isomers retain potency and selectivity for the human α9α10 subtype.

  • The ‘bead’ isomer has the greatest affinity for hα9α10 nAChRs.

Acknowledgments

This work was supported, in part, by the Major International Joint Research Project of National Natural Science Foundation of China (81420108028), National Natural Science Foundation of China (41366002), the Major Science and Technology Project of Hainan Province (grant No. ZDKJ2016002), and Changjiang Scholars and Innovative Research Team in University Grant (IRT_15R15). This work was also supported by National Institutes of Health Grants GM103801 and GM48677 and a grant from the Australian Research Council (ARC; DP150103990). DJC is supported by an ARC Australian Laureate Fellowship (FL150100146).

We thank Layla Azam, Cheryl Dowell, Sean Christensen, Baldomero Olivera, Doju Yoshikami for their advice and help.

Abbreviations

CTx

conotoxin

nAChRs

nicotinic acetylcholine receptors

hα9α10

human α9α10

rα9α10

rat α9α10

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Conflict of interest

The sequence of GeXIVA is patented by Hainan University, with Sulan Luo, Dongting Zhangsun, Yong Wu, Xiaopeng Zhu, Yuanyan Hu and J. Michael McIntosh listed as inventors.

References

  1. Abraham MJ, Murtola T, Schulz R, Páll S, Smith JC, Hess B, Lindahl E. GROMACS: High performance molecular simulations through multi-level parallelism from laptops to supercomputers. SoftwareX. 2015;1–2:19–25. [Google Scholar]
  2. Adams DJ, Berecki G. Mechanisms of conotoxin inhibition of N-type (Ca(v)2.2) calcium channels. Biochim. Biophys. Acta. 2013;1828:1619–1628. doi: 10.1016/j.bbamem.2013.01.019. [DOI] [PubMed] [Google Scholar]
  3. Armishaw CJ, Dutton JL, Craik DJ, Alewood PF. Establishing regiocontrol of disulfide bond isomers of alpha-conotoxin ImI via the synthesis of N-to-C cyclic analogs. Biopolymers. 2010;94:307–313. doi: 10.1002/bip.21360. [DOI] [PubMed] [Google Scholar]
  4. Azam L, McIntosh JM. Molecular basis for the differential sensitivity of rat and human alpha9alpha10 nAChRs to alpha-conotoxin RgIA. J. Neurochem. 2012;122:1137–1144. doi: 10.1111/j.1471-4159.2012.07867.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Azam L, Papakyriakou A, Zouridakis M, Giastas P, Tzartos SJ, McIntosh JM. Molecular interaction of alpha-conotoxin RgIA with the rat alpha9alpha10 nicotinic acetylcholine receptor. Mol. Pharmacol. 2015;87:855–864. doi: 10.1124/mol.114.096511. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Berecki G, McArthur JR, Cuny H, Clark RJ, Adams DJ. Differential Cav2.1 and Cav2.3 channel inhibition by baclofen and alpha-conotoxin Vc1.1 via GABAB receptor activation. J. Gen. Physiol. 2014;143:465–479. doi: 10.1085/jgp.201311104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Callaghan B, Adams DJ. Analgesic alpha-conotoxins Vc1.1 and RgIA inhibit N-type calcium channels in sensory neurons of alpha9 nicotinic receptor knockout mice. Channels. 2010;4:51–54. doi: 10.4161/chan.4.1.10281. [DOI] [PubMed] [Google Scholar]
  8. Callaghan B, Haythornthwaite A, Berecki G, Clark RJ, Craik DJ, Adams DJ. Analgesic alpha-conotoxins Vc1.1 and Rg1A inhibit N-type calcium channels in rat sensory neurons via GABAB receptor activation. J. Neurosci. 2008;28:10943–10951. doi: 10.1523/JNEUROSCI.3594-08.2008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cartier GE, Yoshikami D, Gray WR, Luo S, Olivera BM, McIntosh JM. A new alpha-conotoxin which targets alpha3beta2 nicotinic acetylcholine receptors. J. Biol. Chem. 1996;271:7522–7528. doi: 10.1074/jbc.271.13.7522. [DOI] [PubMed] [Google Scholar]
  10. Craik DJ, Adams DJ, Brierley SM. alpha-Conotoxin Vc1.1 inhibits human dorsal root ganglion neuroexcitability and mouse colonic nociception via GABAB receptors. [17 February 2016];Gut. 2016 doi: 10.1136/gutjnl-2015-310971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Chen CS, Lee CH, Hsieh CD, Ho CT, Pan MH, Huang CS, Tu SH, Wang YJ, Chen LC, Chang YJ, Wei PL, Yang YY, Wu CH, Ho YS. Nicotine-induced human breast cancer cell proliferation attenuated by garcinol through down-regulation of the nicotinic receptor and cyclin D3 proteins. Breast Cancer Res. Treat. 2011;125:73–87. doi: 10.1007/s10549-010-0821-3. [DOI] [PubMed] [Google Scholar]
  12. Chikova A, Bernard HU, Shchepotin IB, Grando SA. New associations of the genetic polymorphisms in nicotinic receptor genes with the risk of lung cancer. Life Sci. 2012;91:1103–1108. doi: 10.1016/j.lfs.2011.12.023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Cuny H, de Faoite A, Huynh TG, Yasuda T, Berecki G, Adams DJ. gamma-Aminobutyric acid type B (GABAB) receptor expression is needed for inhibition of N-type (Cav2.2) calcium channels by analgesic alpha-conotoxins. J. Biol. Chem. 2012;287:23948–23957. doi: 10.1074/jbc.M112.342998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Davis J, Jones A, Lewis RJ. Remarkable inter- and intra-species complexity of conotoxins revealed by LC/MS. Peptides. 2009;30:1222–1227. doi: 10.1016/j.peptides.2009.03.019. [DOI] [PubMed] [Google Scholar]
  15. Dowell C, Olivera BM, Garrett JE, Staheli ST, Watkins M, Kuryatov A, Yoshikami D, Lindstrom JM, McIntosh JM. Alpha-conotoxin PIA is selective for alpha6 subunit-containing nicotinic acetylcholine receptors. J. Neurosci. 2003;23:8445–8452. doi: 10.1523/JNEUROSCI.23-24-08445.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Dutton JL, Bansal PS, Hogg RC, Adams DJ, Alewood PF, Craik DJ. A new level of conotoxin diversity, a non-native disulfide bond connectivity in alpha-conotoxin AuIB reduces structural definition but increases biological activity. J. Biol. Chem. 2002;277:48849–48857. doi: 10.1074/jbc.M208842200. [DOI] [PubMed] [Google Scholar]
  17. Gehrmann J, Alewood PF, Craik DJ. Structure determination of the three disulfide bond isomers of alpha-conotoxin GI: a model for the role of disulfide bonds in structural stability. J. Mol. Biol. 1998;278:401–415. doi: 10.1006/jmbi.1998.1701. [DOI] [PubMed] [Google Scholar]
  18. Grishin AA, Wang CI, Muttenthaler M, Alewood PF, Lewis RJ, Adams DJ. Alpha-conotoxin AuIB isomers exhibit distinct inhibitory mechanisms and differential sensitivity to stoichiometry of alpha3beta4 nicotinic acetylcholine receptors. J. Biol. Chem. 2010;285:22254–22263. doi: 10.1074/jbc.M110.111880. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Halai R, Clark RJ, Nevin ST, Jensen JE, Adams DJ, Craik DJ. Scanning mutagenesis of alpha-conotoxin Vc1.1 reveals residues crucial for activity at the alpha9alpha10 nicotinic acetylcholine receptor. J. Biol. Chem. 2009;284:20275–20284. doi: 10.1074/jbc.M109.015339. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Halai R, Craik DJ. Conotoxins: natural product drug leads. Nat. Prod. Rep. 2009;26:526–536. doi: 10.1039/b819311h. [DOI] [PubMed] [Google Scholar]
  21. Huynh TG, Cuny H, Slesinger PA, Adams DJ. Novel mechanism of voltage-gated N-type (Cav2.2) calcium channel inhibition revealed through alpha-conotoxin Vc1.1 activation of the GABA(B) receptor. Mol. Pharmacol. 2015;87:240–250. doi: 10.1124/mol.114.096156. [DOI] [PubMed] [Google Scholar]
  22. Kaas Q, Yu R, Jin AH, Dutertre S, Craik DJ. ConoServer: updated content, knowledge, and discovery tools in the conopeptide database. Nucleic Acids Res. 2012;40:D325–330. doi: 10.1093/nar/gkr886. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Klimis H, Adams DJ, Callaghan B, Nevin S, Alewood PF, Vaughan CW, Mozar CA, Christie MJ. A novel mechanism of inhibition of high-voltage activated calcium channels by alpha-conotoxins contributes to relief of nerve injury-induced neuropathic pain. Pain. 2011;152:259–266. doi: 10.1016/j.pain.2010.09.007. [DOI] [PubMed] [Google Scholar]
  24. Lewis RJ, Dutertre S, Vetter I, Christie MJ. Conus venom peptide pharmacology. Pharmacol. Rev. 2012;64:259–298. doi: 10.1124/pr.111.005322. [DOI] [PubMed] [Google Scholar]
  25. Lavergne V, Harliwong I, Jones A, Miller D, Taft RJ, Alewood PF. Optimized deep-targeted proteotranscriptomic profiling reveals unexplored Conus toxin diversity and novel cysteine frameworks. Proc.Natl. Acad. Sci. U. S. A. 2015;112:E3782–3791. doi: 10.1073/pnas.1501334112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Li X, Hu Y, Wu Y, Huang Y, Yu S, Ding Q, Zhangsun D, Luo S. Anti-hypersensitive effect of intramuscular administration of alphaO-conotoxin GeXIVA[1,2] and GeXIVA[1,4] in rats of neuropathic pain. Prog. Neuropsychopharmacol. Biol. Psychiatry. 2016;66:112–119. doi: 10.1016/j.pnpbp.2015.12.005. [DOI] [PubMed] [Google Scholar]
  27. Lindorff-Larsen K, Piana S, Palmo K, Maragakis P, Klepeis JL, Dror RO, Shaw DE. Improved side-chain torsion potentials for the Amber ff99SB protein force field. Proteins. 2010;78:1950–1958. doi: 10.1002/prot.22711. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Livett BG, Sandall DW, Keays D, Down J, Gayler KR, Satkunanathan N, Khalil Z. Therapeutic applications of conotoxins that target the neuronal nicotinic acetylcholine receptor. Toxicon. 2006;48:810–829. doi: 10.1016/j.toxicon.2006.07.023. [DOI] [PubMed] [Google Scholar]
  29. Luo S, Christensen S, Zhangsun D, Wu Y, Hu Y, Zhu X, Chhabra S, Norton RS, McIntosh JM. A novel inhibitor of alpha9alpha10 nicotinic acetylcholine receptors from Conus vexillum delineates a new conotoxin superfamily. PLoS One. 2013;8:e54648. doi: 10.1371/journal.pone.0054648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Luo S, Zhangsun D, Harvey PJ, Kaas Q, Wu Y, Zhu X, Hu Y, Li X, Tsetlin VI, Christensen S, Romero HK, McIntyre M, Dowell C, Baxter JC, Elmslie KS, Craik DJ, McIntosh JM. Cloning, synthesis, and characterization of alphaO-conotoxin GeXIVA, a potent alpha9alpha10 nicotinic acetylcholine receptor antagonist. Proc.Natl. Acad. Sci. U. S. A. 2015;112:E4026–4035. doi: 10.1073/pnas.1503617112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. McIntosh JM, Absalom N, Chebib M, Elgoyhen AB, Vincler M. Alpha9 nicotinic acetylcholine receptors and the treatment of pain. Biochem. Pharmacol. 2009;78:693–702. doi: 10.1016/j.bcp.2009.05.020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. McIntosh JM, Plazas PV, Watkins M, Gomez-Casati ME, Olivera BM, Elgoyhen AB. A novel alpha-conotoxin, PeIA, cloned from Conus pergrandis, discriminates between rat alpha9alpha10 and alpha7 nicotinic cholinergic receptors. J. Biol. Chem. 2005;280:30107–30112. doi: 10.1074/jbc.M504102200. [DOI] [PubMed] [Google Scholar]
  33. Mohammadi S, Christie MJ. alpha9-nicotinic acetylcholine receptors contribute to the maintenance of chronic mechanical hyperalgesia, but not thermal or mechanical allodynia. Mol. Pain. 2014;10:64. doi: 10.1186/1744-8069-10-64. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Mohammadi SA, Christie MJ. Conotoxin Interactions with alpha9alpha10-nAChRs: Is the alpha9alpha10-Nicotinic Acetylcholine Receptor an Important Therapeutic Target for Pain Management? Toxins. 2015;7:3916–3932. doi: 10.3390/toxins7103916. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Nevin ST, Clark RJ, Klimis H, Christie MJ, Craik DJ, Adams DJ. Are alpha9alpha10 nicotinic acetylcholine receptors a pain target for alpha-conotoxins? Mol. Pharmacol. 2007;72:1406–1410. doi: 10.1124/mol.107.040568. [DOI] [PubMed] [Google Scholar]
  36. Puillandre N, Duda TF, Meyer C, Olivera BM, Bouchet P. One, four or 100 genera? A new classification of the cone snails. J. Molluscan. Stud. 2015;81:1–23. doi: 10.1093/mollus/eyu055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Sali A, Blundell TL. Comparative protein modelling by satisfaction of spatial restraints. J. Mol. Biol. 1993;234:779–815. doi: 10.1006/jmbi.1993.1626. [DOI] [PubMed] [Google Scholar]
  38. Satkunanathan N, Livett B, Gayler K, Sandall D, Down J, Khalil Z. Alpha-conotoxin Vc1.1 alleviates neuropathic pain and accelerates functional recovery of injured neurones. Brain Res. 2005;1059:149–158. doi: 10.1016/j.brainres.2005.08.009. [DOI] [PubMed] [Google Scholar]
  39. Uribe JE, Puillandre N, Zardoya R. Beyond Conus: Phylogenetic relationships of Conidae based on complete mitochondrial genomes. Mol. Phylogenet. Evol. 2017;107:142–151. doi: 10.1016/j.ympev.2016.10.008. [DOI] [PubMed] [Google Scholar]
  40. van Lierop BJ, Robinson SD, Kompella SN, Belgi A, McArthur JR, Hung A, MacRaild CA, Adams DJ, Norton RS, Robinson AJ. Dicarba alpha-conotoxin Vc1.1 analogues with differential selectivity for nicotinic acetylcholine and GABAB receptors. ACS Chem. Biol. 2013;8:1815–1821. doi: 10.1021/cb4002393. [DOI] [PubMed] [Google Scholar]
  41. Vincler M, Wittenauer S, Parker R, Ellison M, Olivera BM, McIntosh JM. Molecular mechanism for analgesia involving specific antagonism of alpha9alpha10 nicotinic acetylcholine receptors. Proc. Natl. Acad. Sci. U. S. A. 2006;103:17880–17884. doi: 10.1073/pnas.0608715103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Walker CS, Steel D, Jacobsen RB, Lirazan MB, Cruz LJ, Hooper D, Shetty R, DelaCruz RC, Nielsen JS, Zhou LM, Bandyopadhyay P, Craig AG, Olivera BM. The T-superfamily of conotoxins. J. Biol. Chem. 1999;274:30664–30671. doi: 10.1074/jbc.274.43.30664. [DOI] [PubMed] [Google Scholar]
  43. Wright AB, Norimatsu Y, McIntosh JM, Elmslie KS. Limited efficacy of alpha-conopeptides, Vc1.1 and RgIA, to inhibit sensory neuron CaV current. eNeuro. 2015 doi: 10.1523/ENEURO.0057-14.2015. 2.0057-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Wu CH, Lee CH, Ho YS. Nicotinic acetylcholine receptor-based blockade: applications of molecular targets for cancer therapy. Clin. Cancer Res. 2011;17:3533–3541. doi: 10.1158/1078-0432.CCR-10-2434. [DOI] [PubMed] [Google Scholar]
  45. Xu S, Zhang T, Kompella SN, Yan M, Lu A, Wang Y, Shao X, Chi C, Adams DJ, Ding J, Wang C. Conotoxin alphaD-GeXXA utilizes a novel strategy to antagonize nicotinic acetylcholine receptors. Sci. Rep. 2015;5:14261. doi: 10.1038/srep14261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Yu R, Kompella SN, Adams DJ, Craik DJ, Kaas Q. Determination of the alpha-conotoxin Vc1.1 binding site on the alpha9alpha10 nicotinic acetylcholine receptor. J. Med. Chem. 2013;56:3557–3567. doi: 10.1021/jm400041h. http://www.evaluategroup.com/Universal/View.aspx?type=Story&id=135083. [DOI] [PubMed] [Google Scholar]

RESOURCES