alpha-conotoxin AuIB selectively blocks alpha3 beta4 nicotinic acetylcholine receptors and nicotine-evoked norepinephrine release

Abstract

Neuronal nicotinic acetylcholine receptors (nAChRs) with putative alpha3 beta4-subunits have been implicated in the mediation of signaling in various systems, including ganglionic transmission peripherally and nicotine-evoked neurotransmitter release centrally. However, progress in the characterization of these receptors has been hampered by a lack of alpha3 beta4-selective ligands. In this report, we describe the purification and characterization of an alpha3 beta4 nAChR antagonist, alpha-conotoxin AuIB, from the venom of the "court cone," Conus aulicus. We also describe the total chemical synthesis of this and two related peptides that were also isolated from the venom. alpha-Conotoxin AuIB blocks alpha3 beta4 nAChRs expressed in Xenopus oocytes with an IC50 of 0.75 microM, a kon of 1.4 x 10(6) min-1 M-1, a koff of 0.48 min-1, and a Kd of 0.5 microM. Furthermore, alpha-conotoxin AuIB blocks the alpha3 beta4 receptor with >100-fold higher potency than other receptor subunit combinations, including alpha2 beta2, alpha2 beta4, alpha3 beta2, alpha4 beta2, alpha4 beta4, and alpha1 beta1 gamma delta. Thus, AuIB is a novel, selective probe for alpha3 beta4 nAChRs. AuIB (1-5 microM) blocks 20-35% of the nicotine-stimulated norepinephrine release from rat hippocampal synaptosomes, whereas nicotine-evoked dopamine release from striatal synaptosomes is not affected. Conversely, the alpha3 beta2-specific alpha-conotoxin MII (100 nM) blocks 33% of striatal dopamine release but not hippocampal norepinephrine release. This suggests that in the respective systems, alpha3 beta4-containing nAChRs mediate norepinephrine release, whereas alpha3 beta2-containing receptors mediate dopamine release.

Fig. 1.

A, C. aulicus. Cone snails are venomous marine predators. C. aulicus is found in coral reefs and sand substrates in the Indo-Pacific (except Hawaii) and hunts primarily gastropods but also small fish. For the first time, nicotinic antagonists were isolated from its venom.B, Purification of AuI α-conotoxins by RPLC.Panel 1, Filtrate of venom extract was loaded onto a semipreparative Vydac C₁₈ column with 100% buffer A and eluted with a gradient of 5–65% buffer B per hour. Flow rate was 5 ml/min. Panel 2, Sixteen percent of the material eluting in the position indicated by the arrow in panel 1 was diluted with 2 vol of 0.1% TFA and repurified on an analytical Vydac C₁₈ column, using a flow rate of 1 ml/min. The gradient was 25–30% buffer B for 5 min and then 30–55% buffer B for 50 min. Panel 3, Fractions indicated in panel 2 were rechromatographed as described to obtain the final purified products. Although AuIB is well separated, AuIA and AuIC nearly co-elute. A 5-ml-sample loading loop was used in all chromatography. Buffer A = 0.1% TFA; buffer B = 0.1% TFA, 90% acetonitrile (panel 1) and 0.1% TFA, 60% acetonitrile for all other purifications steps. Absorbance was monitored at 280 nm.

Fig. 2.

A, α-Conotoxin AuIA, AuIB, and AuIC block ACh responses in oocytes expressing α3β4 nAChRs.Xenopus oocytes expressing α3β4 nAChRs were voltage-clamped, and the responses to 1 sec pulses of ACh were monitored before exposure to toxin and during equilibrium exposure to 3 μm α-conotoxins. Note that the block by α-conotoxin AuIB is the greatest of the three peptides. B, AuIB (10 μm) reversibly blocks 95% of the ACh response. Peptide application and washout are indicated by the bars.

Fig. 3.

Kinetics of block by α-conotoxin AuIB.A, α-Conotoxin AuIB (300 nm) was perfused onto an oocyte expressing α3β4 receptors while the responses to 1 sec applications of ACh were measured. B, After maximal block was achieved α-conotoxin AuIB was washed out. Solid lines are single exponential curves that best fit the data.

Fig. 4.

Selectivity of α-conotoxin AuIB.A, AuIB blocks α3β4 but not muscle nAChR. The peptide AuIB (3 μm) blocks 84 ± 2% of the ACh response of α3β4 receptors (n = 10). In contrast, it fails to inhibit the α1β1γδ (muscle) receptor. B, α-conotoxin AuIB preferentially blocks α3β4 versus other nAChR subunit combinations. The dose–response curve shows that α-conotoxin AuIB blocks α3β4 receptors with an IC₅₀ of 0.75 μm, (n_H is 1.05). By comparison, AuIB is ∼10-fold (α7) and >100-fold less potent on other nAChR subtypes. Each data point represents the average of three to five oocytes. Error bars are SEM. SEM is not shown for thetop right five data points for figure clarity, but is less than 5% of the mean in each of these cases.

Fig. 5.

AuIB blocks nicotine-stimulated norepinephrine, but not dopamine, release. A, AuIB blocks norepinephrine release from rat hippocampal synaptosomes. In contrast, α-CTx MII (α3β2-selective), α-CTx ImI (α7-selective), and α-CTx MI (α1βγδ-selective) all fail to block release. B,Conversely, α-CTx MII, but not α-CTx AuIB, α-CTx ImI, or α-CTx MI, blocks dopamine release from rat striatal synaptosomes. *p ≤ 0.001. Data are from 3–10 experiments with three to six replicates within each experiment. Norepinephrine release: 1 μm AuIB, p = 0.001; 5 μm AuIB, p < 0.001; MII,p = 0.75; ImI, p = 0.64; MI,p = 0.5. Dopamine release: 1 μm AuIB,p = 0.93; 5 μm AuIB,p = 0.68; MII, p < 0.001; ImI,p = 0.24; MI, p = 0.85.