Luo S, Kulak J M, Cartier G E, Jacobsen R B, Yoshikami D, Olivera B M, McIntosh J M
Department of Biology, University of Utah, Salt Lake City, Utah 84112, USA.
J Neurosci. 1998 Nov 1;18(21):8571-9. doi: 10.1523/JNEUROSCI.18-21-08571.1998.
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.
具有假定的α3β4亚基的神经元烟碱型乙酰胆碱受体（nAChRs）已被认为参与多种系统中的信号传导，包括外周神经节传递以及中枢尼古丁诱发的神经递质释放。然而，由于缺乏α3β4选择性配体，这些受体的特性研究进展受到阻碍。在本报告中，我们描述了从“宫廷芋螺”（Conus aulicus）毒液中纯化和鉴定一种α3β4 nAChR拮抗剂α-芋螺毒素AuIB的过程。我们还描述了该肽以及另外两种也从毒液中分离出的相关肽的全化学合成。α-芋螺毒素AuIB阻断非洲爪蟾卵母细胞中表达的α3β4 nAChRs，其IC50为0.75微摩尔，kon为1.4×10⁶分钟⁻¹摩尔⁻¹，koff为0.48分钟⁻¹, Kd为0.5微摩尔。此外，α-芋螺毒素AuIB阻断α3β4受体的效力比其他受体亚基组合（包括α2β2、α2β4、α3β2、α4β2、α4β4和α1β1γδ）高100倍以上。因此，AuIB是一种新型的、针对α3β4 nAChRs的选择性探针。AuIB（1 - 5微摩尔）阻断大鼠海马突触体中尼古丁刺激的去甲肾上腺素释放的20 - 35%，而纹状体突触体中尼古丁诱发的多巴胺释放不受影响。相反，α3β2特异性的α-芋螺毒素MII（100纳摩尔）阻断33%的纹状体多巴胺释放，但不影响海马去甲肾上腺素释放。这表明在各自的系统中，含α3β4的nAChRs介导去甲肾上腺素释放，而含α3β2的受体介导多巴胺释放。
