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首页> 外文期刊>Biochemistry >Critical residues influence the affinity and selectivity of alpha-conotoxin MI for nicotinic acetylcholine receptors.
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Critical residues influence the affinity and selectivity of alpha-conotoxin MI for nicotinic acetylcholine receptors.

机译：关键残基影响α-芋螺毒素MI对烟碱乙酰胆碱受体的亲和力和选择性。

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摘要

The mammalian skeletal muscle acetylcholine receptor contains two nonequivalent acetylcholine binding sites, one each at the alpha/delta and alpha/gamma subunit interfaces. Alpha-Conotoxin MI, a 14-amino acid competitive antagonist, binds at both interfaces but has approximately 10(4) higher affinity for the alpha/delta site. We performed an "alanine walk" to identify the residues in alpha-MI that contribute to this selective interaction with the alpha/delta site. Electrophysiological measurements with Xenopus oocytes expressing normal receptors or receptors lacking either the gamma or delta subunit were made to assay toxin-receptor interaction. Alanine substitutions in most amino acid positions had only modest effects on toxin potency at either binding site. However, substitutions in two positions, proline-6 and tyrosine-12, dramatically reduced toxin potency at the high-affinity alpha/delta site while having comparatively little effect on low-affinity alpha/gamma binding. When tyrosine-12 was replaced by alanine, the toxin's selectivity for the high-affinity site (relative to that for the low-affinity site) was reduced from 45,000- to 30-fold. A series of additional amino acid substitutions in this position showed that increasing side chain size/hydrophobicity increases toxin potency at the alpha/delta site without affecting alpha/gamma binding. In contrast, when tyrosine-12 is diiodinated, toxin binding is nearly irreversible at the alpha/delta site but also increases by approximately 500-fold at the alpha/gamma site. The effects of position 12 substitutions are accounted for almost entirely by changes in the rate of toxin dissociation from the high-affinity alpha/delta binding site.
机译：哺乳动物骨骼肌乙酰胆碱受体含有两个非等价的乙酰胆碱结合位点，每个在α/δ和α/γ亚基界面。 Alpha-芋螺毒素MI，一种14个氨基酸的竞争性拮抗剂，在两个界面上均结合，但对α/δ位点的亲和力大约高10（4）。我们进行了“丙氨酸行走”，以鉴定α-MI中有助于与α/δ位点选择性相互作用的残基。用表达正常受体或缺乏γ或δ亚基的受体的爪蟾卵母细胞进行电生理学测量以测定毒素-受体相互作用。在大多数氨基酸位置上的丙氨酸取代对任一结合位点上的毒素效力仅具有中等影响。但是，脯氨酸6和酪氨酸12这两个位置的取代显着降低了高亲和力α/δ位点的毒素效力，而对低亲和力α/γ结合的影响却很小。当酪氨酸12被丙氨酸替代时，毒素对高亲和力位点的选择性（相对于低亲和力位点的选择性）从45,000降低到30倍。在该位置的一系列其他氨基酸取代表明，增加侧链大小/疏水性可增加α/δ位点的毒素效力，而不会影响α/γ结合。相反，当酪氨酸12被二碘化时，毒素结合在α/δ位点几乎是不可逆的，但在α/γ位点也增加了约500倍。位置12取代的影响几乎完全由毒素从高亲和力α/δ结合位点解离的速率所引起。

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