Chimeric analysis of a neuronal nicotinic acetylcholine receptor reveals amino acids conferring sensitivity to α-bungarotoxin
Chimeric analysis of a neuronal nicotinic acetylcholine receptor reveals amino acids conferring sensitivity to α-bungarotoxin
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DOI：
10.1074/jbc.274.37.26113
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发表时间：
1999-09-10
影响因子：
4.8
通讯作者：
Hawrot, E
中科院分区：
文献类型：
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作者：
Levandoski, MM;Lin, YX;Hawrot, E
我们通过嵌合分析两个同源的α亚基，一个对α-BGTX高度敏感(α1)，另一个不敏感(α3)，研究了Cy-银环蛇毒素(αBGTX)与烟碱型乙酰胆碱受体结合的分子决定因素。通过用鱼雷α1亚基的相应区域替换RAT(α3)残基184-191，我们在(α3亚基)中引入了5个α1残基(Trp-184、Trp-187、Val-188、Tyr-189和Thr-Lgl)的簇。在非洲爪哇卵母细胞中共表达嵌合的α3亚基(α3/α1[5])和大鼠β2或β4亚基后，评估其功能活性和αBGTX敏感性。含α3/α1[5]受体的激动剂诱导的反应可被纳摩尔亲和力IC50值为41 nM的αBGTX阻断(α3/α1[5]β2为41 nM，α3/α1[5]β4为19 nM)。此外，含有单点突变α3K189Y的受体对αBGTX阻断ICS具有显著的敏感性，值：α3K189Yβ2为186 nM，(α3K189Yβ4)为179 nM，另一个α3嵌合亚单位，α3/α7[6]，类似于α3/α1[5]，但整合了来自αBGTX敏感Cur亚单位的相应残基，当与β4亚单位共表达时，也赋予了αBGTX对嵌合受体的高度敏感性(IC50值=31 nM)。我们的研究结果表明，在α-BGTX与烟碱型乙酰胆碱受体的相互作用中，α-BGTX敏感亚基A1和α7之间的第184-191位残基起着关键的功能作用。
We have investigated the molecular determinants responsible for cy-bungarotoxin (alpha Bgtx) binding to nicotinic acetylcholine receptors through chimeric analysis of two homologous alpha subunits, one highly sensitive to alpha Bgtx block (alpha 1) and the other, alpha Bgtx-insensitive (alpha 3). By replacing rat (alpha 3) residues 184-191 with the corresponding region from the Torpedo alpha 1 subunit, we introduced a cluster of five alpha 1 residues (Trp-184, Trp-187, Val-188, Tyr-189, and Thr-lgl) into the (alpha 3 subunit. Functional activity and alpha Bgtx sensitivity were assessed following co-expression in Xenopus oocytes of the chimeric alpha 3 subunit (alpha 3/alpha 1[5]) with either rat beta 2 or beta 4 subunits. Agonist-evoked responses of alpha 3/alpha 1[5]-containing receptors were blocked by alpha Bgtx with nanomolar affinity IC50 values: 41 nM for (alpha 3/alpha 1[5]beta 2 and 19 nM for alpha 3/alpha 1[5]beta 4). Furthermore, receptors containing the single point mutation alpha 3K189Y acquire significant sensitivity to alpha Bgtx block ICS, values: 186 nM for alpha 3K189Y beta 2 and 179 nM for (alpha 3K189Y beta 4), Another alpha 3 chimeric subunit, alpha 3/alpha 7[6], similar to alpha 3/alpha 1[5] but incorporating the corresponding residues from the alpha Bgtx-sensitive cur subunit, also conferred potent alpha Bgtx sensitivity to chimeric receptors when co-expressed with the beta 4 subunit (IC50 value = 31 nM). Our findings demonstrate that the residues between positions 184 and 191 of the alpha Bgtx-sensitive subunits al and alpha 7 play a critical functional role in the interaction of alpha Bgtx with nicotinic acetylcholine receptors sensitive to this toxin.