Analogs of α-conotoxin MII are selective for α6-containing nicotinic acetylcholine receptors
Analogs of α-conotoxin MII are selective for α6-containing nicotinic acetylcholine receptors
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DOI：
10.1124/mol.65.4.944
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发表时间：
2004-04-01
影响因子：
3.6
通讯作者：
Whiteaker, P
中科院分区：
文献类型：
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作者：
McIntosh, JM;Azam, L;Whiteaker, P
神经元烟碱乙酰胆碱受体（nAChRs）既介导直接胆碱能突触传递，又通过其他神经递质调节突触传递。需要新的配体作为探针来区分结构相关的nAChR亚型。α-芋螺毒素MII是一种能区分多种nAChR亚型的选择性配体，但不能很好地区分含有密切相关的α 3和α 6亚基的某些亚型。进行α-芋螺毒素MII的结构-功能分析以试图产生优先于含α 6的[α 6 *（标记指示可能存在另外的亚基）] nAChR的类似物。丙氨酸取代导致在非洲爪蟾卵母细胞中异源表达的α 3 * 与α 6 * nAChRs活性降低的几种类似物。从最初的类似物，合成了一系列具有两个丙氨酸取代的突变。His 9和Leu 15（MII[H9 A; L15 A]）的取代导致α 6 β 4的IC 50比α 3 β 4 nAChR低29倍。该肽对α 6/α 3 β 2的IC 50比α 3 β 2低590倍，对α 6/α 3 β 2 β 3的IC 50比α 3 β 2 nAChR低2020倍。MII[H9 A; L15 A]对α 2 β 2、α 2 β 4、α 3 β 4、α 4 β 2、α 4 β 4和α 7 nAChR几乎没有活性或没有活性。MII[H9 A; L15 A]对大鼠α 6/α 3 β 2 β 3 nAChR的功能性阻断（IC 50 = 2.4 nM）与MII[H9 A; L15 A]对[I-125] α-芋螺毒素MII与小鼠脑匀浆中推定的α 6 β 2 * nAChR结合的抑制常数（Ki = 3.3 nM）密切相关。因此，α-芋螺毒素MII的结构-功能分析使得能够创建新型选择性拮抗剂，用于区分含有α 3和α 6亚基的nAChR。
Neuronal nicotinic acetylcholine receptors (nAChRs) both mediate direct cholinergic synaptic transmission and modulate synaptic transmission by other neurotransmitters. Novel ligands are needed as probes to discriminate among structurally related nAChR subtypes. alpha-Conotoxin MII, a selective ligand that discriminates among a variety of nAChR subtypes, fails to discriminate well between some subtypes containing the closely related alpha3 and alpha6 subunits. Structure-function analysis of alpha-conotoxin MII was performed in an attempt to generate analogs with preference for alpha6-containing [alpha6* (asterisks indicate the possible presence of additional subunits)] nAChRs. Alanine substitution resulted in several analogs with decreased activity at alpha3* versus alpha6* nAChRs heterologously expressed in Xenopus laevis oocytes. From the initial analogs, a series of mutations with two alanine substitutions was synthesized. Substitution at His9 and Leu15 (MII[H9A;L15A]) resulted in a 29-fold lower IC50 at alpha6beta4 versus alpha3beta4 nAChRs. The peptide had a 590-fold lower IC50 for alpha6/alpha3beta2 versus alpha3beta2 and a 2020-fold lower IC50 for alpha6/alpha3beta2beta3 versus alpha3beta2 nAChRs. MII[H9A;L15A] had little or no activity at alpha2beta2, alpha2beta4, alpha3beta4, alpha4beta2, alpha4beta4, and alpha7 nAChRs. Functional block by MII[H9A;L15A] of rat alpha6/alpha3beta2beta3 nAChRs (IC50 = 2.4 nM) correlated well with the inhibition constant of MII[H9A;L15A] for [I-125]alpha-conotoxin MII binding to putative alpha6beta2* nAChRs in mouse brain homogenates (K-i = 3.3 nM). Thus, structure- function analysis of alpha-conotoxin MII enabled the creation of novel selective antagonists for discriminating among nAChRs containing alpha3 and alpha6 subunits.