McIntosh J Michael, Azam Layla, Staheli Sarah, Dowell Cheryl, Lindstrom Jon M, Kuryatov Alexander, Garrett James E, Marks Michael J, Whiteaker Paul
Department of Psychiatry, University of Utah, Salt Lake City, UT 84112, USA.
Mol Pharmacol. 2004 Apr;65(4):944-52. doi: 10.1124/mol.65.4.944.
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 IC(50) at alpha6beta4 versus alpha3beta4 nAChRs. The peptide had a 590-fold lower IC(50) for alpha6/alpha3beta2 versus alpha3beta2 and a 2020-fold lower IC(50) 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 (IC(50) = 2.4 nM) correlated well with the inhibition constant of MII[H9A;L15A] for [(125)I]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.
神经元烟碱型乙酰胆碱受体（nAChRs）既介导直接的胆碱能突触传递，也调节其他神经递质的突触传递。需要新型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，但在区分一些含有密切相关的α3和α6亚基的亚型时效果不佳。对α-芋螺毒素MII进行了结构-功能分析，试图生成对含α6的[α6()（星号表示可能存在其他亚基）]nAChRs具有偏好性的类似物。丙氨酸取代产生了几种在非洲爪蟾卵母细胞中异源表达的α3()与α6() nAChRs上活性降低的类似物。从最初的类似物开始，合成了一系列有两个丙氨酸取代的突变体。His9和Leu15处的取代（MII[H9A;L15A]）导致α6β4相对于α3β4 nAChRs的半数抑制浓度（IC(50)）降低了29倍。该肽对α6/α3β2相对于α3β2的IC(50)低590倍，对α6/α3β2β3相对于α3β2 nAChRs的IC(50)低2020倍。MII[H9A;L15A]在α2β2、α2β4、α3β4、α4β2、α4β4和α7 nAChRs上几乎没有活性。MII[H9A;L15A]对大鼠α6/α3β2β3 nAChRs的功能阻断（IC(50) = 2.4 nM）与MII[H9A;L15A]对小鼠脑匀浆中假定的α6β2() nAChRs上[(125)I]α-芋螺毒素MII结合的抑制常数（K(i) = 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用于区分含α3和α6亚基的nAChRs的新型选择性拮抗剂。
