Probing function in ligand-gated ion channels without measuring ion transport.
Probing function in ligand-gated ion channels without measuring ion transport.
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DOI：
10.1085/jgp.202213082
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发表时间：
2022-06-06
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影响因子：
--
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Godellas和Grosman重新使用配体结合测定来研究五聚体配体门控离子通道（pLGIC）。它们表明配体结合亲和力不受结合位点占用的影响，并且跨膜结构域的变化不太可能影响与细胞外结构域的结合。虽然离子通道的功能特性最准确地评估使用电生理方法，一些实验情况下，需要替代方法。在这里，工作的五聚体配体门控离子通道（pLGIC）超家族的成员，我们专注于实际的实施，并从平衡型配体结合试验的结果的解释。配体结合的研究pLGICs绝不是新的，但缺乏统一的出版协议，不同的群体获得的结果之间的巨大差异，并放置在实验观察的简单的理论考虑的约束一般无视，这一经典技术的彻底分析是为了。为此，我们提出了一个详细的实践和理论研究，这种类型的测定使用放射性标记的α-银环蛇毒素，未标记的小分子胆碱能配体，人同聚体α7-AChR，并在一个现实的五个结合位点的反应方案的框架内进行了广泛的计算。此外，我们展示了这种方法的实际应用的例子，以解决两个长期存在的问题，在该领域：我们的研究结果表明，配体结合亲和力是不敏感的结合位点占用和突变的跨膜结构域中的氨基酸残基是不太可能影响通道的亲和力的配体结合到胞外结构域。
Godellas and Grosman revisit the use of ligand-binding assays to study pentameric ligand-gated ion channels (pLGICs). They show that ligand-binding affinity is unaffected by binding-site occupancy and that changes to the transmembrane domain are unlikely to affect binding to the extracellular domain. Although the functional properties of ion channels are most accurately assessed using electrophysiological approaches, a number of experimental situations call for alternative methods. Here, working on members of the pentameric ligand-gated ion channel (pLGIC) superfamily, we focused on the practical implementation of, and the interpretation of results from, equilibrium-type ligand-binding assays. Ligand-binding studies of pLGICs are by no means new, but the lack of uniformity in published protocols, large disparities between the results obtained for a given parameter by different groups, and a general disregard for constraints placed on the experimental observations by simple theoretical considerations suggested that a thorough analysis of this classic technique was in order. To this end, we present a detailed practical and theoretical study of this type of assay using radiolabeled α-bungarotoxin, unlabeled small-molecule cholinergic ligands, the human homomeric α7-AChR, and extensive calculations in the framework of a realistic five-binding-site reaction scheme. Furthermore, we show examples of the practical application of this method to tackle two longstanding questions in the field: our results suggest that ligand-binding affinities are insensitive to binding-site occupancy and that mutations to amino-acid residues in the transmembrane domain are unlikely to affect the channel’s affinities for ligands that bind to the extracellular domain.
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影响因子：
64.5
作者：
Noviello CM;Gharpure A;Mukhtasimova N;Cabuco R;Baxter L;Borek D;Sine SM;Hibbs RE
通讯作者：
Hibbs RE
影响因子：
1.7
作者：
Bertrand, S;DevillersThiery, A;Bertrand, D
通讯作者：
Bertrand, D
影响因子：
16.2
作者：
Gu, Shenyan;Matta, Jose A.;Bredt, David S.
通讯作者：
Bredt, David S.
影响因子：
3.3
作者：
LEE, CY
通讯作者：
LEE, CY
DOI：
10.1073/pnas.251402498
发表时间：
2001-11-20
影响因子：
11.1
作者：
Grosman, C;Auerbach, A
通讯作者：
Auerbach, A