Vafa B, Lewis T M, Cunningham A M, Jacques P, Lynch J W, Schofield P R
The Garvan Institute of Medical Research, Sydney, New South Wales, Australia.
J Neurochem. 1999 Nov;73(5):2158-66.
Four discontinuous extracellular sequence domains have been proposed to form the ligand binding sites of the ligand-gated ion channel receptor superfamily. In this study, we investigated the role of 12 contiguous residues of the inhibitory glycine receptor that define the proposed "loop A" ligand binding domain. Using the techniques of site-directed mutagenesis and patch-clamp electrophysiology, four of the 12 residues were shown to have impaired ligand binding. Three mutants, 193A, A101H, and N102A, resulted in significant (17-44-fold) increases in the agonist EC50 values as compared with the wild-type glycine receptor, whereas Hill coefficients, ImaX values, and antagonist affinity remained largely unaffected. Consideration of receptor efficacy values indicates that these residues are involved in ligand binding rather than channel activation. A fourth mutant, W94A, failed to give rise to any glycine-activated currents, although cell-surface expression was observed, suggesting that this residue may also be involved in agonist binding. These data provide the most extensive characterization of the loop A ligand binding domain available to date and define two new residue locations, Ile93 and Asn102, as contributing to the four-loop model of ligand binding.
四个不连续的细胞外序列结构域被认为构成了配体门控离子通道受体超家族的配体结合位点。在本研究中，我们研究了抑制性甘氨酸受体中12个连续残基的作用，这些残基定义了所谓的“A环”配体结合结构域。使用定点诱变和膜片钳电生理学技术，发现12个残基中的4个残基的配体结合受损。与野生型甘氨酸受体相比，三个突变体193A、A101H和N102A导致激动剂EC50值显著（17 - 44倍）增加，而希尔系数、最大电流值和拮抗剂亲和力基本未受影响。对受体效能值的考虑表明，这些残基参与配体结合而非通道激活。第四个突变体W94A尽管观察到细胞表面表达，但未能产生任何甘氨酸激活电流，这表明该残基也可能参与激动剂结合。这些数据提供了迄今为止对A环配体结合结构域最全面的表征，并确定了两个新的残基位置，即Ile93和Asn102，它们有助于配体结合的四环模型。
