Delta selective compound 2 (DS2; 4-chloro-N-[2-(2-thienyl)imidazo[1,2-a]pyridin-3-yl]benzamide) is one of the most widely used tools to study selective actions mediated by δ-subunit–containing GABA_A receptors. DS2 was discovered over 10 years ago, but despite great efforts, the precise molecular site of action has remained elusive. Using a combination of computational modeling, site-directed mutagenesis, and cell-based pharmacological assays, we probed three potential binding sites for DS2 and analogs at α₄β₁δ receptors: an α₄⁽⁺⁾δ⁽⁻⁾ interface site in the extracellular domain (ECD), equivalent to the diazepam binding site in αβγ₂ receptors, and two sites in the transmembrane domain (TMD) - one in the α₄⁽⁺⁾β₁⁽⁻⁾ and one in the α₄⁽⁻⁾β₁⁽⁺⁾ interface, with the α₄⁽⁻⁾β₁⁽⁺⁾ site corresponding to the binding site for etomidate and a recently disclosed low-affinity binding site for diazepam. We show that mutations in the ECD site did not abrogate DS2 modulation. However, mutations in the TMD α₄⁽⁺⁾β₁⁽⁻⁾ interface, either α₄(S303L) of the α₄⁽⁺⁾ side or β₁(I289Q) of the β₁⁽⁻⁾ side, convincingly disrupted the positive allosteric modulation by DS2. This was consistently demonstrated both in an assay measuring membrane potential changes and by whole-cell patch-clamp electrophysiology and rationalized by docking studies. Importantly, general sensitivity to modulators was not compromised in the mutated receptors. This study sheds important light on the long-sought molecular recognition site for DS2, refutes the misconception that the selectivity of DS2 for δ-containing receptors is caused by a direct interaction with the δ-subunit, and instead points toward a functional selectivity of DS2 and its analogs via a surprisingly well conserved binding pocket in the TMD.

中文翻译：

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基于 δ 选择性化合物 2 活性的分子决定因素，含 δ 的 GABAA 受体

Delta 选择性化合物 2（DS2；4-氯-N- [2-（2-噻吩基）咪唑并[1,2 - a ]吡啶-3-基]苯甲酰胺）是研究介导的选择性作用的最广泛使用的工具之一通过含有δ亚基的 GABA _A受体。DS2 是在 10 多年前被发现的，但尽管付出了巨大的努力，但其作用的精确分子位点仍然难以捉摸。结合计算建模、定点诱变和基于细胞的药理学分析，我们在α ₄ β ₁ δ受体上探测了 DS2 和类似物的三个潜在结合位点：α ₄ ⁽⁺⁾ δ ^(-)细胞外域 (ECD) 中的界面位点，相当于αβγ ₂受体中的地西泮结合位点，以及跨膜域 (TMD) 中的两个位点 - 一个位于α ₄ ⁽⁺⁾ β ₁ ^(-)，一个位于α ₄ ^(-) β ₁ ⁽⁺⁾界面，其中α ₄ ^(-) β ₁ ⁽⁺⁾位点对应于依托咪酯的结合位点和最近公开的地西泮的低亲和力结合位点。我们表明 ECD 位点的突变并没有取消 DS2 调制。然而，TMD α ₄ ⁽⁺⁾ β _{1 中的}突变^(-)界面，α ₄ ⁽⁺⁾侧的α ₄ (S303L)或β ₁ ^(-)侧的β ₁ (I289Q) ，令人信服地破坏了 DS2 的正变构调制。这在测量膜电位变化的测定和全细胞膜片钳电生理学中得到一致证明，并通过对接研究合理化。重要的是，突变受体对调节剂的一般敏感性并未受到影响。这项研究揭示了长期以来寻找的 DS2 分子识别位点，驳斥了 DS2 对δ的选择性的误解含β-亚基的受体是由与δ-亚基的直接相互作用引起的，而是通过TMD中令人惊讶的高度保守的结合口袋指向DS2及其类似物的功能选择性。