Receptor Biology Laboratory, Department of Physiology and Biomedical Engineering, Mayo Clinic College of Medicine, Rochester, MN, 55905, USA.
School of Life Sciences, Oxford Brookes University, Oxford, OX3 OBP, UK.
Cell Mol Life Sci. 2019 Mar;76(6):1151-1167. doi: 10.1007/s00018-018-2993-7. Epub 2019 Jan 1.
Neuronal nicotinic receptors containing α4 and β2 subunits assemble in two pentameric stoichiometries, (α4)(β2) and (α4)(β2), each with distinct pharmacological signatures; (α4)(β2) receptors are strongly potentiated by the drug NS9283, whereas (α4)(β2) receptors are unaffected. Despite this stoichiometry-selective pharmacology, the molecular identity of the target for NS9283 remains elusive. Here, studying (α4)(β2) receptors, we show that mutations at either the principal face of the β2 subunit or the complementary face of the α4 subunit prevent NS9283 potentiation of ACh-elicited single-channel currents, suggesting the drug targets the β2-α4 pseudo-agonist sites, the α4-α4 agonist site, or both sites. To distinguish among these possibilities, we generated concatemeric receptors with mutations at specified subunit interfaces, and monitored the ability of NS9283 to potentiate ACh-elicited single-channel currents. We find that a mutation at the principal face of the β2 subunit at either β2-α4 pseudo-agonist site suppresses potentiation, whereas mutation at the complementary face of the α4 subunit at the α4-α4 agonist site allows a significant potentiation. Thus, monitoring potentiation of single concatemeric receptor channels reveals that the β2-α4 pseudo-agonist sites are required for stoichiometry-selective drug action. Together with the recently determined structure of the (α4)(β2) receptor, the findings have implications for structure-guided drug design.
神经元烟碱型受体包含α4 和β2 亚基，以两种五聚体计量组装，(α4)(β2)和(α4)(β2)，每种都具有独特的药理学特征；药物 NS9283 强烈增强(α4)(β2)受体，而(α4)(β2)受体不受影响。尽管这种计量比选择性药理学存在，但 NS9283 的靶标分子身份仍然难以捉摸。在这里，通过研究(α4)(β2)受体，我们表明β2 亚基的主表面或α4 亚基的互补表面的突变会阻止 NS9283 增强 ACh 引发的单通道电流，这表明该药物的靶标是β2-α4 拟似激动剂位点、α4-α4 激动剂位点或两个位点。为了区分这些可能性，我们生成了具有特定亚基界面突变的串联受体，并监测 NS9283 增强 ACh 引发的单通道电流的能力。我们发现，β2 亚基主表面上的突变（位于β2-α4 拟似激动剂位点之一）抑制了增强作用，而α4 亚基互补表面上的突变（位于α4-α4 激动剂位点）允许显著增强。因此，监测单个串联受体通道的增强作用表明β2-α4 拟似激动剂位点是计量比选择性药物作用所必需的。与最近确定的(α4)(β2)受体结构相结合，这些发现对基于结构的药物设计具有启示意义。
