Ecole Normale Supérieure, Institut de Biologie de l' Ecole Normale Supérieure, Institut National de la Santé et de la Recherche Médicale U1024, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 8197, F-75005 Paris, France.
Proc Natl Acad Sci U S A. 2014 Apr 22;111(16):6081-6. doi: 10.1073/pnas.1318808111. Epub 2014 Apr 8.
Reprogramming receptors to artificially respond to light has strong potential for molecular studies and interrogation of biological functions. Here, we design a light-controlled ionotropic glutamate receptor by genetically encoding a photoreactive unnatural amino acid (UAA). The photo-cross-linker p-azido-L-phenylalanine (AzF) was encoded in NMDA receptors (NMDARs), a class of glutamate-gated ion channels that play key roles in neuronal development and plasticity. AzF incorporation in the obligatory GluN1 subunit at the GluN1/GluN2B N-terminal domain (NTD) upper lobe dimer interface leads to an irreversible allosteric inhibition of channel activity upon UV illumination. In contrast, when pairing the UAA-containing GluN1 subunit with the GluN2A subunit, light-dependent inactivation is completely absent. By combining electrophysiological and biochemical analyses, we identify subunit-specific structural determinants at the GluN1/GluN2 NTD dimer interfaces that critically dictate UV-controlled inactivation. Our work reveals that the two major NMDAR subtypes differ in their ectodomain-subunit interactions, in particular their electrostatic contacts, resulting in GluN1 NTD coupling more tightly to the GluN2B NTD than to the GluN2A NTD. It also paves the way for engineering light-sensitive ligand-gated ion channels with subtype specificity through the genetic code expansion.
重新编程受体以人工响应光具有很强的分子研究和生物功能检测潜力。在这里，我们通过基因编码光反应性非天然氨基酸（UAA）设计了一种光控离子型谷氨酸受体。光交联剂 p-叠氮-L-苯丙氨酸（AzF）被编码在 NMDA 受体（NMDAR）中，NMDAR 是一类谷氨酸门控离子通道，在神经元发育和可塑性中发挥关键作用。AzF 在 GluN1 亚基的必需 GluN1/GluN2B N 端结构域（NTD）上的结合导致通道活性的不可逆变构抑制，当在 UV 照射下。相比之下，当将含有 UAA 的 GluN1 亚基与 GluN2A 亚基配对时，光依赖性失活完全不存在。通过结合电生理和生化分析，我们确定了 GluN1/GluN2 NTD 二聚体界面上的亚基特异性结构决定因素，这些决定因素对 UV 控制的失活至关重要。我们的工作表明，两种主要的 NMDAR 亚型在其外域-亚基相互作用方面存在差异，特别是它们的静电接触，导致 GluN1 NTD 与 GluN2B NTD 的偶联比与 GluN2A NTD 的偶联更紧密。它还为通过遗传密码扩展工程具有亚型特异性的光敏感配体门控离子通道铺平了道路。
