Institute for Clinical Neurobiology, University Hospital, Julius-Maximilians-University of Würzburg, 97070 Würzburg, Germany.
Center for Biopharmaceuticals, University of Copenhagen, 2100 Copenhagen, Denmark.
J Neurosci. 2020 Jun 17;40(25):4954-4969. doi: 10.1523/JNEUROSCI.2490-19.2020. Epub 2020 Apr 30.
Glycine receptors (GlyRs) are the major mediators of fast synaptic inhibition in the adult human spinal cord and brainstem. Hereditary mutations to GlyRs can lead to the rare, but potentially fatal, neuromotor disorder hyperekplexia. Most mutations located in the large intracellular domain (TM3-4 loop) of the GlyRα1 impair surface expression levels of the receptors. The novel mutation P366L, located in the TM3-4 loop, showed normal surface expression but reduced chloride currents, and accelerated whole-cell desensitization observed in whole-cell recordings. At the single-channel level, we observed reduced unitary conductance accompanied by spontaneous opening events in the absence of extracellular glycine. Using peptide microarrays and tandem MS-based analysis methods, we show that the proline-rich stretch surrounding P366 mediates binding to syndapin I, an F-BAR domain protein involved in membrane remodeling. The disruption of the noncanonical Src homology 3 recognition motif by P366L reduces syndapin I binding. These data suggest that the GlyRα1 subunit interacts with intracellular binding partners and may therefore play a role in receptor trafficking or synaptic anchoring, a function thus far only ascribed to the GlyRβ subunit. Hence, the P366L GlyRα1 variant exhibits a unique set of properties that cumulatively affect GlyR functionality and thus might explain the neuropathological mechanism underlying hyperekplexia in the mutant carriers. P366L is the first dominant mutation identified within the GlyRα1 TM3-4 loop that affects GlyR physiology without altering protein expression at the whole-cell and surface levels. We show that the intracellular domain of the inhibitory glycine receptor α1 subunit contributes to trafficking and synaptic anchoring. A proline-rich stretch in this receptor domain forms a noncanonical recognition motif important for the interaction with syndapin I (PACSIN1). The disruption of this motif, as present in a human patient with hyperekplexia led to impaired syndapin I binding. Functional analysis revealed that the altered proline-rich stretch determines several functional physiological parameters of the ion channel (e.g., faster whole-cell desensitization) reduced unitary conductance and spontaneous opening events. Thus, the proline-rich stretch from the glycine receptor α1 subunit represents a multifunctional intracellular protein motif.
甘氨酸受体（GlyRs）是成人脊髓和脑干中快速突触抑制的主要介质。GlyRs 的遗传突变可导致罕见但潜在致命的神经运动障碍——发作性肌张力障碍。大多数位于 GlyRα1 大细胞内域（TM3-4 环）的突变会降低受体的表面表达水平。位于 TM3-4 环中的新型突变 P366L 表现出正常的表面表达，但氯离子电流减少，并且在全细胞记录中观察到全细胞脱敏加速。在单通道水平上，我们观察到在没有细胞外甘氨酸的情况下，单位电导降低伴随着自发开放事件。使用肽微阵列和串联 MS 分析方法，我们表明 P366 周围富含脯氨酸的伸展与 syndapin I 结合，syndapin I 是一种参与膜重塑的 F-BAR 结构域蛋白。P366L 破坏非典型Src 同源 3 识别基序会减少 syndapin I 的结合。这些数据表明 GlyRα1 亚基与细胞内结合伴侣相互作用，因此可能在受体运输或突触锚定中发挥作用，迄今为止，这一功能仅归因于 GlyRβ 亚基。因此，P366L GlyRα1 变体表现出一组独特的特性，这些特性累积影响 GlyR 的功能，从而可能解释突变载体中发作性肌张力障碍的神经病理学机制。P366L 是在 GlyRα1 TM3-4 环内鉴定的第一个影响 GlyR 生理学而不改变全细胞和表面水平蛋白表达的显性突变。我们表明，抑制性甘氨酸受体α1 亚基的细胞内域有助于运输和突触锚定。该受体结构域中的富含脯氨酸的伸展形成与 syndapin I（PACSIN1）相互作用的非典型识别基序。在患有发作性肌张力障碍的人类患者中存在的这种基序的破坏会导致 syndapin I 结合受损。功能分析表明，改变的富含脯氨酸的伸展决定了离子通道的几个功能生理参数（例如，全细胞脱敏更快）、降低的单位电导和自发开放事件。因此，甘氨酸受体α1 亚基的富含脯氨酸的伸展代表了一种多功能的细胞内蛋白基序。
