Startle disease is a rare disorder associated with mutations in GLRA1 and GLRB, encoding glycine receptor (GlyR) α1 and β subunits, which enable fast synaptic inhibitory transmission in the spinal cord and brainstem. The GlyR β subunit is important for synaptic localization via interactions with gephyrin and contributes to agonist binding and ion channel conductance. Here, we have studied three GLRB missense mutations, Y252S, S321F, and A455P, identified in startle disease patients. For Y252S in M1 a disrupted stacking interaction with surrounding aromatic residues in M3 and M4 is suggested which is accompanied by an increased EC₅₀ value. By contrast, S321F in M3 might stabilize stacking interactions with aromatic residues in M1 and M4. No significant differences in glycine potency or efficacy were observed for S321F. The A455P variant was not predicted to impact on subunit folding but surprisingly displayed increased maximal currents which were not accompanied by enhanced surface expression, suggesting that A455P is a gain-of-function mutation. All three GlyR β variants are trafficked effectively with the α1 subunit through intracellular compartments and inserted into the cellular membrane. In vivo, the GlyR β subunit is transported together with α1 and the scaffolding protein gephyrin to synaptic sites. The interaction of these proteins was studied using eGFP-gephyrin, forming cytosolic aggregates in non-neuronal cells. eGFP-gephyrin and β subunit co-expression resulted in the recruitment of both wild-type and mutant GlyR β subunits to gephyrin aggregates. However, a significantly lower number of GlyR β aggregates was observed for Y252S, while for mutants S321F and A455P, the area and the perimeter of GlyR β subunit aggregates was increased in comparison to wild-type β. Transfection of hippocampal neurons confirmed differences in GlyR-gephyrin clustering with Y252S and A455P, leading to a significant reduction in GlyR β-positive synapses. Although none of the mutations studied is directly located within the gephyrin-binding motif in the GlyR β M3-M4 loop, we suggest that structural changes within the GlyR β subunit result in differences in GlyR β-gephyrin interactions. Hence, we conclude that loss- or gain-of-function, or alterations in synaptic GlyR clustering may underlie disease pathology in startle disease patients carrying GLRB mutations.

惊恐症是一种与基因突变相关的罕见疾病GLRA1和GLRB ，编码甘氨酸受体 (GlyR) α1 和 β 亚基，可实现脊髓和脑干中的快速突触抑制传递。 GlyR β 亚基通过与 gephyrin 相互作用对于突触定位非常重要，并有助于激动剂结合和离子通道电导。在这里，我们研究了三个GLRB在惊恐症患者中发现了错义突变 Y252S、S321F 和 A455P。对于 M1 中的 Y252S，表明与 M3 和 M4 中周围芳香族残基的堆积相互作用被破坏，并伴随着 EC ₅₀值的增加。相比之下，M3 中的 S321F 可能会稳定与 M1 和 M4 中芳香族残基的堆积相互作用。 S321F 的甘氨酸效力或功效没有观察到显着差异。预计 A455P 变体不会影响亚基折叠，但令人惊讶的是，它显示出最大电流增加，但并未伴随表面表达增强，这表明 A455P 是一种功能获得性突变。所有三种 GlyR β 变体均与 α1 亚基有效地通过细胞内区室运输并插入细胞膜中。体内，GlyR β 亚基与 α1 和支架蛋白 gephyrin 一起转运至突触位点。使用 eGFP-gephyrin 研究这些蛋白质的相互作用，在非神经元细胞中形成胞质聚集体。 eGFP-gephyrin 和 β 亚基共表达导致野生型和突变型 GlyR β 亚基募集到 gephyrin 聚集体中。 然而，Y252S 中观察到的 GlyR β 聚集体数量显着减少，而对于突变体 S321F 和 A455P，GlyR β 亚基聚集体的面积和周长与野生型 β 相比有所增加。海马神经元的转染证实了 GlyR-gephyrin 与 Y252S 和 A455P 聚类的差异，导致 GlyR β 阳性突触显着减少。尽管所研究的突变均不直接位于 GlyR β M3-M4 环中的 gephyrin 结合基序内，但我们认为 GlyR β 亚基内的结构变化会导致 GlyR β-gephyrin 相互作用的差异。因此，我们得出结论，功能丧失或获得，或者突触 GlyR 簇的改变可能是携带GLRB突变。