Haddad Sabrin, Ablinger Cornelia, Stanika Ruslan, Hessenberger Manuel, Campiglio Marta, Ortner Nadine J, Tuluc Petronel, Obermair Gerald J
Institute of Physiology, Medical University Innsbruck, Innsbruck, Austria.
Division of Physiology, Department of Pharmacology, Physiology, and Microbiology, Karl Landsteiner University of Health Sciences, Krems, Austria.
J Neurochem. 2025 Jan;169(1):e16197. doi: 10.1111/jnc.16197. Epub 2024 Aug 19.
αδ proteins serve as auxiliary subunits of voltage-gated calcium channels and regulate channel membrane expression and current properties. Besides their channel function, αδ proteins regulate synapse formation, differentiation, and synaptic wiring. Considering these important functions, it is not surprising that CACNA2D1-4, the genes encoding for αδ-1 to -4 isoforms, have been implicated in neurological, neurodevelopmental, and neuropsychiatric disorders. Mutations in CACNA2D2 have been associated with developmental and epileptic encephalopathy (DEE) and cerebellar atrophy. In our present study, we performed a detailed functional characterization of the p.R593P mutation in αδ-2, a homozygous mutation previously identified in two siblings with DEE. Importantly, we analyzed both calcium channel-dependent as well as synaptic functions of αδ-2. Our data show that the corresponding p.R596P mutation in mouse αδ-2 drastically decreases membrane expression and synaptic targeting of αδ-2. This defect correlates with altered biophysical properties of postsynaptic Ca1.3 channel but has no effect on presynaptic Ca2.1 channels upon heterologous expression in tsA201 cells. However, homologous expression of αδ-2_R596P in primary cultures of hippocampal neurons affects the ability of αδ-2 to induce a statistically significant increase in the presynaptic abundance of endogenous Ca2.1 channels and presynaptic calcium transients. Moreover, our data demonstrate that in addition to lowering membrane expression, the p.R596P mutation reduces the trans-synaptic recruitment of GABA receptors and presynaptic synapsin clustering in glutamatergic synapses. Lastly, the αδ-2_R596P reduces the amplitudes of glutamatergic miniature postsynaptic currents in transduced hippocampal neurons. Taken together, our data strongly link the human biallelic p.R593P mutation to the underlying severe neurodevelopmental disorder and highlight the importance of studying αδ mutations not only in the context of channelopathies but also synaptopathies.
αδ蛋白作为电压门控钙通道的辅助亚基，调节通道膜表达和电流特性。除了其通道功能外，αδ蛋白还调节突触形成、分化和突触连接。考虑到这些重要功能，编码αδ-1至-4亚型的基因CACNA2D1-4与神经、神经发育和神经精神疾病有关也就不足为奇了。CACNA2D2的突变与发育性和癫痫性脑病（DEE）以及小脑萎缩有关。在我们目前的研究中，我们对αδ-2中的p.R593P突变进行了详细的功能表征，该纯合突变先前在两名患有DEE的兄弟姐妹中被鉴定出来。重要的是，我们分析了αδ-2的钙通道依赖性以及突触功能。我们的数据表明，小鼠αδ-2中相应的p.R596P突变显著降低了αδ-2的膜表达和突触定位。这种缺陷与突触后Ca1.3通道生物物理特性的改变相关，但在tsA201细胞中异源表达时对突触前Ca2.1通道没有影响。然而，αδ-2_R596P在海马神经元原代培养物中的同源表达影响了αδ-2诱导内源性Ca2.1通道突触前丰度和突触前钙瞬变统计学显著增加的能力。此外，我们的数据表明，除了降低膜表达外，p.R596P突变还减少了GABA受体的跨突触募集以及谷氨酸能突触中突触前突触素的聚集。最后，αδ-2_R596P降低了转导的海马神经元中谷氨酸能微小突触后电流的幅度。综上所述，我们的数据有力地将人类双等位基因p.R593P突变与潜在的严重神经发育障碍联系起来，并强调了不仅在通道病背景下而且在突触病背景下研究αδ突变的重要性。
