A biallelic mutation in CACNA2D2 associated with developmental and epileptic encephalopathy affects calcium channel-dependent as well as synaptic functions of αδ-2.

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.