A biallelic mutation in <scp>CACNA2D2</scp> associated with developmental and epileptic encephalopathy affects calcium channel‐dependent as well as synaptic functions of α ₂ δ‐2

α₂δ proteins serve as auxiliary subunits of voltage-gated calcium channels and regulate channel membrane expression and current properties. Besides their channel function, α₂δ proteins regulate synapse formation, differentiation, and synaptic wiring. Considering these important functions, it is not surprising that CACNA2D1-4, the genes encoding for α₂δ-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 α₂δ-2, a homozygous mutation previously identified in two siblings with DEE. Importantly, we analyzed both calcium channel-dependent as well as synaptic functions of α₂δ-2. Our data show that the corresponding p.R596P mutation in mouse α₂δ-2 drastically decreases membrane expression and synaptic targeting of α₂δ-2. This defect correlates with altered biophysical properties of postsynaptic Ca_V1.3 channel but has no effect on presynaptic Ca_V2.1 channels upon heterologous expression in tsA201 cells. However, homologous expression of α₂δ-2_R596P in primary cultures of hippocampal neurons affects the ability of α₂δ-2 to induce a statistically significant increase in the presynaptic abundance of endogenous Ca_V2.1 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_A receptors and presynaptic synapsin clustering in glutamatergic synapses. Lastly, the α₂δ-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 α₂δ mutations not only in the context of channelopathies but also synaptopathies.