THE EPILEPSY-ASSOCIATED R785C MUTATION IN ALPHA<SUB>2</SUB>DELTA-2 IMPAIRS NEURONAL MEMBRANE EXPRESSION WHILE PARTIALLY MAINTAINING MODULATION OF VOLTAGE-GATED CALCIUM CHANNELS

Voltage-gated calcium channels (VGCCs) trigger neurotransmitter release and regulate gene transcription and neuronal plasticity. They are modulated by extracellular auxiliary α₂δ subunits which also mediate critical and channel-independent synaptic functions. It is therefore not surprising that mutations in CACNA2D genes encoding α₂δ proteins have been linked to neurological (epilepsy, movement disorders) and neurodevelopmental (autism spectrum disorder, schizophrenia) impairment. However, the underlying pathophysiological mechanisms are insufficiently understood. Here, we present the preliminary functional characterization of the newly identified R785C mutation in α₂δ-2 (R788C in mouse) found in two siblings with developmental and epileptic encephalopathy presenting with drug-resistant seizures and developmental slowing. This condition is associated with severe outcomes such as intellectual disability, psychiatric disorders, multisystem complications, and increased mortality. Structure-homology modeling suggests a destabilizing effect of R785C on the dCache domains of α₂δ-2 due to a loss of ionic interactions. Electrophysiological recordings in transfected tsA201 cells show that mouse α₂δ-2_R788C fails to increase the current density of postsynaptic Ca_V1.3 channels and to induce the characteristic left shift in the voltage-dependence of activation. In contrast, α₂δ-2_R788C modulates presynaptic Ca_V2.1 channels similar to wildtype α₂δ-2. Immunocytochemistry of cultured hippocampal neurons transfected with wildtype or mutated epitope-tagged α₂δ-2 shows a strong reduction of membrane expression without concomitantly reduced total protein levels, suggesting a failure of membrane trafficking. Furthermore, presynaptic expression of α₂δ-2_R788C does not regulate postsynaptic GABA_AR clustering, a previously identified transsynaptic function of α₂δ-2. Together, this suggests pathophysiological mechanisms involving aberrant, isoform-specific VGCC modulation and altered synaptic function due to impaired membrane trafficking.