Autism-Linked Mutations in α₂δ-1 and α₂δ-3 Reduce Protein Membrane Expression but Affect Neither Calcium Channels nor...

α₂δ proteins regulate membrane trafficking and biophysical properties of voltage-gated calcium channels. Moreover, they modulate axonal wiring, synapse formation, and trans-synaptic signaling. Several rare missense variants in CACNA2D1 (coding for α₂δ-1) and CACNA2D3 (coding for α₂δ-3) genes were identified in patients with autism spectrum disorder (ASD). However, the pathogenicity of these variants is not known, and the molecular mechanism by which α₂δ proteins may contribute to the pathophysiology of autism is, as of today, not understood. Therefore, in this study we functionally characterized two heterozygous missense variants in α₂δ-1 (p.R351T) and α₂δ-3 (p.A275T), previously identified in patients with ASD.

Electrophysiological recordings in transfected tsA201 cells were used to study specific channel-dependent functions of mutated α₂δ proteins. Membrane expression, presynaptic targeting, and trans-synaptic signaling of mutated α₂δ proteins were studied upon expression in murine cultured hippocampal neurons.

Homologous expression of both mutated α₂δ proteins revealed a strongly reduced membrane expression and synaptic localization compared to the corresponding wild type α₂δ proteins. Moreover, the A275T mutation in α₂δ-3 resulted in an altered glycosylation pattern upon heterologous expression. However, neither of the mutations compromised the biophysical properties of postsynaptic L-type (Ca_V1.2 and Ca_V1.3) and presynaptic P/Q-type (Ca_V2.1) channels when co-expressed in tsA201 cells. Furthermore, presynaptic expression of p.R351T in the α₂δ-1 splice variant lacking exon 23 did not affect trans-synaptic signaling to postsynaptic GABA_A receptors.

Our data provide evidence that the pathophysiological mechanisms of ASD-causing mutations of α₂δ proteins may not involve their classical channel-dependent and trans-synaptic functions. Alternatively, these mutations may induce subtle changes in synapse formation or neuronal network function, highlighting the need for future α₂δ protein-linked disease models.