Autism-Linked Mutations in α(2)δ-1 and α(2)δ-3 Reduce Protein Membrane Expression but Affect Neither Calcium Channels nor Trans-Synaptic Signaling.

BACKGROUND: α(2)δ 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 α(2)δ-1) and CACNA2D3 (coding for α(2)δ-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 α(2)δ 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 α(2)δ-1 (p.R351T) and α(2)δ-3 (p.A275T), previously identified in patients with ASD. METHODS: Electrophysiological recordings in transfected tsA201 cells were used to study specific channel-dependent functions of mutated α(2)δ proteins. Membrane expression, presynaptic targeting, and trans-synaptic signaling of mutated α(2)δ proteins were studied upon expression in murine cultured hippocampal neurons. RESULTS: Homologous expression of both mutated α(2)δ proteins revealed a strongly reduced membrane expression and synaptic localization compared to the corresponding wild type α(2)δ proteins. Moreover, the A275T mutation in α(2)δ-3 resulted in an altered glycosylation pattern upon heterologous expression. However, neither of the mutations compromised the biophysical properties of postsynaptic L-type (Ca(V)1.2 and Ca(V)1.3) and presynaptic P/Q-type (Ca(V)2.1) channels when co-expressed in tsA201 cells. Furthermore, presynaptic expression of p.R351T in the α(2)δ-1 splice variant lacking exon 23 did not affect trans-synaptic signaling to postsynaptic GABA(A) receptors. CONCLUSIONS: Our data provide evidence that the pathophysiological mechanisms of ASD-causing mutations of α(2)δ 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 α(2)δ protein-linked disease models.