Autism-Linked Mutations in αδ-1 and αδ-3 Reduce Protein Membrane Expression but Affect Neither Calcium Channels nor Trans-Synaptic Signaling.

BACKGROUND

αδ 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 αδ-1) and CACNA2D3 (coding for αδ-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 αδ 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 αδ-1 (p.R351T) and αδ-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 αδ proteins. Membrane expression, presynaptic targeting, and trans-synaptic signaling of mutated αδ proteins were studied upon expression in murine cultured hippocampal neurons.

RESULTS

Homologous expression of both mutated αδ proteins revealed a strongly reduced membrane expression and synaptic localization compared to the corresponding wild type αδ proteins. Moreover, the A275T mutation in αδ-3 resulted in an altered glycosylation pattern upon heterologous expression. However, neither of the mutations compromised the biophysical properties of postsynaptic L-type (Ca1.2 and Ca1.3) and presynaptic P/Q-type (Ca2.1) channels when co-expressed in tsA201 cells. Furthermore, presynaptic expression of p.R351T in the αδ-1 splice variant lacking exon 23 did not affect trans-synaptic signaling to postsynaptic GABA receptors.

CONCLUSIONS

Our data provide evidence that the pathophysiological mechanisms of ASD-causing mutations of αδ 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 αδ protein-linked disease models.