Electrophysiological classification of CACNA1G gene variants associated with neurodevelopmental and neurological disorders

This study highlights the complementarity of automated patch-clamp (APC) and manual patch-clamp (MPC) approaches to describe the electrophysiological properties of eighteen Ca _v 3.1 calcium channel variants associated with various neurological conditions. Current density was measured efficiently for all variants in APC experiments, with four variants (p.V184G, p.N1200S, p.S1263A and p.D2242N) showing high current densities, compared to wild-type Ca _v 3.1 channel, while six variants (p.M197R, p.V392M, p.F956del, p.I962N, p.I1412T, and p.G1534D) displayed low current densities, and were therefore preferentially studied using MPC. The electrophysiological properties were well conserved in APC (e.g. inactivation and deactivation kinetics, steady-state properties), with only the APC-MPC correlation for the activation kinetics being less robust. In addition, neuronal modeling, using a deep cerebellar neuron (DCN) environment, revealed that most of the variants localized in the intracellular gate (S5 and S6 segments) could increase DCN spike frequencies. This DCN firing was critically dependent on the current density and further pointed to the gain-of-function (GOF) properties of p.A961T and p.M1531V, the recurrent variants associated with Spinocerebellar Ataxia type-42 with Neurodevelopmental Deficit (SCA42ND). Action-potential (AP) clamp experiments performed using cerebellar and thalamic neuron activities further established the GOF properties of p.A961T and p.M1531V variants. Overall, this study demonstrates that APC is well-suited to high-throughput analysis of Ca _v 3.1 channel variants, and that MPC complements APC for characterizing low-expression variants. Furthermore, in silico modeling and AP clamp experiments establish that the gain- or loss-of-function properties of the variants are determined by how the Ca _v 3.1 channel decodes the electrophysiological context of a neuron.