OBJECTIVE: Developmental epileptic encephalopathies (DEEs) are genetically 
heterogeneous severe childhood-onset epilepsies with developmental delay or 
cognitive deficits. In this study, we explored the pathogenic mechanisms of 
DEE-associated de novo mutations in the CACNA1A gene.
METHODS: We studied the functional impact of four de novo DEE-associated CACNA1A 
mutations, including the previously described p.A713T variant and three novel 
variants (p.V1396M, p.G230V, and p.I1357S). Mutant cDNAs were expressed in 
HEK293 cells, and whole-cell voltage-clamp recordings were conducted to test the 
impacts on CaV 2.1 channel function. Channel localization and structure were 
assessed with immunofluorescence microscopy and three-dimensional (3D) modeling.
RESULTS: We find that the G230V and I1357S mutations result in loss-of-function 
effects with reduced whole-cell current densities and decreased channel 
expression at the cell membrane. By contrast, the A713T and V1396M variants 
resulted in gain-of-function effects with increased whole-cell currents and 
facilitated current activation (hyperpolarized shift). The A713T variant also 
resulted in slower current decay. 3D modeling predicts conformational changes 
favoring channel opening for A713T and V1396M.
SIGNIFICANCE: Our findings suggest that both gain-of-function and 
loss-of-function CACNA1A mutations are associated with similarly severe DEEs and 
that functional validation is required to clarify the underlying molecular 
mechanisms and to guide therapies.