Abstract
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 CaV2.1 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.
Original language | English (US) |
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Pages (from-to) | 1881-1894 |
Number of pages | 14 |
Journal | Epilepsia |
Volume | 60 |
Issue number | 9 |
DOIs | |
State | Published - Sep 1 2019 |
Externally published | Yes |
Keywords
- CACNA1A
- Ca2.1
- Lennox-Gastaut syndrome
- de novo mutations
- epilepsy
- epileptic encephalopathies
- immunofluorescence
- patch-clamp
- structural modeling
ASJC Scopus subject areas
- Neurology
- Clinical Neurology