Both human neuroblastoma SH-SY5Y cells and HEK293T cells have been used in functional studies of epilepsy-related variants.25-27 Considering the substantial difference in UNC13A expression levels between SH-SY5Y cells (nTPM: 9.2) and HEK293T cells (nTPM: 0.1; Human Protein Atlas),28 we overexpressed plasmid containing wildtype or UNC13A variants in HEK293T cells. Ca2+ indicators (GCaMPs) or Ca2+ channel subunits (CaV1.2, CaVβ2a) have been overexpressed in HEK293T cells to study Ca2+ influx or Ca2+ currents in previous studies.29-32 Additionally, calcium imaging in HEK293T cells has been utilized to investigate the function of genes and variants associated with Alzheimer's disease.33
pLVX plasmids encoding GCaMP6s and lentiviral packaging components were transfected into HEK293T cells using Lipofectamine 2000 (Invitrogen). Lentivirus was collected 48-72 h post-transfection, purified, and concentrated. To generate a stable GCaMP6s-expressing cell line, 2 μL purified lentivirus was added to HEK293T cells, and cells were selected with puromycin. Media was refreshed every 2 days for 2 weeks until clone formation. One day before imaging, GCaMP6s-expressing HEK293T cells were plated on 35 mm Mattek dishes. Wildtype or mutant UNC13A constructs were transfected using Lipofectamine 2000. Two days post-transfection, cells were imaged using an Axio Observer Z1 (Zeiss) at 5 s intervals to detect spontaneous calcium influx (Supplemental Video 3-6). Calcium fluctuations were measured using the GCaMP6s calcium sensor in HEK293T cells.
Supplementary Video2-siUNC13A.mp4
Supplementary Video2-siUNC13A.mp4
Supplementary Video4-UNC13A-M631K.mp4
GCaMP6s 是一类基因编码型钙离子指示器（GECI），用来把细胞内 Ca²⁺ 浓度变化直接翻译成绿色荧光强度变化。它不是“染料”，而是一段能在细胞里表达的蛋白。名字里那个 s = slow，指的是它的动力学偏慢，但灵敏度很高。
GCaMP6s 是一个“三明治”结构：