Aspartic acid 272 of VMP1 is critical for its regulation of ER Ca ²⁺ release and naive T cell survival. (A) Putative topology of VMP1 protein. Negatively charged amino acids on the ER lumen side that are also close to a transmembrane segment are indicated, including E67, D272, E340/343, E354, and E361. (B) VMP1-KO HEK293T cells were transfected with indicated plasmids. The expression of VMP1 and mutants was examined by immunoblot. (C) VMP1-KO HEK293T cells were transfected with indicated plasmids. ER Ca²⁺ and SOCE were measured by flow cytometry. (D) ER Ca²⁺ stored in T cells from mice with indicated genotypes was examined by flow cytometry. (E) Representative flow cytometry plots and absolute number of T cells in spleen (SPL) and peripheral lymph nodes (pLN) from mice with indicated genotypes. Mice were 4–6 wk old. T cell number of Cd4^Cre and Cd4^CreVmp1^fl/fl mice were from the same mice in Fig. 3 C. (B) Representative blots from one of two independent experiments. (C) Representative plots and statistics from one of three independent experiments. (D and E) Representative plots and statistics from one of two independent experiments. n = 3 in C, n = 3 mice per genotype in D, n = 3–4 mice per genotype in E; each symbol represents one sample or an individual mouse; data represent mean ± SEM, two-way ANOVA in C, one-way ANOVA in D and E; * <0.05, ** <0.01, *** <0.001, **** <0.001. Source data are available for this figure: SourceData F4.