Figure 5

Neuroplastin is required for PMCA stabilization and Ca²⁺ clearance. (A) Western blot analysis of PMCA1, PMCA4 and Neuroplastin in wt and Nptn ^−/− samples from CD4 T cells and thymus. Representative blots show lack of Neuroplastin and strong reduction of PMCA1 and PMCA4 in Nptn ^−/− CD4 T cells and thymus. Signal intensities were quantified by densitometry using β-actin signals for normalization. Bar graphs show mean ± SEM from 4 wt and 4 Nptn ^−/− replicate thymus samples, and from 3 samples of CD4 T cell extracts each, **p < 0.01, ***p < 0.001, unpaired two-tailed t-test. (B) qRT-PCR of PMCA1 transcript levels in lymph node cells. B2m and Hprt served as references for normalization. Graph shows mean relative transcript levels ± SD derived from 7 samples per group. (C–E) Flow cytometric ratiometric measurement of post-stimulatory Ca²⁺ clearance in wt or Nptn ^−/− naïve CD4 T cells. After baseline recording, Ca²⁺ release from internal stores was induced in Ca²⁺-free buffer by 1 μg/ml ionomycin. Kinetics of the mean Fluo-3/FuraRed ratio ± SD from 3 experiments is shown (C). Ca²⁺ release from the ER was induced by 1 μg/ml thapsigargin in Ca²⁺ -free buffer. Mean ratios ± SD from 4 experiments are shown (D). Ca²⁺ clearance across the plasma membrane, investigated after thapsigargin treatment as in (D) and consecutive induction of SOCE by resuspending cells in buffer containing 1 mM Ca²⁺. High Fluo-3/FuraRed ratios were recorded for 1 min before Ca²⁺ clearance was induced by resuspending cells in Ca²⁺ -free buffer again, and recording was immediately continued. Mean ratios ± SD from 5 experiments are shown (E). (F,G) Ca²⁺ levels in Pmca1 haploinsufficient T cells. Ca²⁺ release from internal stores induced by ionomycin (F) or thapsigargin (G) were repeated exactly as in (C) and (D), respectively, but with T cells from Pmca1 ^f/+ mice expressing either a T cell-specific Cre recombinase (cre+) or no Cre (cre−). Mean Fluo-3/FuraRed ratios ± SD were derived from 3 experiments each. Quantifications of the Ca²⁺ decay shown in (E) and (H) by fitting exponential curves are summarized in Supplementary Tables 1 and 2, respectively.