Figure 5

PMCA expression and [iCa²⁺] depend on Np. (a,b) Total homogenates from HEK cells were analyzed by Western blot. Identity of proteins is indicated on the right. Antibodies against GFP and GAPDH/actin were used to control protein over-expression and equal loading of the samples, respectively. Graphs displayed below each representative experiment show blot densitometries expressed as mean ± SEM (four independent experiments per series. *P < 0.05 vs. GFP, unpaired two-tailed t-test). (a) Endogenous PMCA levels in cells transfected 24 hours with GFP, rat Np65-GFP (rNp65-GFP), rNp55-GFP, or human Np65-GFP (hNp65-GFP) were assessed using a pan-PMCA antibody. (b) HEK cells were transfected with hPMCA2 or co-transfected with GFP or rNp65-GFP additionally. hPMCA2 was detected with an anti-PMCA2 antibody. (c) Single or double transfection in HEK cells with plasmids encoding rNp55-RFP and hPMCA1-GFP or hPMCA2-GFP. Arrow heads point to PMCA1-Np55 or PMCA2-Np55 co-localization in the cell plasma membrane of co-transfected cells. Representative pictures from 3 independent transfections. (d) Immature neurons (5 DIV) were transfected with hPMCA2 (red) or co-transfected with hPMCA2 and GFP-GPI or hPMCA2 and hNp65-GFP for 24 hours and photographed using confocal microscopy. hPMCA2 over-expression was revealed using an anti-PMCA2 antibody and a Cy3-congugated secondary antibody. Representative pictures from 3 independent transfections. (e) Mature wild-type and Nptn−/− neurons (16 DIV) were stained with antibodies against Np65 (magenta) and pan-PMCAs (green) and photographed using a confocal microscope (representative pictures from at least 5 independent experiments) or (f,g) loaded with Fluo-4 and Fura-Red fluorescent probes to measure [iCa²⁺]. (f) Nptn−/− intensity ratio was normalized against wild-type ratio and demonstrates the stability of the single-cell recordings in the presence of 1 µM TTX. (g) The graph shows the baseline intensity ratio expressed as mean ± SEM (16 wild-type and 18 Nptn−/− neurons (*P < 0.05, unpaired two-tailed t-test). (h, i) Fluo-4-loaded mature wild-type and Nptn−/− neurons (16 DIV) were stimulated with KCl (30 mM, 15 sec). (h) The spikes are the mean ± SEM of 11 wild-type and 12 Nptn−/− neurons. (i) Time required to recover to baseline levels of [iCa²⁺] (peak-to-baseline) was quantified (*P < 0.05, unpaired two-tailed t-test). (f,g,h,i) Measurements were performed in pyramidal neurons as identified morphologically in a confocal microscope under controlled conditions of temperature and pH. Upper graph Lower Scale bars = 20 µm.