Figure 5
Pmr1p is involved in αSyn-induced dysregulation of Ca2+ homeostasis. (a) Aequorin-luminescence-based determination of basal cytosolic Ca2+ levels in WT cells and indicated deletion mutants expressing αSyn for 20 h. Data was normalized to corresponding isogenic vector control. Mean±S.E.M., n=12; ***P<0.001; NS, not significant. (b) Aequorin-equipped WT and Δpmr1 yeast cells expressing αSyn or harbouring the vector control were challenged with 150 mM CaCl2 and transient [Ca2+]cyt responses were observed for 50 s. Mean±S.E.M., n=6. (c) Western blot analysis of aequorin expression and αSyn expression in WT cells and indicated deletion mutants. Blots were probed with antibodies directed against aequorin, against FLAG-epitope to detect FLAG-tagged αSyn and against glyceraldehyd-3-phosphate dehydrogenase (GAPDH) as loading control. (d) Aequorin-equipped WT and Δpmr1 cells constitutively expressing αSyn (using the expression vector pGGE181) or harbouring the empty pGGE181 vector (Ctrl.) were starved for glucose, supplemented with low doses of Ca2+ (10 mM) and subsequently challenged with 80 mM glucose. Transient [Ca2+]cyt responses were monitored. Data represent average recordings, n≥9. (e) Maximum [Ca2+]cyt peak amplitude after addition of 80 mM glucose as depicted in (d) in aequorin-equipped WT cells and indicated deletion mutants upon expression of αSyn. Mean±S.E.M., n≥9; ***P<0.001; NS, not significant
