Figure 4.

Monitoring the subplasma membrane Ca²⁺ concentration with pmAEQ. (A) Response to ATP (100 μM) of CHO cells transiently expressing pmAEQ alone (control, light gray trace), or together with PrP^c (black trace) or Dpl (dark gray trace), and maintained in 1 mM CaCl₂. pmAEQ was reconstituted by incubating cells with wild-type coelenterazine (5 μM; see Materials and Methods). Addition of ATP provoked a [Ca²⁺]_pm transient that in cells with PrP^c was by far more elevated than in the other two cell types. With respect to the control, however, the resting [Ca²⁺] level remained significantly higher in the presence of both PrP^{c pm}and Dpl (in the inset, resting [Ca²⁺]_pm are reported on an expanded scale). (B) Response to CaCl₂ addition (1 mM) in the same cell types described in A, but maintained in (Ca²⁺-free) KRB supplemented with EGTA (100 μM). After pmAEQ reconstitution, addition of 1 mM CaCl₂ induced a response that mimicked that shown in A with respect to both peak and resting [Ca²⁺]_pm levels. The latter are also shown in the inset on an expanded scale. Presented data are typical of at least six independent experiments, which yielded equivalent results.