The prion protein and its paralogue Doppel affect calcium signaling in Chinese hamster ovary cells

Abstract

The function of the prion protein (PrP(c)), implicated in transmissible spongiform encephalopathies (TSEs), is largely unknown. We examined the possible influence of PrP(c) on Ca(2+) homeostasis, by analyzing local Ca(2+) fluctuations in cells transfected with PrP(c) and Ca(2+)-sensitive aequorin chimeras targeted to defined subcellular compartments. In agonist-stimulated cells, the presence of PrP(c) sharply increases the Ca(2+) concentration of subplasma membrane Ca(2+) domains, a feature that may explain the impairment of Ca(2+)-dependent neuronal excitability observed in TSEs. PrP(c) also limits Ca(2+) release from the endoplasmic reticulum and Ca(2+) uptake by mitochondria, thus rendering unlikely the triggering of cell death pathways. Instead, cells expressing Doppel, a PrP(c) paralogue, display opposite effects, which, however, are abolished by the coexpression of PrP(c). These findings are consistent with the functional interplay and antagonistic role attributed to the proteins, whereby PrP(c) protects, and Doppel sensitizes, cells toward stress conditions.

Figure 1.

Localization (A) and expression level (B) of PrP^c and Dpl in transiently transfected CHO cells. (A) Distribution of PrP^c and Dpl in intact cells expressing each protein alone, after treatment (4°C) with anti-PrP mAb 8H4 (left), or anti-Dpl mAb Dpl-79 (right), followed by fluorescein isothiocyanate-conjugated secondary antibody. Both proteins are present on the cell surface, correctly exposed to the exoplasmic space. Bar, 20 μm. (B) Western blots of PrP^c (left) and Dpl (right) of cells expressing each protein alone (second lanes), or together (third lanes), show that both proteins exhibit high mass diffuse bands typical of their complex N-linked glycosylation (Massimino et al., 2004). Cell lysate SDS-PAGE separated proteins were immunoblotted with the above-described mAbs, and immunoreactive bands were then subjected to densitometric analysis. Comparison between the density of PrP^c- and Dpl-containing bands and the band density elicited by a known quantity of PrP_rec or Dpl_rec (0.5 ng, first lane of the left and right panel, respectively) yielded a value of 3.8 and 4.0 ng (in singly transfected cells, second lanes of both panels), and 2.1 and 2.3 ng (in doubly transfected cell, third lanes of both panels) for PrP^c and Dpl amounts, respectively.

Figure 2.

Effects of the presence of PrP^c or Dpl (A), and of GFP-GPI_PrP (B), on the Ca²⁺ filling of the ER lumen and on the agonist-stimulated ER Ca²⁺ discharge, and effects of the presence of PrP^c, or/and Dpl, on the ER membrane Ca²⁺ passive efflux (C), and SERCA expression (D). (A–C) CHO cells, transiently expressing erAEQ alone (control, light gray trace), or together with PrP^c (black trace) or Dpl (dark gray trace; A and C), or GFP-GPI_PrP (dark gray trace; B), were first depleted of Ca²⁺ (see Materials and Methods and text) and then incubated with 5 μM coelenterazine n (in EGTA-supplemented KRB), and finally, after extensive washings, transferred to the thermostatted chamber of the luminometer and perfused with EGTA-containing KRB. As monitored by erAEQ, EGTA replacement by CaCl₂(1 mM) at the indicated time points induced the Ca²⁺ accumulation into the ER lumen, whereas addition of InsP₃-generating agonist ATP (100 μM; A and B), or of the SERCA inhibitor, tBuBHQ (20 μM; C), stimulated the ion discharge. Note that, although cells with either PrP^c or Dpl maintained a similar resting [Ca²⁺]_er (by ∼15% higher than in controls; A and C), ATP-induced Ca²⁺ efflux from the ER was more extensive in Dpl-containing cells (A), followed by a partial refilling of ER stores. Conversely, the levels of resting and discharged [Ca²⁺]_er in cells with GFP-GPI_PrP were as in the control (B). When cells were challenged with tBuBHQ, no difference was observed in the ER membrane passive permeability of the three cell types (C). Presented data are typical of at least seven independent experiments, which yielded equivalent results. (D) Western blots of the endogenous SERCA and β-actin in controls (first lane) or in cells expressing PrP^c (second lane) and Dpl (third lane) separately, or together (fourth lane), using mAbs against the ubiquitous SERCA 2 isoform and β-actin, respectively. By normalizing the density of each SERCA band to the corresponding β-actin band, cells with PrP^c resulted to express the highest, and those expressing Dpl (alone or together with PrP^c) the lowest, quantity of SERCA.

Figure 3.

Resting [Ca²⁺]_c (A) and ATP-induced effects on [Ca²⁺]_c (B and C), in CHO cells transiently expressing cytAEQ alone (control), or together with PrP^c, or Dpl. (A) Resting [Ca²⁺]_c, monitored by the Ca²⁺-indicator fura-2, is expressed as the ratio of the fluorescence emitted by fura-2 after cell excitation at 340 and 380 nm (see Materials and Methods). No statistically significant difference was found in the three cell types. (B) [Ca²⁺]_c transients, after ATP (100 μM) addition at the indicated time point, were monitored with cytAEQ reconstituted with wild-type coelenterazine (5 μM; see Materials and Methods). Although a similar peak was found in controls (light gray trace) and Dpl-containing cells (dark gray trace), cells with PrP^c (black trace) gave rise to a transient of smaller magnitude. (C) Using the above described cytAEQ, a protocol was applied so as to evaluate the contribution to the ATP-induced [Ca²⁺]_c movements shown in B, first of the InsP₃-induced Ca²⁺ mobilization (first peak, ATP added in the presence of 100 μM EGTA, i.e., with no external Ca²⁺) and then of the Ca²⁺ influx from the external medium (second peak, in the presence of 1 mM CaCl₂). On Ca²⁺ release from the ER (first peak), a transient of smaller magnitude was again observed in cells with PrP^c, whereas no difference in the second peak was evident in the three cell types. Presented data are typical of at least seven independent experiments, which yielded equivalent results.

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.

Figure 5.

ATP-induced effects on [Ca²⁺]_m in CHO cells transiently expressing mtAEQ alone (control, light gray trace), or together with PrP^c (black trace) or Dpl (dark gray trace). mtAEQ was reconstituted by incubating cells with wild-type coelenterazine (5 μM; see Materials and Methods). The addition of ATP (100 μM) induced a rise in the [Ca²⁺]_m that was higher in Dpl-expressing cells than in the other two cell types. Presented data are typical of at least seven independent experiments, which yielded equivalent results.

Figure 6.

Monitoring [Ca²⁺]_er (A), [Ca²⁺]_c (B), [Ca²⁺]_pm (C), and [Ca²⁺]_m (D) in CHO cells transiently expressing the corresponding AEQ alone (control, light gray trace), or together with both PrP^c and Dpl (black trace). Experimental conditions were as reported in the legend to Figures 2A, 3B, 4B, and 5, respectively. Clearly, the copresence of PrP^c and Dpl abolished the divergent Ca²⁺ signaling observed in the various compartments of cells expressing PrP^c, or Dpl, alone. Presented data are typical of at least nine independent experiments, which yielded equivalent results.