Calcium Dependent Prion Protein Toxic Conformation Figure 1. Comparison between PrP27–30 protein and hPrP90–231 peptide in Tip Dip electrophysiological experiments. Fifteen consecutive current recordings obtained at 80 mV membrane potential are shown for infected- brain-extracted protein (panel A) and recombinant PrP peptide (panel B). Panels C and D depict corresponding current amplitude histograms. Histograms were built at each test potential and used to calculate the ionic pathway conductance in the current/voltages relationship showed in the bottom of the figure (panels E and F). From the linear regression of the experimental points (dotted lines) we calculate a conductance of 7260.23 to 2460.7 pS for PrP27–30 protein and hPrP90–231 peptide respectively (n = 5). doi:10.1371/journal.pone.0038314.g001 denaturation were compared with those elicited following the incubation with increasing calcium concentrations. The results showed that the pre-incubation of hPrP90–231 with 1.8 mM Ca++ was peculiarly devoid of any effect on SH-SY5Y cell viability while 5 mM Ca++ resulted in moderate cell death. Indeed, preincuba- tion with 10 mM Ca++ did induce a cell toxicity comparable to the thermal denatured peptide, after 2 days of treatment. These data are all the more meaningful if we consider that the culture medium itself contains high [Ca++]. Interestingly, the level of in vitro Figure 2. Calcium concentration modulates the ionic conduc- tance induced by hPrP90–231 recombinant peptide. A: current/ voltage relationship obtained from Tip Dip experiment using the peptide in 5 different calcium concentrations. B: the current/voltage relationships were obtained from experiments performed in 1.8 mM calcium concentration but the peptide was prior incubated for 1 hour in 4 different calcium concentrations. C: the histogram shows the conductance values versus calcium concentrations either present in the external solution during the experiment or only used during hPrP90–231 incubation. The conductance in the two different conditions show similar values and reaches its maximum at 10 mM calcium. Black and white bars visualize the calcium increase (n = 5). doi:10.1371/journal.pone.0038314.g002 cytotoxicity induced by thermally denatured and calcium treated hPrP90–231 is similar with that elicited by brain extracted Syrian hamster PrP27–30 (Fig. 3A). In the histograms of Figure 3B we compare the membrane conductance obtained using the manipulated hPrP90–231 peptide in Tip Dip experiments, using the same treatment reported in the cytotoxicity experiments depicted in Figure 3A. After mild thermal denaturation as well as after pre-incubation in high [Ca++] hPrP90–231 drastically increases membrane conductance to a level similar to that induced by PrP27–30. These results suggest that mild thermal partial denaturation and high [Ca++] play a similar role on the functional activity of the peptide that seems to be not linked to an increase of calcium ion permeability. We hypothesized that Ca++ binding to the peptide is responsible of a rearrangement of its conformation to acquire similar characteris- tics to those obtained during the thermal denaturation procedure. To probe this hypothesis, we pre-incubated hPrP90–231 in a solution containing 10 mM CaCl2. Just before testing the peptide in the Tip Dip apparatus, we diluted (1:500) the solution to reach 1.8 mM [Ca++] (Fig. 3B). Also in these experimental conditions hPrP90–231 greatly affected ionic conductance. Even if thermal denaturation procedure showed a bigger impact on membrane conductance, pre-incubation with 10 mM Ca++ caused a substan- tial increase of ion membrane permeability and the two values that were not statistically different (n = 14). Similarly, applying the same protocol, we demonstrate that hPrP90–231 preincubated with 10 mM Ca++and then diluted to 1.8 mM, retains also its toxic effects (Fig. 3A). Finally the effects of incubation with 10 mM Ca++ and thermal denaturation were not additive, as far as both cell toxicity and membrane conductance (Fig. 3A and 3B), supporting the hypothesis that the two protocols may induce the same structural alterations. The effects of 10 calcium was prevented by addition of EGTA into the incubation medium. 25 mM of the divalent ion chelator mixed with 10 mM calcium results in a final calcium concentration of 1.6 mM (Clampex, Molecular Device, Novato CA) (Fig. 3A and B). As a control, both in toxicity and lipid bilayer conductance experiments, we use a purified GST protein at the same concentration of hPrP901–231. In three different experimental conditions, 1.8 and 10 mM Ca++ as well as after thermal denaturation at 53uC, there was no significant cell toxicity (2% 65, 4% 63 and 0% 69, respectively) neither evident transmembrane ionic flow activity. In Figure 3A and B we report only the results of GST preincubated with 10 mM Ca++. The general idea coming out from our experiments is that a direct link exists between membrane ionic conductance, promoted by the PrP after different treatments, and the ability of the same peptide to be harmful for the cells: larger conductance results in a higher level of toxicity. Relationship between cytotoxicity and perturbation on the membrane ion conductivity was also tested for single point mutated forms hPrP90–231(E200K), that has been demonstrated [17] to be much more toxic in its native conformation than the wt recombinant peptide or the hPrP90–231(D202N) form, bearing a different point mutation (Fig. 3C and D). Partial denaturation procedure or the exposure to high calcium level did not change the E200K mutant powerful effects on cell viability while it causes PLoS ONE | www.plosone.org 4 July 2012 | Volume 7 | Issue 7 | e38314