Calcium Dependent Prion Protein Toxic Conformation Figure 3. Toxicity versus membrane ionic conductance of the hPrP90–231 peptide. Histograms in panel A and B compare neuroblastoma SH-SY5Y cell death percentage and Tip Dip membrane ionic conductance of PrP27–30 protein (scrapie) with hPrP90–231 peptide in different experimental condition reported below each histogram. In panel C and D the same parallel study was done using two different hPrP90–231 mutants: D202N and E200K. Cell death was measured using the MTT assay after 48 hours of treatment. * = p,0.01 vs. scrapie-induced effects. doi:10.1371/journal.pone.0038314.g003 drastic change in D202N. Conductance values calculated here by Tip Dip technique are 2462.1 and 8062.2 pS for D202N and E200K mutants, respectively. Thereby, the membrane conduc- tance change induced by the two mutations proved that while the hPrP90–231(D202N) shows a same conductance value of the w.t. peptide, hPrP90–231(E200K) induces a high level of ion conductivity already in its native conformation paralleling its effects on SHSY5Y cell survival. The exposure to high calcium level did not change the E200K mutant powerful effects on membrane conductance; on the contrary, mimicking the native recombinant, the peptide bearing the D202N mutation increases cytotoxicity and conductance in the presence of both partial denaturation procedure or high calcium exposure (Fig. 3C and D). Relationships between PrP27–30- and hPrP90–231 Forms Induced Ionic Current and Cell Toxicity From all these experiments cell toxicity and membrane conductance appear to be strictly correlated. To demonstrate this relationship we performed a linear regression analysis comparing the amount of cell death and the ionic conductance of hPrP90– 231 w.t. and the mutants using the data obtained in all the previous experiments. The plot depicted in Figure 4 shows 18 different data points obtained in parallel electrophysiological and cell survival experiments in several different conditions, including the non pathogenic GST protein. There is a good correlation between the toxicity data and the increase of membrane conductivity operated by the diverse PrP peptides tested in all the different conditions, showing a highly statistical significance (regression coefficient, R2: 0.93, P,0.0001, Fig. 4). Finally, to demonstrate the relationship between ionic conduc- tance and cell toxicity in experimental conditions in which the structural alterations of hPrP90–231 may spontaneously occur, we evaluated the effects of the peptide after its prolonged incubation at 37uC before being added to the cells (toxicity experiments) or to the lipid bilayer (electrophysiology experiments). In fact, we previously suggested that thermal denaturation at 53uC catalyzes a spontaneous process already occurring at 37uC, reducing the time required for the structural alterations responsible of the gain of toxicity of hPrP90–231 [7]. In line with this hypothesis, we demonstrated that prolonged incubation at 37uC (5 to 24 hrs) favored the acquisition a conformation highly toxic for SH-SY5Y cells in culture (Fig. 5A). Importantly, the same treatment also increased ionic conductance (Fig. 5B), further correlating the two phenomena. For comparison to the two plots were added grey horizontal bands representing the range of high toxicity associated with a large conductance obtained treating hPrP90–231 for a very short time in different conditions (see Fig. 3). Longer pre- incubations at 37uC (5, 15, 24, and 48 hrs) showed a lower response in terms of cell death and higher ionic conductance (Fig. 5A, B). At 72 hours the almost total recovery of cell viability is not followed by a decrease in ionic membrane conductance. This is probably due to the few highly reactive oligomers left during the PLoS ONE | www.plosone.org 5 July 2012 | Volume 7 | Issue 7 | e38314