completely elucidated. However, in the past years, several studies using recombinant PrP molecules highlighted potential mecha- nisms by which misfolded PrP may cause cell death and potential molecular determinants of such effects [51]. We developed an experimental model to study PrP neurotoxicity using an amino- terminally truncated PrP isoform (PrP90–231) that can transit from a PrPC-like to a PrPSc-like conformation by mild thermal denaturation. In these conditions hPrP90–231 become b-sheet structured, highly hydrophobic, amyloidogenic and neurotoxic. In this study we analyzed different and possibly more physiological mechanisms of hPrP90–231 transition toward the neurotoxic conformation, evaluating the role of Ca++ in this process. Moreover, comparing the effects of the recombinant protein with PrP27–30 isolated from infected hamster brain, we identified an altered ion current in synthetic lipid bilayers as marker of the interaction of neurotoxic PrP molecules with cell membranes. It is a whispered characteristic of amyloid compounds to be able to interact with lipid bilayers [52–54]. Not only amyloid compounds are found in close association with cell membrane but also they are responsible to cause a vast variety of electrophysiological transmembrane signals [55]. PrP is not an exception. Several investigations report the ability of PrP to induce changes in ionic conductance both in artificial [56,57] and cell [58] membranes. Whether PrP is able to form structures similar to conventional ion channels is still debatable. Whether the induction of ion flux is part of the toxicity mechanism responsible for cells death during insurgency of the pathological condition it is even more controversial [59]. In a previous report we demonstrate that PrPSc purified from infected hamster brain is able to open ionic conductance in which the most probable current level is around 80 pS. On the contrary the recombinant peptide, hPrP90–231, composed by the same amino acid primary sequence shows an average conductance of 20 pS [23]. In the present investigation we confirm these data and correlate the ionic conductance value with the toxicity level obtained in neuroblastoma cell culture. Cell death percentages follow proportionally the measured conductance calculated for the different conditions in which we treated the PrP90–231. Patho- logical PrPSc appears to be 5 folds more harmful when compared with recombinant peptide in its native, a-helix structured conformation. The arrangement of the secondary structure of PrP peptides is known to determine their cytotoxicity. Recombi- nant hPrP90–231 peptide is a versatile model to study the conformation-dependent toxicity [7,10]. In a previous study it was demonstrated that both mild thermal denaturation and the conformational changes induced by TSE-related point mutations drastically increases cell toxicity [17]. Here, using hPrP90–231 in Tip Dip experiments, we demonstrate that a higher level of cell death correspond to a higher membrane conductance values reaching values comparable to those induced by PrP 27–30. Importantly, similar results were obtained incubating w.t. and mutant hPrP90–231 molecules with increasing Ca++ concentra- tions. In these experimental conditions PrP truncated peptides increased ionic conductance and toxicity, with a highly significant direct relationship. Since these experimental conditions determine a rearrangement of the tridimensional structure of the peptides, we can conclude that the key conformational features present in the hamster prion responsible of the membrane interaction are likely reproduced in hPrP90–231. This is the first evidence that biological and electrophysiological characteristics of brain extracted PrP27–30 can be entirely reproduced by recombinant PrP fragments. Moreover, these results further support the use of hPrP90–231 for identification of the mechanisms of neuronal dysfunction and neurotoxicity in TSE Calcium Dependent Prion Protein Toxic Conformation patients and for the identification of potential new pharmacolog- ical approaches to be translate in clinical setting [22,49]. The second relevant observation coming out from this study is that the same neurotoxic conformation we previously described to be induced by mild thermal denaturation can be reproduced in the presence of high [Ca++]. In particular, accordingly to our molecular modeling, to induce hPrP90–231 gain of toxicity, Ca++ binding to the recombinant peptide might induce hPrP90– 231 aggregation that, in turn, is responsible of alterations in the peptide three-dimensional structure, increasing of its hydropho- bicity, and, finally inducing a partial resistance to protease K digestion. These observations suggest two additional consider- ations: 1) hPrP90–231, likely due to the N-terminal truncation, show reduced energy barrier and thus is more prone to adopt a neurotoxic conformation in different environmental conditions (mild thermal denaturation, high divalent ion concentrations); 2) the highly reproducible induction of the same biological effects by hPrP90–231, independently of the means used to induce the conformation change, suggest that this transition may represent the (or one of the) neurotoxic PrP conformation(s) in vivo. In this scenario the role of Ca++ on PrP peptide function it is of fundamental importance. Increase of [Ca++] from physiological levels up to 10 mM is directly responsible of the conformation changes that allow acquisition of the two main biological features identified in hPrP90–231: activation ionic conductance and increased toxicity. To understand how Ca++ binding can induce such a dramatic structural alteration, we performed in silico structural analysis comparing the amino acid charge distribution of PrP w.t. in the absence or presence of Ca++ binding in a specific coordination site, with the model of PrP E200K. In fact, from previous studies we identified two TSE-related PrP mutations (D202N, E200K) that differently affected PrP a-helix 3 stability [35]. Importantly, E200K mutation induces in hPrP90–231 a spontaneous acquisition of the toxic and ionotropic conformation, independently from thermal denaturation [17] or Ca++ binding. Thus, we verified were the three-dimensional alteration induced by the E200K mutation could be mimicked by Ca++ binding to PrP90–231. Our in silico study confirmed this hypothesis showing that Ca++ binding can occur in proximity of PrP pavement, in an interdomain region connecting H1, H2 and H3 domains. The analysis of electrostatic similarity in model I, II and III showed also that both Ca++-bound w.t. protein and the E200K mutant are characterized by a significant similarity in the pavement region. This result indicates that either the mutation or Ca++ binding exert similar effects in the charge distribution on protein surface. Importantly, our results indicated that both E200K substitution and Ca++ binding may reduce the strength of H1–H2 and H1–H3 interactions by a similar mechanism of electrostatic destabilization affecting the conformational stability of the protein and, thus, facilitating the PrPC RPrPSc transition. Consistently with this electrostatic interpretation, it is expected that the binding of other bivalent metal ions, such as Mg++, Zn++ or Cu++ (whose coordination features can be considered similar to Ca++) can also induce analogous effects on w.t. protein. This observation could be particularly relevant for Cu++ considering the known modulator role of this ion on PrP folding and, possibly, function [60–63]. The evaluation of Cu++ effects on hPrP90–231 structural, biochemical and biological function is currently in progress. Thus, using several different experimental approaches, we demonstrated that Ca++ binding to hPrP90–231 may favor the conversion of hPrP90–231 in a neurotoxic conformer. However, the experimental conditions responsible to convert PrP peptide in the harmful form are rather extreme (maximum effect at [Ca++] PLoS ONE | www.plosone.org 11 July 2012 | Volume 7 | Issue 7 | e38314