We have shown previously that the protease-resistant and neurotoxic prion peptide fragment PrP[106–126] of human PrP incorporates into lipid bilayer membranes to form heterogeneous ion channels, one of which is a Cu2+-sensitive fast cation channel. To investigate the role of PrP[106–126]’s hydrophobic core, AGAAAAGA, on its ability to form ion channels and their regulation with Cu2+, we used the lipid-bilayer technique to examine membrane currents induced as a result of PrP[106–126] (AA/SS) and PrP[106–126] (VVAA/SSSS) interaction with lipid membranes and channel formation. Channel analysis of the mutant (VVAAA/SSS), which has a reduced hydrophobicity due to substitution of hydrophobic residues with the hydrophilic serine residue, showed a significant change in channel activity, which reflects a decrease in the β-sheet structure, as shown by CD spectroscopy. One of the channels formed by the PrP[106–126] mutant has fast kinetics with three modes: burst, open and spike. The biophysical properties of this channel are similar to those of channels formed with other aggregation-prone amyloids, indicating their ability to form the common β sheet-based channel structure. The current-voltage (I–V) relationship of the fast cation channel, which had a reversal potential, E rev , between -40 and -10 mV, close to the equilibrium potential for K+ (E K = - 35 mV), exhibited a sigmoidal shape. The value of the maximal slope conductance (gmax) was 58 pS at positive potentials between 0 and 140 mV. Cu2+ shifted the kinetics of the channel from being in the open and “burst” states to the spike mode. Cu2+ reduced the probability of the channel being open (P o ) and the mean open time (To) and increased the channel’s opening frequency (Fo) and the mean closed time (Tc) at a membrane potential (Vm) between + 20 and +140 mV. The fact that Cu2+ induced changes in the kinetics of this channel with no changes in its conductance, indicates that Cu2+ binds at the mouth of the channel via a fast channel block mechanism. The Cu2+-induced changes in the kinetic parameters of this channel suggest that the hydrophobic core is not a ligand Cu2+ site, and they are in agreement with the suggestion that the Cu2+-binding site is located at M109 and H111 of this prion fragment. Although the data indicate that the hydrophobic core sequence plays a role in PrP[106–126] channel formation, it is not a binding site for Cu2+. We suggest that the role of the hydrophobic region in modulating PrP toxicity is to influence PrP assembly into neurotoxic channel conformations. Such conformations may underlie toxicity observed in prion diseases. We further suggest that the conversions of the normal cellular isoform of prion protein (PrPc) to abnormal scrapie isoform (PrPSc) and intermediates represent conversions to protease-resistant neurotoxic channel conformations.
The Journal of Membrane Biology – Springer Journals
Published: Mar 25, 2002
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