A major prion protein (PrP) mutant that forms amyloid fibrils in the diseased brain of patients with Gerstmann-Sträussler-Scheinker syndrome (GSS) is a fragment of 7 kDa spanning from residues 81-82 to 144-153 of PrP. Analysis of ionic membrane currents, recorded with a lipid bilayer technique, revealed that the wild-type fragment PrP(82-146) WT and the partially scrambled PrP(82-146) (127-146) SC are capable of forming heterogeneous ion channels that are similar to those channels formed with PrP(106-126). In contrast, PrP(82-146) peptides in which the region from residue 106 to 126 had been scrambled (SC) showed a reduction in interaction with lipid membranes and did not form channels. The PrP(82-146) WT- and PrP(82-146) (127-146) SC-formed cation channels with fast kinetics are Cu2+ sensitive and rifampicin (RIF) insensitive, whereas the time-dependent inactivating channels formed by these same peptides are both Cu2+ and RIF insensitive. The presence of RIF in the solution before the addition of PrP(82-146) WT or PrP(82-146) (127-146) SC affected their incorporation into the lipid bilayers. PrP(82-146) WT and PrP(82-146) (127-146) SC fast cation channels formed in the presence of RIF appeared in an electrically semisilent state or an inactivated state. Increasing [Cd2+]cis enhanced the incorporation of PrP(82-146) WT and PrP(82-146) (127-146) SC channels formed in the presence of RIF. We conclude that the major PrP mutant fragment in the diseased brain of GSS patients is prone to form channels in neuronal membranes, causing their dysfunction. We propose that Cd2+ may accentuate the neurotoxicity of this channel-forming PrP fragment by enhancing its incorporation into the membrane.
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 Cu(2+)-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 Cu(2+), 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 beta-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 beta 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 (g(max)) was 58 pS at positive potentials between 0 and 140 mV. Cu(2+) shifted the kinetics of the channel from being in the open and "burst" states to the spike mode. Cu(2+) reduced the probability of the channel being open (P(o)) and the mean open time (T(o)) and increased the channel's opening frequency (F(o)) and the mean closed time (T(c)) at a membrane potential ( V(m)) between +20 and + 140 mV. The fact that Cu(2+) induced changes in the kinetics of this channel with no changes in its conductance, indicates that Cu(2+) binds at the mouth of the channel via a fast channel block mechanism. The Cu(2+)-induced changes in the kinetic parameters of this channel suggest that the hydrophobic core is not a ligand Cu(2+) site, and they are in agreement with the suggestion that the Cu(2+)-binding site is located at M(109) and H(111) 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 Cu(2+). 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 (PrP(c)) to abnormal scrapie isoform (PrP(Sc)) and intermediates represent conversions to protease-resistant neurotoxic channel conformations.
We found that the amyloid  peptide A(1-42) is capable of interacting with membrane and forming heterogeneous ion channels in the absence of any added Cu 2ϩ or biological redox agents that have been reported to mediate A(1-42) toxicity. binds to the histidine residues located at the mouth of the channel. It is proposed that the Cu 2ϩ -binding site of the A(1-42)-formed channels is modulated with Cu 2ϩ in a similar way to those of channels formed with the prion protein fragment PrP(106-126), suggesting a possible common mechanism for Cu 2ϩ modulation of A and PrP channel proteins linked to neurodegenerative diseases. neurodegenerative diseases; transitional metals; ion channel pathologies; membrane injuries; calcium homeostasis ALZHEIMER'S DISEASE (AD) is a neurodegenerative disorder that affects the cognitive function of the brain. Pathological changes in AD are characterized by the formation of amyloid plaques and neurofibrillary tangles as well as extensive neuronal loss. The plaques, which accumulate extracellularly in the brain, are composed of aggregates and cause direct neurotoxic effects and/or increase neuronal vulnerability to excitotoxic insults. The major components of the extracellular neurofibrillar bundles are polymerized amyloid  (A) peptides A(1-40), A(1-42), and A(1-43). It has been shown that A familial AD-linked mutations of the amyloid protein precursors presenilin-1 and presenilin-2 increase the concentration of A(1-42) (53), which has been shown to be toxic in primary neuronal culture at micromolar concentrations (56). The major mechanisms proposed for A-induced cytotoxicity involve the loss of Ca 2ϩ homeostasis (see Refs. 46 and 47) and the generation of reactive oxygen species (see Refs. 9,11,12,and 25). The changes in Ca 2ϩ homeostasis could be the result of 1) alterations in endogenous ion transport systems and 2) formation of heterogeneous ion channels (see Refs. 32,33,and 35). Several laboratories have found that A(1-40) and other fragments of amyloid precursor protein that contain A also possess the ability to form ion channels in both artificial and biological membranes. Electrophysiological studies have shown that A fragments, e.g., A(25-35), A(1-40), and A(1-42), elicit cation-selective currents when reconstituted into lipid bilayers (1-4, 23, 24, 30, 35, 37, 41, 55) and in the plasma membrane of neurons (28,29,48) as well as in Xenopus oocytes (21). A(1-42) and A(1-40) increase Ca 2ϩ uptake in liposomes in a dose-dependent manner (38, 49), and soluble As induce Ca 2ϩ influx in neurons and nonneuronal cells (10,50,51,57).In addition to the A being linked to AD, a role for transition metals has also been recognized. Cu 2ϩ and Zn 2ϩ have been implicated in AD (11,12,42), Parkinson's disease (54), prion protein (PrP) (26), and immunoglobulin light chain amyloidosis (17). The mechanisms underlying the interaction between A and these metals may mediate their role in neurotoxicity. There is also evidence to show that A, and also PrP, binds Cu 2ϩ (5, 6) to a si...
We investigated the action of the acridine derivative, quinacrine (QC), which has been shown to act as a noncompetitive channel inhibitor. The main effects of QC are voltage- and concentration-dependent changes in the kinetics of the prion protein fragment (PrP[106-126])-formed cation channels. The current-voltage relationships show that the maximal current (I) was not affected whereas the physiologically important mean current (I') was reduced as a result of changes in channel kinetics. These findings suggest that QC acts on the open state of the channels. The half-inhibitory concentration (IC50) for the dose-dependent effects of [QC]cis on the kinetic parameters of the PrP(106-126)-formed cation channel shows a reduction in the ratios Po(QC)/Po, Fo(QC)/Fo, and To(QC)/To, whereas Tc(QC)/Tc increases. Of these ratios, Po(QC)/Po was more sensitive than the others. The corresponding IC50 for these ratios were 51, 94, 86, and 250 microM QC, respectively. The QC-induced changes in the kinetic parameters were more apparent at positive voltages. IC50 values for Po were 95, 75, and 51 microM at +20, +80, and +140 mV, respectively. The fact that QC induced changes in the kinetics of this channel, although the conductance of the channel remained unchanged, indicates that QC may bind at the mouth of the channel via a mechanism known as fast channel block. The QC-induced changes in the kinetic parameters of this channel suggest that they are pathophysiologically significant because these channels could be the mechanisms by which amyloids induce membrane damage in vivo.
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