Abstract
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.
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Abbreviations
- Aβ:
-
amyloid β-peptide
- CD:
-
circular dichroism spectroscopy
- PrP:
-
prion protein
- PrPc :
-
normal cellular form of prion proteins
- TSE:
-
transmissible spongiform encephalopathy
References
Arispe, N., Pollard, H.B., Rojas, E. 1996. Zn2+ interaction with Alzheimer amyloid beta protein calcium channels. Proc. Natl. Acad. Sci. USA 93:1710–1715
Barrow, C.J., Yasuda, A., Kenny, P.T., Zagorski, M.G. 1992. Solution conformations and aggregational properties of synthetic amyloid beta-peptides of Alzheimer’s disease. Analysis of circular dichroism spectra. J. Mol. Biol. 225:1075–1093
Barrow, C.J., Zagorski, M.G. 1991. Solution structures of beta peptide and its constituent fragments: relation to amyloid deposition. Science 253:79–182
Barrow, P.A., Holmgren, C.D., Tapper, A.J., Jefferys, J.G. 1999. Intrinsic physiological and morphological properties of principal cells of the hippocampus and neocortex in hamsters infected with scrapie. Neurobiol. Dis. 6:406–423
Barry, P.H. 1994. JPCalc, a Software package for calculating liquid junction potential corrections in patch-clamp, intracellular, epithelial and bilayer measurements and for correcting junction potential measurements. J. Neurosci. Meth. 51:107–116
Brown, D.R., Clive, C., Haswell, S.J. 2001. Antioxidant activity related to copper binding of native prion protein. J. Neurochem. 76:69–76
Brown, D.R., Herms, J., Kretzschmar, H.A. 1994. Mouse cortical cells lacking cellular PrP survive in culture with a neurotoxic PrP fragment. Neuroreport 5:2057–2060
Bucciantini, M., Giannoni, E., Chiti, F., Baroni, F., Formigli, L., Zurdo, J., Taddei, N., Ramponi, G., Dobson, C.M., Stefani, M. 2002. Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases. Nature 416:507–511
Bush, A.I. 2000. Metals and neuroscience. Curr. Opin. Chem. Biol. 4:184–191
Chapron, Y., Peyrin, J.M., Crouzy, S., Jaegly, A., Dormont, D. 2000. Theoretical analysis of the implication of PrP in neuronal death during transmissible subacute spongiform encephalopathies: hypothesis of a PrP oligomeric channel. J. Theor. Biol. 204:103–111
Colquhoun, D., Hawkes, A.G. 1983. Fitting and statistical analysis of single-channel recording. In: Single Channel Recording. B. Sakmann and E. Neher, editors. pp. 135–175. Plenum, New-York
Durell, S.R., Guy, H.R., Arispe, N., Rojas, E., Pollard, H.B. 1994. Theoretical models of the ion channel structure of amyloid beta-protein. Biophys. J. 67:2137–2145
Florio, T., Grimaldi, M., Scorziello, A., Salmona, M., Bugiani, O., Tagliavini, F., Forloni, G., Schettini, G. 1996. Intracellular calcium rise through L-type calcium channels, as molecular mechanism for the prion protein fragment 106–126-induced astroglial proliferation. Biochem. Biophys. Res. Commun. 228:397–405
Forloni, G., Del Bo, R., Angeretti, N., Smiroldo, S., Gabellini, N., Vantini, G. 1993. Nerve growth factor does not influence the expression of beta amyloid precursor protein mRNA in rat brain: in vivo and in vitro studies. Brain. Res. 620:292–296
Gu, Y., Fujioka, H., Mishra, R.S., Li, R., Singh, N. 2002. Prion peptide 106–126 modulates the aggregation of cellular prion protein and induces the synthesis of potentially neurotoxic transmembrane PrP. J. Biol. Chem. 277:2275–2286
Hilbich, C., Kisters-Woike, B., Reed, J., Masters, C.L., Beyreuther, K. 1992. Substitutions of hydrophobic amino acids reduce the amyloidogenicity of Alzheimer’s disease beta A4 peptides. J. Mol. Biol. 228:460–473
Hirakura, Y., Yiu, W.W., Yamamoto, A., Kagan, B.L. 2000. Amyloid peptide channels: blockade by zinc and inhibition by Congo red (amyloid channel block). Amyloids 3:194–199
Jobling, M.F., Huang, X., Stewart, L.R., Barnham, K.J., Curtain, C.C., Volitakis, I., Perugini, M., White, A.R., Cherny, R.A., Masters, C.L., Barrow, C.J., Collins, S.J., Bush, A.I., Cappai, R. 2001. Copper and zinc binding modulates the aggregation and neurotoxic properties of the prion peptide PrP 106–126. Biochem. 40:8073–8084
Jobling, M.F., Stewart, L.R., White, A.R., McLean, C., Friedhuber, A., Maher, F., Beyreuther, K., Masters, C.L., Barrow, C.J., Collins, S.J., Cappai, R. 1999. The hydrophobic core sequence modulates the neurotoxic and secondary structure properties of the prion peptide 106-126. J. Neurochem. 73:1557–1565
Kawahara, M., Kuroda, Y., Arispe, N., Rojas, E. 2000. Alzheimer’s disease-amyloid, human islet amylin, and prion protein fragment evoke intracellular free calcium elevations by a common mechanism in hypothalamic GnRH neuronal cell line. J. Biol. Chem. 275:14077–14083
Kourie, J.I. 1996. Vagaries of artificial bilayers and gating modes of the SCl channel from the sarcoplasmic reticulum muscle. J. Membr. Sci. 116:221–227
Kourie, J.I. 1999. Calcium dependence of C-type natriuretic peptide-formed fast K+ channel. Am. J. Physiol. 277:C43-C50
Kourie, J.I. 2001. Mechanisms of prion-induced modification in membrane transport systems. Chemico-Biological Interactions 138:1–26
Kourie, J.I., Culverson, A. 2000. Prion peptide fragment PrP[106–126] forms distinct cation channel types. J. Neurosci. Res. 62:120–133
Kourie, J.I., Farrelly, P.V., Henry, C.L. 2001a. Channel activity of deamidated isoforms of prion protein fragment 106–126 in planar lipid bilayers. J. Neurosci. Res. 66:214–220
Kourie, J.I., Henry, C.L. 2001. Protein aggregation and deposition: Implications for ion channel formation and membrane damage. Crot. Med. Res. 42:358–373
Kourie, J.L., Henry, C.L. 2002. Ion channel formation and membrane-linked pathologies of misfolded hydrophobic proteins: The role of dangerous unchaperoned molecules. Clin. Exp. Pharmacol. Physiol. 29:741–753
Kourie, J.I., Henry, C.L., Farrelly, P.V. 2001b. Diversity of amyloid β protein fragment [l–40]-formed channels. Cell. Mol. Neurobiol. 21:255–284
Kourie, J.I., Laver, D.R., Junankar, P.R., Gage, P.W., Dulhunty, A.F. 1996. Characteristic of two types of chloride channel in sarcoplasmic reticulum vesicles from rabbit skeletal muscle. Biophys. J. 70:202–221
Lashuel, H.A., Hartley, D., Petre, B.M., Walz, T., Lansbury P.T. Jr. 2002. Neurodegenerative disease: amyloid pores from pathogenic mutations. Nature 418:291
Lin, M.-C, Mirzabekov, T., Kagan, B.L. 1997. Channel formation by a neurotoxic prion protein fragment. J. Biol. Chem. 272:44–47
Miller, C., Racker, E. 1976. Ca+ +-induced fusion of fragmented sarcoplasmic reticulum with artificial planar bilayers. J. Membrane Biol. 30:283–300
Mobashery, N., Nielsen, C., Andersen, O.S. 1997. The conformational preference of gramicidin channels is a function of lipid bilayer thickness. FEBS Lett. 412:15–20
Pan, K.-M., Baldwin, M., Nguyen, J., Gasset, M., Serban A., Groth D., Melhorn I., Huang Z., Flettenck R.J., Cohen F.E., Prusiner S.B. 1993. Conversion of α-helices into β-sheets features in the formation of the scrapie prion proteins. Proc. Natl. Acad. Sci. USA 90:10962–10966
Patlak, J.B. 1993. Measuring kinetics of complex single ion channel data using mean-variance histograms. Biophys. J. 65:29–42
Pike, C.J., Walencewicz-Wasserman, A.J., Kosmoski, J., Cribbs, D.H., Glabe, C.G., Cotman, C.W. 1995. Structure-activity analyses of β-amyloid peptides: contributions of the β25–35 region to aggregation and neurotoxicity. J. Neurochem. 64:253–265
Prusiner, S.B., Scott, M.R., DeArmond, S.J., Cohen, F.E. 1998. Prion protein biology. Cell 93:337–348
Singh, N., Gu, Y., Bose, S., Kalepu, S., Mishra, R.S., Verghese, S. 2002. Prion peptide 106–126 as a model for prion replication and neurotoxicity. Front. Biosci. 7:60–71
Stewart, L.R., White, A.R., Jobling, M.F., Needham, B.E., Maher, F., Thyer, J., Beyreuther, K., Masters, C.L., Collins, S.J., Cappai, R. 2001. Involvement of the 5-lipoxygenase pathway in the neurotoxicity of the prion peptide PrP106–126. J. Neurosci. 65:565–572
Tagliavini, F., Forloni, G., Bugiani, O., Salmona, M. 2001. Studies on peptide fragments of prion proteins. Adv. Protein Chem. 57:171–201
Warwicker, J. 1999. Modelling charge interactions in prion protein: predictions for pathogenesis. FEBS Lett. 450:144–148
White, A.R., Guirguis, R., Brazier, M.W., Jobling, M.F., Hill, A.F., Beyreuther, K., Barrow, C.J., Masters, C.L., Collins, S.J., Cappai, R. 2001. Sublethal concentrations of prion peptide PrP106–126 or the amyloid beta peptide of Alzheimer’s disease activates expression of proapoptotic markers in primary cortical neurons. Neurobiol. Dis. 8:299–316
Wong, B.-S., Pan, T., Liu, T., Li, R., Petersen, R.B., Jones, I.M., Gambetti, P., Brown, D.R., Sy, M.-S. 2000. Prion disease: A loss of antioxidant function. Biochem. Biophys. Res. Commun. 275:249–252
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Kourie, J.I., Kenna, B.L., Tew, D. et al. Copper Modulation of Ion Channels of PrP[106–126] Mutant Prion Peptide Fragments. J. Membrain Biol. 193, 35–45 (2003). https://doi.org/10.1007/s00232-002-2005-5
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DOI: https://doi.org/10.1007/s00232-002-2005-5