Biophys J, August 2000, p. 934-944, Vol. 79, No. 2

Displacement Currents Associated with the Insertion of Alzheimer Disease Amyloid -Peptide into Planar Bilayer Membranes

J. Vargas,^* J. M. Alarcón,^* and E. Rojas^*

 ^*Institute of Biomedical Sciences, Faculty of Medicine, University of Chile, Santiago, Chile,  Laboratory of Biochemistry and Cell Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892 USA and  Sansum Medical Research Institute, Santa Barbara, CA 93105 USA

The role of endogenous amyloid -peptides as causal factors of neurodegenerative diseases is largely unknown. We have previously reported that interactions between Alzheimer's disease AP[1-40] peptide in solution and planar bilayer membranes made from anionic phospholipids lead to the formation of cation-selective channels. We now find and report here that the spontaneous insertion of free AP[1-40] across the bilayer can be detected as an increase in bilayer capacity. To this end we recorded the displacement currents across planar bilayers (50 mM KCl on both sides) in response to sudden displacements of the membrane potential, from 300 to 300 mV in 20-mV increments. To monitor the AP[1-40]-specific displacement currents, we added AP[1-40] (1-5 µM) to the solution on either side of the membrane and noted that the direction of the displacement current depended on the side with AP[1-40]. The size of the AP[1-40]-specific charge displaced during a pulse was always equal to the charge returning to the original configuration after the pulse, suggesting that the dipole molecules are confined to the membrane. As a rule, the steady-state distribution of the AP[1-40]-specific charges within the bilayer could be fit by a Boltzmann distribution. The potential at which the charges were found to be equally distributed (V_o) were ~ 135 mV (peptide added to the solution in the compartment electrically connected to earth) and 135 mV (peptide added to the solution connected to the input of the amplifier). The AP[1-40]-specific transfer of charge reached a maximum value (Q_max) when the electrical potential of the side containing the amyloid -protein was taken to either 300 or 300 mV. For a circular membrane of 25-µm radius (~2000 µm²), the total AP[1-40]-specific charge Q_max was estimated as 55 fC, corresponding to some 170 e.c./µm². Regardless of the side selected for the addition of AP[1-40], at V_o the charge displaced underwent an e-fold change for a ~27-mV change in potential. The effective valence (a) of the AP[1-40] dipole (i.e., the actual valence Z multiplied by the fraction of the electric field  acting on the dipole) varied from 1 to 2 electronic charges. We also tested, with negative results, the amyloid peptide with the reverse sequence (AP[40-1]). These data demonstrate that AP[1-40] molecules can span the low dielectric domain of the bilayer, exposing charged residues (D₁, E₃, R₅, H₆, D₇, E₁₁, H₁₃, and H₁₄) to the electric field. Thus the AP[1-40] molecules in solution must spontaneously acquire suitable conformations (-pleated sheet) allowing specific interactions with charged phospholipids. Interestingly, the domain from residues 676 to 704 in the APP₇₅₁ is homologous with the consensus sequence for lipid binding found in other membrane proteins regulated by anionic phospholipids.

Biophys J, August 2000, p. 934-944, Vol. 79, No. 2
© 2000 by the Biophysical Society   0006-3495/00/08/934/11  $2.00

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