Soluble amyloid oligomers increase bilayer conductance by altering dielectric structure

Abstract

The amyloid hypothesis of Alzheimer's toxicity has undergone a resurgence with increasing evidence that it is not amyloid fibrils but a smaller oligomeric species that produces the deleterious results. In this paper we address the mechanism of this toxicity. Only oligomers increase the conductance of lipid bilayers and patch-clamped mammalian cells, producing almost identical current-voltage curves in both preparations. Oligomers increase the conductance of the bare bilayer, the cation conductance induced by nonactin, and the anion conductance induced by tetraphenyl borate. Negative charge reduces the sensitivity of the membrane to amyloid, but cholesterol has little effect. In contrast, the area compressibility of the lipid has a very large effect. Membranes with a large area compressibility modulus are almost insensitive to amyloid oligomers, but membranes formed from soft, highly compressible lipids are highly susceptible to amyloid oligomer-induced conductance changes. Furthermore, membranes formed using the solvent decane (instead of squalane) are completely insensitive to the presence of oligomers. One simple explanation for these effects on bilayer conductance is that amyloid oligomers increase the area per molecule of the membrane-forming lipids, thus thinning the membrane, lowering the dielectric barrier, and increasing the conductance of any mechanism sensitive to the dielectric barrier.

Figure 1.

A high sensitivity recording of the current induced by Aβ oligomers. The current was recorded at 100 mV. The “control” trace was recorded before the addition of Aβ oligomers. Successive traces above the control trace were recorded at 10–20-s intervals in the 2-min period following the addition of Aβ oligomers to a concentration of 0.1 μM. Note that there is no increase in current noise as the current increases to ∼80 pA. Bilayer composition was PC/PE (1/1, molar).

Figure 2.

Amyloid-induced conductance depends on the electrolyte concentration of the bathing solution as shown by these three dose–response curves in electrolytes ranging in concentration from 100 mM KCL to 1 mM HEPES-Tris. Bilayer composition was PC/PS (1/1, molar), voltage was 150 mV.

Figure 3.

The reversal potentials measured for asymmetric solutions of different ionic composition in the presence of 4 μM Aβ oligomers added to one side. Trace 1, 100 mM KCl, cis; 100 mM CaCl₂, trans. Trace 2, 100 mM KCl, cis; 50 mM CaCl₂, trans. Bilayer composition was PC/PE (1/1, molar).

Figure 4.

The effect of Aβ oligomers on the conductance induced by nonactin. (A) The bottom trace shows the current measured at 150 mV induced by Aβ oligomers. Additions of Aβ oligomers and the resulting concentrations (μM) are shown by the arrows. The top trace shows a similar experiment performed in the presence of 10⁻⁸ M nonactin but with only two additions of amyloid, corresponding to the two lowest concentrations in the bottom trace. The bath solution contained 10 mM KCl in both cases, lipid composition was PC/PS (1/1, molar). (B) Normalized nonactin conductance and bare bilayer conductance as a function of Aβ oligomer concentration. Conductance at a given Aβ oligomer concentration divided by the conductance at 0 Aβ oligomer concentration is plotted against Aβ oligomer concentration. The initial conductance of the nonactin-K⁺ membrane was >200 times that of bare bilayer. Note that the dose–response curve for nonactin is essentially a multiple of the bare bilayer dose–response curve.

Figure 5.

The effect of Aβ oligomers on the rate of tetraphenyl borate translocation. (A) The currents induced by tetraphenyl borate in a PC/PS bilayer as a response to voltage pulses of +300 mV. The tetraphenyl borate concentration was 10⁻⁶ M on both sides of the membrane in 10 mM KCl. Solid, control; dashed, 4 μM Aβ oligomers; dotted, 16 μM Aβ oligomers. (B) The translocation rate of tetraphenyl borate ions deduced from the time constants as a function of Aβ oligomer concentration.

Figure 6.

Amyloids act on mammalian RBL cells in almost exactly the same fashion in which they act on lipid bilayers. Three current voltage curves are shown. A control trace was taken before the addition of Aβ oligomers. The second trace, taken after addition of Aβ oligomers to a concentration of 12 μM, shows a current–voltage curve nearly identical in shape to those obtained after addition of Aβ oligomers to solutions bathing planar lipid bilayers. A final trace was taken after washout.

Figure 7.

(A) Current recorded in patch-clamped RBL cells during wash in and wash out of 12 mM Aβ oligomers. (B) Increase of bilayer conductivity after addition of 4 μM Aβ oligomers to the cis compartment and its decrease after the perfusion of this compartment with Aβ oligomer-free solution. The break in the record is due to high noise of bilayer current during the perfusion. The current was measured at 150 mV. The bath solution was 10 mM KCl, bilayer composition was PC/PE (1/1, molar).

Figure 8.

Immunological specificity of the Aβ oligomer–induced conductance. The filled circles show the current increase of a lipid bilayer clamped at 150 mV after the addition of Aβ oligomers to a concentration of 0.25 μM. In this case there was only a single addition. The open circles show the effect of addition of 0.25 μM Aβ oligomers preincubated with an excess (4:1 antibody molar ratio to Aβ) of antibody. Subsequent additions (arrows) of Aβ and antibody in the same ratio raise the aqueous concentration first to 0.5 μM and then to 1.0 μM. This result demonstrates the same immunospecificity of the Aβ oligomer–induced conductance seen when antibody is added after the establishment of an elevated conductance by the addition of oligomer (Kayed et al., 2004).

Figure 9.

Effect of charge and cholesterol on the amyloid-induced conductance. Currents measured at a voltage of 150 mV in bilayers of different lipid compositions versus Aβ oligomer concentration in 10 mM KCl.

Figure 10.

Effect of lipid composition on the Aβ oligomer–induced conductance measured in symmetric and asymmetric bilayers. The top panel shows the effect of Aβ oligomers additions to the cis and trans sides of bilayers formed with pure PS and PC. Note that both cis and trans addition produced approximately the same increase in conductance. Scales of the left and right figures differ. The bottom panel shows the effect of Aβ oligomers on asymmetric bilayers with one leaflet containing PC and another PS. Note that addition of Aβ to the PC side of bilayer produced a much greater effect.

Figure 11.

The effect of decane on the conductance and capacitance increase induced by Aβ oligomers. Time course of both current (open circles) and capacitance (closed circles) with successive additions of Aβ followed by the addition of a suspension containing decane. The current was measured at +150 mV. Bilayer composition: PC/ PE = 1/1 (molar). Bath solution 10 mM KCl.

Figure 12.

Aβ oligomers are ineffective in sphingomyelin bilayers. A series of dose–response curves for Aβ oligomers in membranes ranging from pure PC to pure SM. Currents were measured at 150 mV in 10 mM KCl.