Amyloid ion channels: a common structural link for protein-misfolding disease

Abstract

Protein conformational diseases, including Alzheimer's, Huntington's, and Parkinson's diseases, result from protein misfolding, giving a distinct fibrillar feature termed amyloid. Recent studies show that only the globular (not fibrillar) conformation of amyloid proteins is sufficient to induce cellular pathophysiology. However, the 3D structural conformations of these globular structures, a key missing link in designing effective prevention and treatment, remain undefined as of yet. By using atomic force microscopy, circular dichroism, gel electrophoresis, and electrophysiological recordings, we show here that an array of amyloid molecules, including amyloid-beta(1-40), alpha-synuclein, ABri, ADan, serum amyloid A, and amylin undergo supramolecular conformational change. In reconstituted membranes, they form morphologically compatible ion-channel-like structures and elicit single ion-channel currents. These ion channels would destabilize cellular ionic homeostasis and hence induce cell pathophysiology and degeneration in amyloid diseases.

Fig. 1.

CD spectra of mutant amyloid molecules in solution. CD spectrometry analysis of ABri, ADan, α-synuclein, amylin, SAA, and Aβ(1–40) in 5 mM Tris (pH 7.4) was carried out on a Jasco J-720 spectropolarimeter at 1-nm intervals over the wavelength range 190–260 nm at 24°C in a 0.1-cm path-length cell. Results are expressed in molar ellipticity (deg·cm²·mol^-1).

Fig. 2.

Electrophoresis of ABri, ADan, α-synuclein, amylin, SAA, and Aβ(1–40) on 16.5% SDS/PAGE under reducing conditions. The right lane shows peptide in aqueous solution; the left lane shows peptide in DOPC membrane after solubilization in 2% SDS. Positions of molecular mass markers are indicated on the left. Amylin and Aβ(1–40) were cross-linked both in solution and in membrane. In solution, for all peptides the monomers are observed. Small amounts of dimers are observed in solution for Aβ(1–40), ABri, and amylin. In the membrane, besides monomers and dimers, trimers are observed for amylin and Aβ(1–40), and tetramers are observed for ABri, amylin, Aβ(1–40), and α-synuclein. Also observed are pentamers for amylin and Aβ(1–40); hexamers for α-synuclein, SAA, ADan, amylin, and Aβ(1–40); and heptamers and octamers for α-synuclein and SAA.

Fig. 3.

Single-channel records of amyloid channels. Current traces as a function of time under voltage-clamp conditions are shown. Current jumps corresponding to the opening or closing of individual ion channels can be observed for all six amyloid peptides. Solutions contained 100 mM KCL (except B, which contained 10 mM KCl, and F, which contained 1 M KCl), buffered to pH 7.4. Peptide concentrations were as follows. (A) Aβ(1–40): 21 μg/ml, V = -30mV. (B) Amylin: 3 μg/ml, V =+50 mV. (C) ABri: 50 μg/ml, V =-50 mV. (D) ADan: 100 μg/ml, V =-50 mV. (E) NAC: 15 nM, V =-50mV. (F) SAA: 1 μg/ml, V = -60 mV.

Fig. 4.

AFM images of freshly dissolved peptide molecules. Green arrows indicate surface-adsorbed peptides with a width of 8–12 nm and a height of 1–2 nm (consistent with the size of monomers). Blue arrows indicate higher-order oligomers and clusters. For ABri, most observed features are monomers and dimers; for other peptides, larger multimers and aggregates are observed. For ADan and amylin, the amount of monomers adsorbed is small, and mostly multimers and clusters are observed. In one experiment for α-synuclein, fiber-like structures were observed, indicated by green dotted lines. Peptides were adsorbed on mica for 20–40 min and then washed to remove unadsorbed peptides and imaged in buffer (see Materials and Methods). (Scale bars: 500 nm for ADan and 100 nm for all other peptides.)

Fig. 5.

AFM images of amyloid peptides reconstituted in membrane bilayers. Inset shows lipid bilayer with thickness of ≈5 nm. For Aβ(1–40), ADan, and α-synuclein, channel-like structures with a central pore can be easily resolved. For ABri, SAA, and amylin, the central pore is only resolved on some multimer structures. Arrows indicate locations where annular structures can be observed clearly. For those indicated channels, channel sizes (outer diameter) are 16 nm for Aβ(1–40), 14 nm for ABri, 14 nm for ADan, 16 nm for α-synuclein, 12 nm for SAA, and 15 nm for amylin. (Scale bars: 100 nm.)

Fig. 6.

Individual channel-like structures at high resolution. Two examples are shown for each molecule, in which a central pore can be observed. The number of subunits observed protruding from the surface varies from four to eight subunits. Resolution of AFM images is not enough to resolve individual subunit structures. [Image sizes are 25 nm for Aβ(1–40), 25 nm for α-synuclein, 35 nm for ABri, 20 nm for ADan, 25 nm for amylin, and 20 nm for SAA.]