Abstract

A central hallmark of Alzheimer's disease is the presence of extracellular amyloid plaques chiefly consisting of amyloid-β (Aβ) peptides in the brain interstitium. Aβ largely exists in two isoforms, 40 and 42 amino acids long, but a large body of evidence points to Aβ(1-42) rather than Aβ(1-40) as the cytotoxic form. One proposed mechanism by which Aβ exerts toxicity is the formation of ion channel pores that disrupt intracellular Ca2+ homeostasis. However, previous studies using membrane mimetics have not identified any notable difference in the channel forming properties between Aβ(1-40) and Aβ(1-42). Here, we tested whether a more physiological environment, membranes excised from HEK293 cells of neuronal origin, would reveal differences in the relative channel forming ability of monomeric, oligomeric, and fibrillar forms of both Aβ(1-40) and Aβ(1-42). Aβ preparations were characterized with transmission electron microscopy and thioflavin T fluorescence. Aβ was then exposed to the extracellular face of excised membranes, and transmembrane currents were monitored using patch clamp. Our data indicated that Aβ(1-42) assemblies in oligomeric preparations form voltage-independent, non-selective ion channels. In contrast, Aβ(1-40) oligomers, fibers, and monomers did not form channels. Ion channel conductance results suggested that Aβ(1-42) oligomers, but not monomers and fibers, formed three distinct pore structures with 1.7-, 2.1-, and 2.4-nm pore diameters. Our findings demonstrate that only Aβ(1-42) contains unique structural features that facilitate membrane insertion and channel formation, now aligning ion channel formation with the differential neurotoxic effect of Aβ(1-40) and Aβ(1-42) in Alzheimer's disease.

Highlights

  • A central hallmark of Alzheimer’s disease is the presence of extracellular amyloid plaques consisting of amyloid-␤ (A␤) peptides in the brain interstitium

  • Our findings demonstrate that only A␤(1– 42) contains unique structural features that facilitate membrane insertion and channel formation, aligning ion channel formation with the differential neurotoxic effect of A␤(1– 40) and A␤(1– 42) in Alzheimer’s disease

  • The ability of A␤ to form membrane-spanning ion channels is a mechanism by which A␤ might exert synaptic neurotoxicity (17–19)

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Summary

Results

A␤ Ion Channels Are Observed with Oligomeric A␤(1– 42) but Not Oligomeric A␤(1– 40)—Patch pipettes were backfilled with 5 ␮M A␤ preparations. We exposed the excised membranes to A␤(1– 40) and found that A␤(1– 40) monomer (n ϭ 20), oligomer (n ϭ 20), and fiber (n ϭ 20) preparations did not form any A␤-associated ion channels in a 30-min recording period. Typical current traces recorded for A␤(1– 42) oligomers are shown in Fig. 5a for three representative channels that exhibit three distinct conductances. Channels formed by A␤(1– 42) oligomer and fiber preparations were similar in size with comparable median conductances. Three more channel-incorporated patches were perfused with patch clamp solution that had an 80% volume replacement with equimolar sucrose Under these conditions, the Erev of a purely cation-selective channel would be expected to shift ϩ40 mV (Equation 2). A small Erev of Ϫ8 mV was measured (336, 622, and 642 pS) (Fig. 7b)

Discussion
Experimental Procedures
Size Exclusion Chromatography
Transmission Electron Microscopy
Cell Culture
Patch Clamp Recording
Calculation of Channel Pore Diameter Using Channel Conductance
Calculation of Channel Selectivity
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