Aβ Ion Channels Research Articles

Imaging Amyloid-β Membrane Interactions: Ion-Channel Pores and Lipid-Bilayer Permeability in Alzheimer's Disease.

The accumulation of the amyloid-β peptides (Aβ) is central to the development of Alzheimer's disease. The mechanism by which Aβ triggers a cascade of events that leads to dementia is a topic of intense investigation. Aβ self-associates into a series of complex assemblies with different structural and biophysical properties. It is the interaction of these oligomeric, protofibril and fibrillar assemblies with lipid membranes, or with membrane receptors, that results in membrane permeability and loss of cellular homeostasis, a key event in Alzheimer's disease pathology. Aβ can have an array of impacts on lipid membranes, reports have included: a carpeting effect; a detergent effect; and Aβ ion-channel pore formation. Recent advances imaging these interactions are providing a clearer picture of Aβ induced membrane disruption. Understanding the relationship between different Aβ structures and membrane permeability will inform therapeutics targeting Aβ cytotoxicity.

Imaging Amyloid-β Membrane Interactions: Ion-Channel Pores and Lipid-Bilayer Permeability in Alzheimer's Disease.

The accumulation of the amyloid-β peptides (Aβ) is central to the development of Alzheimer's disease. The mechanism by which Aβ triggers a cascade of events that leads to dementia is a topic of intense investigation. Aβ self-associates into a series of complex assemblies with different structural and biophysical properties. It is the interaction of these oligomeric, protofibril and fibrillar assemblies with lipid membranes, or with membrane receptors, that results in membrane permeability and loss of cellular homeostasis, a key event in Alzheimer's disease pathology. Aβ can have an array of impacts on lipid membranes, reports have included: a carpeting effect; a detergent effect; and Aβ ion-channel pore formation. Recent advances imaging these interactions are providing a clearer picture of Aβ induced membrane disruption. Understanding the relationship between different Aβ structures and membrane permeability will inform therapeutics targeting Aβ cytotoxicity.

NMR Studies of the Ion Channel-Forming Human Amyloid-β with Zinc Ion Concentrations.

Alzheimer’s disease (AD) is classified as an amyloid-related disease. Amyloid beta (Aβ) is a transmembrane protein known to play a major role in the pathogenesis of AD. These Aβ proteins can form ion channels or pores in the cell membrane. Studies have elucidated the structure of the transmembrane domain of Aβ ion channels. In addition, various studies have investigated substances that block or inhibit the formation of Aβ ion channels. Zinc ions are considered as potential inhibitors of AD. In this study, we focused on the transmembrane domain and some external domains of the Aβ protein (hAPP-TM), and solution-state NMR was used to confirm the effect on residues of the protein in the presence of zinc ions. In addition, we sought to confirm the structure and orientation of the protein in the presence of the bicelle using solid-state NMR.

Cholesterol Modulates the Formation of the Aβ Ion Channel in Lipid Bilayers.

The misfolding of amyloid beta (Aβ) is one of the predominant hallmarks in the pathology of Alzheimer's disease (AD). In this study, we showed that the formation of the Aβ ion channel on the membrane depended on the cholesterol concentration. From a mechanical aspect, we found that cholesterol levels affected the stability and assembly of lipid bilayers. Measurements on planar lipid bilayers indicated that a small amount of cholesterol interacted with Aβ proteins and promoted the insertion process. Conversely, high cholesterol integrated the lipid bilayer and exerted an opposite effect on Aβ insertion. The Aβ ion channel was then detected by graphene-based field-effect transistors. Results demonstrated that the Aβ ion channel promoted a Ca2+ flux in the presence of 15% cholesterol but prevented a Ca2+ flux in high cholesterol. Thus, cholesterol had a complex impact on the Aβ ion channel that can be described as two different effects. First, a small amount of cholesterol interacted with Aβ and facilitated the Aβ ion channel formation in the membrane. Second, a large amount of cholesterol did not induce the ion flux in the membrane, which can be explained by the cholesterol damage to the regular distribution of the lipid bilayer. Overall, this study suggested a possible approach to consider cholesterol levels for the treatment of AD patients.

Single-cell screening of cytosolic [Ca(2+)] reveals cell-selective action by the Alzheimer's Aβ peptide ion channel.

Interaction of the Alzheimer's Aβ peptides with the plasma membrane of cells in culture results in chronic increases in cytosolic [Ca(2+)]. Such increases can cause a variety of secondary effects leading to impaired cell growth or cell degeneration. In this investigation, we made a comprehensive study of the changes in cytosolic [Ca(2+)] in single PC12 cells and human neurons stressed by continuous exposure to a medium containing Aβ42 for several days. The differential timing and magnitude of the Aβ42-induced increase in [Ca(2+)] reveal subpopulations of cells with differential sensitivity to Aβ42. These results suggest that the effect produced by Aβ on the level of cytosolic [Ca(2+)] depends on the type of cell being monitored. Moreover, the results obtained of using potent inhibitors of Aβ cation channels such as Zn(2+) and the small peptide NA7 add further proof to the suggestion that the long-term increases in cytosolic [Ca(2+)] in cells stressed by continuous exposure to Aβ is the result of Aβ ion channel activity.

Chronic Cytosolic Calcium Changes Induced by Alzheimer's Peptides Aβ Allows for Sorting of Aβ-Resistant Cell Subpopulations

The Alzheimer's Aβ peptides interaction with the plasma membrane has been shown to result in a chronic increase in the cytosolic calcium concentrations of cells in culture. Such alteration may cause a variety of secondary effects which may lead to cell degeneration and reduced cell culture growth. We have proposed that the alteration in the cytosolic calcium concentrations induced by Aβ is initiated by early formation of Aβ ion channels in the plasma membrane. To study the Aβ-induced cytosolic calcium change dynamics we monitored the level of cytosolic calcium from hundreds of individual cells in culture exposed to Aβ for prolonged period of time. Because of the continuous cytotoxic effect of Aβ on cells, the number of viable cells in the cultures varied with time of exposure. Histograms based on the individual intracellular calcium levels showed that while most cells in the culture remain unaffected, a defined subpopulation of cells show increasingly higher than normal cytosolic calcium levels. After days of continuous exposure to Aβ cells more sensitive to Aβ died, and consequently the averaged cytosolic calcium for remaining cells in the whole culture approached control values. Aβ ion channel blockers prevented the induced cytosolic calcium changes and preserved cell viability, confirming the participation of Aβ ion channels. Confocal microscopic analysis using fluorescent annexin V on cells loaded with fura 2 showed that those cells that remained after prolonged exposure to Aβ did not display the proposed extracellular Aβ receptor phosphatidyl serine. Further addition of Aβ to this sorted cell subpopulation did not induce cytosolic calcium changes and cells continued to grow and divide. Application of this procedure allows for sorting out Aβ resistant subpopulation of cells.

Antihypertensive Antagonists of L-Type Calcium Channel Exert Enhancement of Alzheimer's Aβ Peptides Effect on Cells

The Alzheimer's Aβ peptides are shown to increase the cytosolic calcium concentrations of cells in culture. Such an alteration may cause a variety of secondary effects, leading to cell degeneration and reduced cell culture growth. Cytosolic calcium concentration can be increased by the formation of cation-selective ion channels by the Aβ peptides. This property is well documented and has been extensively studied both in artificial membrane systems as well as in living cells. Here we show that L-type calcium channel antagonists, commonly used in the treatment of hypertension and ischemic heart disease, intensify the capacity of Aβ peptides to increase cytosolic calcium concentrations and consequently enhance the cytotoxicity of the Aβ peptides. Calcium imaging experiments were conducted with the fluorescent indicator Fura-2 to measure at different periods of times the levels of cytosolic calcium during exposure to Aβ peptides and to the L-type calcium channel antagonists. Two different 1,4-dihydropyridines (nifedipine, and nitrendipine), and Diltiazem, a structurally unrelated Ca2+-channel antagonist belonging to the phenylalkylamine class were used and found to exert significant excitatory effects on the toxicity of Aβ. The reduced toxicity observed in solutions of combinations of Aβ40 and Aβ42 is also reversed by L-type calcium channel antagonists. The toxicity of Aβ peptides was weighed by quantifying the growth of cultures of PC12 cells using the colorimetric assay XTT and by measuring the release of LDH to the media. The enhancement of the Aβ peptides toxicity by L-type calcium channel antagonists can be constrained by specific Aβ ion channel blockers suggesting the mediation of the ion channel property of Aβ peptides. The relevance of our finding derives from the widespread clinical use of L-type calcium channel antagonists in the treatment of hypertension and angina pectoris.

Modeling Ion-Permeable β-Amyloid (Aβ) Barrels in the Lipid Bilayer

We modeled Alzheimer's Aβ ion channel in a β-barrel structure embedded in the lipid bilayer using molecular dynamics (MD) simulations. The β-barrel is characterized by the inclination angle of the β-strands with respect to the pore axis. Our Aβ barrels have two β-strand layers, and each layer has 16 or 20 β-strands that are tilted by ∼37° relative to the membrane normal. In the Aβ barrel simulations, we employed the U-shaped β-strand-turn-β-strand peptides using currently available NMR-based coordinates of non-amyloidogenic p3 (Aβ17-42) and Aβ9-42 peptides. The non-amyloidogenic Aβs resulting from α-secretase and BACE cleavage are often found in amyloid plaques, but the biophysical properties and functional role of these non-amyloidogenic peptides are not understood. In good agreement with previously modeled Aβ ion channels that aligned the β-strands parallel to the pore axis, all Aβ barrels break into loosely-associated mobile β-sheet subunits, verifying that membranes do not support intact β-sheet conformations. We obtain ion-permeable pore-like Aβ barrels whose subunit morphologies and shapes are consistent with AFM images, suggesting that the Aβ barrels are polymorphic conformations of Aβ ion channels in the membrane. The emerging picture from our large-scale simulations is that toxic ion channels formed by β-sheets spontaneously break into loosely-interacting dynamic units which associate and dissociate leading to toxic ionic flux. Funded by NCI Contract HHSN261200800001E (RN) and NIH (NIA) extramural program (RL).

Elucidation of Ca2+ Influx through Alzheimer's Aβ Channels in the Aβ-Induced Cellular Ca2+ Response

The Alzheimer's disease Aβ peptide interaction with the plasma membrane of cells results in a response characterized by the elevation of the intracellular (cytoplasmic) Ca2+ concentration which may critically perturb Ca2+ homeostasis. Calcium influx into the cytosol can occur across the plasma membrane via receptor-mediated, voltage-gated, store-operated calcium channels, or from internal stores. Based on the ion channel formation by Aβ peptides on artificial membranes we have proposed that perturbation in Ca2+ homeostasis induced by Aβ could be caused by external calcium entering through ion channels formed by Aβ in the plasma membrane. The activity of these channels would permit the entrance of extracellular calcium ions into the cell, subsequently triggering the release of calcium from internal stores. To elucidate the contribution of calcium influx through the Aβ ion channels to the Aβ-induced calcium response we used specific blockers of plasma membrane channels and specific inhibitors of the mechanisms that permit the release of calcium from the ER, the largest intracellular store. With all those mechanisms blocked and inhibited, we visualized a fast raising, short-lasting calcium entry immediately after PC12 cells were exposed to Aβ. This calcium signal was identified as calcium flowing through the Aβ channels by using specific Aβ channel blockers. The Aβ channel blockers did not affect other mechanisms that contribute to the cytosolic calcium increase, and their use prevented the calcium entry through the Aβ channels and consequent development of the whole cellular Aβ-induced calcium response.

Interactions With Imidazole Side Groups As A Mechanism To Block The Alzheimer'S Aβ Peptide-induced Intracellular Calcium Increase

We have proposed the formation of Aβ ion channels as a mechanism to explain the intracellular calcium increase which occurs after cells are exposed to the cytotoxic Alzheimer's Aβ peptide. We developed highly effective and specific histidine-related Aβ channel blockers which prevent the Aβ-induced intracellular calcium response. Substitution experiments showed that histidine is essential for the blocking process. We hypothesized that the blocking efficiency of these compounds was related to imidazole side chains in the histidine residues. We rationalized that the resonance structure of the imidazole ring would be attracted to a full or partial positively charged form of the Histidine in the Aβ subunits of the Aβ channels. This investigation studied the intracellular calcium increase which occurs after cells are exposed to the Aβ peptide. The role of the imidazole side chains in mechanism of action was determined using histidine-related Aβ channel blockers in which the imidazole side chains were methylated. Additionally, we studied these compounds when modified by amidation and acetylation of the carboxyl and amine end groups, respectively. Our results showed that the efficacy to prevent the Aβ-induced intracellular calcium increase, and the capacity to protect cells from the toxic action of Aβ, is completely abolished when the imidazole side chains of the histidine-related Aβ channel blockers are methylated. On the other hand, the efficacy of the Aβ channel blockers is significantly improved when the ends of the molecules are capped leaving the imidazole side chain as the sole group available for interaction. We conclude that aromatic interactions between the imidazole side chains in the histidine-related blockers, and the charged form of the His residues in the Aβ channels, constitutes a blocking mechanism for the Aβ peptide-induced intracellular calcium increase.

Early and Late Cytotoxic Effects of External Application of the Alzheimer's Aβ Result from the Initial Formation and Function of Aβ Ion Channels

Extracellular application of the Alzheimer's beta-amyloid (Abeta) peptide evokes a series of cellular responses that leads to the death of cells by apoptosis. Some responses to freshly prepared Abeta occur immediately, including changes in intracellular calcium concentration and changes in membrane permeability and phosphatidylserine asymmetry. We show here that the cytotoxic action of externally applied Abeta, such as caspase activation and apoptotic loss of cell viability, occurs and persists even several days after Abeta is removed from the medium. We find that the mechanism for this persistent cytotoxic action of extracellular Abeta is based on the sustained activity of active Abeta ion channels that remain incorporated in the cell membrane. To confirm this assessment, we blocked the late cytotoxic action of Abeta with the classically known Abeta channel blockers zinc and tromethamine. To further validate this conclusion, we developed a specific peptide segment from the sequence forming the mouth of the Abeta channel to block Abeta Ca2+ channels acutely and to block late Abeta effects on caspase activation and apoptosis. This is the first report of a specific Abeta channel blocker compound, NA4, which efficaciously and potently blocks the most known cellular responses to Abeta.

Histidines 13 and 14 in the Aβ sequence are targets for inhibition of Alzheimer's disease Aβ ion channel and cytotoxicity

The fact that Alzheimer's beta amyloid (Abeta) peptides forms cation channels in lipid bilayers was discovered during the course of our experiments in the laboratory of "Guayo" Rojas at NIH in Bethesda, Maryland (USA). Recently, we found that the Abeta ion channel could be blocked selectively with small peptides that copy the amino acid sequence of the predicted mouth region of the Abeta channel pore. We now have searched for the essential amino acid residues required for this blocking effect by mutations. We found that the ability of peptides to block Abeta channel activity could be lost by replacement of histidines 13 and 14 by alanine or lysine. The amino acid substitution also resulted in the loss of the capacity of the peptides to protect cells from Abeta cytotoxicity. These data thus contribute to the definition of the region of the Abeta sequence that participates in the formation of the channel pore. Additionally, these data support the hypothesis that the ion channel activity of Ab contributes significantly to the cytotoxic properties of Abeta. These data also emphasize the potential value in using inhibition of Abeta ion channel activity as an end point for Alzheimer's disease drug discovery.