Demuro A, Smith M, Parker I.

GM48071 NIGMS NIH HHS , P50-AG16573 NIA NIH HHS , R37 GM048071 NIGMS NIH HHS , R01 GM048071 NIGMS NIH HHS , P50 AG016573 NIA NIH HHS

	Figure 1. Western blot analysis of Aβ1−42 oligomer preparations and assay of potency to evoke Ca2+ influx in Xenopus oocytes. (A and B) Western blots showing aggregation profiles of Aβ1–42 at 30 min after initially dissolving Aβ peptide monomer (A) and after 48 h of incubation (B). 4 µg of samples was separated using gel electrophoresis on a 4–20% Tris-HCl gel. The membranes were probed separately with a monoclonal antibody, 6E10, which is sequence specific to Aβ, and with OC antibody, which recognizes a generic epitope associated with the fibrillar amyloid conformation independent of peptide sequence. Black lines indicate that intervening lanes have been spliced out. (C and D) Bioassay of the potency of Aβ1–42 oligomer preparations to induce membrane permeability to Ca2+. The traces in C show representative global Ca2+ fluorescence signals (indicated as F; top) and corresponding voltage-clamped membrane currents (indicated as I; bottom) evoked by 30-s voltage steps from 0 to −100 mV delivered at various times (indicated in minutes) before and after pipette application of Aβ1−42 oligomers to a nonpeeled oocyte loaded with fluo-4 dextran. (D) Measurements of inward currents (without leak subtraction) evoked by voltage steps from 0 to −100 mV taken at 5-min intervals after application of Aβ1−42 (final bath concentration of 1 µg/ml). Data show means ± 1 SEM from four oocytes using two different Aβ1−42 oligomer preparations.
	Figure 2. Aβ1–42 oligomers form Ca2+-permeable ion pores in the plasma membrane of Xenopus oocytes. (A) Representative image showing localized Ca2+ transients (SCCaFTs) imaged by TIRF microscopy during hyperpolarization to −100 mV within a 40 × 40–µm region of a vitelline-stripped oocyte loaded with fluo-4 dextran and EGTA. The imaging region was close to the edge of the footprint of the oocyte on the cover glass, and 1 µg/ml Aβ1−42 oligomers was applied to the bathing solution ∼18 min beforehand from a pipette positioned near the bottom right corner of the image frame. No activity was observed before application of Aβ1−42. The grayscale image of fluorescence ratio changes (ΔF/Fo; black = 0, white = 1.6) shows a mean of six consecutive video frames (2 ms per frame) from within a sequence of 15,000 frames. (B) Map showing the locations of all SCCaFTs (Aβ pores) identified during a 20-s recording from the membrane region in A. The map was automatically constructed by CellSpecks by identifying the coordinates of each SCCaFT. (C) Representative fluorescence profiles from regions of interest (1 µm2) centered on two pore locations.
	Figure 3. Ca2+ influx through Aβ1−42 pores is voltage dependent and blocked by Zn2+. (A) Amplitudes of SCCaFTs from Aβ1−42 pores plotted as function of membrane potential. Points show mean amplitudes (ΔF/F0) ± 1 SEM from >30 events from 10 different pores. The fitted regression line extrapolates to 0 at about 30 mV. (B) Superimposed records of SCCaFTs recorded from 26 Aβ pore sites at the voltages indicated. (C) Representative traces from three regions of interest showing control records of SCCaFTs induced by Aβ1−42 (top) and records from the same regions 5 min after adding 300 µM Zn2+ to the bathing solution.
	Figure 4. SCCaFTs generated by Aβ1−42 display multiple Ca2+ flux levels. (A) The trace at the left shows a representative record of SCCaFTs induced by Aβ1−42 oligomers, and the trace at the right shows the segment marked by the gray box on an expanded timescale. The bars indicate Ca2+ fluorescence levels (ΔF/F0) corresponding to different amplitude dwell states during SCCaFTs. (B) Distribution of dwell-state amplitude levels during Aβ1−42 SCCaFTs measured by visual inspection of traces from 76 pores selected from a single image record. (C) Distribution of the maximum SCCaFT amplitude observed at a given site generated by CellSpecks from an image sequence that encompassed 2,820 total sites. Data are representative of results from three or more image records obtained in each of four oocytes.
	Figure 5. Aβ1−42 pores display wide variability in kinetics and Po. (A) Channel-chip representation of the activity of Aβ1−42 pores. Pore openings (SCCaFTs) are represented as pseudocolored streaks, with warmer colors representing higher fluorescence signals, as indicated by the color bar; time runs from left to right. The top panel illustrates the activity of 170 different pores showing the highest Po values among a total of 2,820 pores detected by CellSpecks within a single imaging frame (40 × 40 µm2). Pores are depicted top to bottom in order of decreasing Po. The bottom panels show consecutive expanded views of the regions marked by the white boxes. (B) Representative traces showing activity from six different pores, illustrating those with high (top two traces), medium (middle two traces), and low (bottom two traces) Po.
	Figure 6. Gating kinetics of Aβ1−42 pores. (A) Distribution of mean open durations (SCCaFT durations) among the 2,820 pores. (B) Corresponding distribution of mean closed times (intervals between SCCaFTs). (C) Corresponding distribution of mean open probabilities (Po; measured as proportion of time for which the fluorescence exceeded a threshold level above the baseline). (D) The Po of pores strongly correlates with their calcium permeability. The scatter plot shows the Po of pores as a function of the amplitude (ampl.) of the largest SCCaFT observed from that pore. (A–C) The blue curves are double exponential fits, with decay constants of 5.3 and 16.3 ms (A), 3.8 and 54 s (B), and 0.0017 and 0.0096 s (C). Data are representative of results from three or more image records obtained in each of four oocytes.
	Figure 7. Aβ1−42 pores with high open probabilities contribute disproportionately to the total cellular Ca2+ load. The Ca2+ load contributed by a given pore was determined by integrating the area under the fluorescence trace (30-s duration), during which the fluorescence exceeded a threshold of 0.2 ΔF/Fo. (A–F) The abscissa represents the number of pores within a 10 × 10–µm imaging field ranked in order of increasing Ca2+ load and normalized to 100%. (A–C) Data obtained 20 min after application of Aβ1−42 are shown. Plots of pore Po (B) and amplitude of the largest SCCaFT observed at each site (C) on the same abscissa as in A are shown. (D–F) Corresponding analyses from the same membrane region as in A–C obtained 10 min later, showing a marked increase in proportion of pores contributing a high Ca2+ load. Blue bars (righthand ordinate; shown in A) show the Ca2+ load contributed by each individual pore expressed as an integral of the fluorescence signals during all SCCaFTs detected at that site. The curve (left ordinate; red) plots the cumulative contribution of pores toward the normalized total Ca2+ load.

Alberdi, Amyloid beta oligomers induce Ca2+ dysregulation and neuronal death through activation of ionotropic glutamate receptors. 2010, Pubmed

Alberdi, Amyloid beta oligomers induce Ca2+ dysregulation and neuronal death through activation of ionotropic glutamate receptors. 2010, Pubmed
Arispe, Abeta ion channels. Prospects for treating Alzheimer's disease with Abeta channel blockers. 2007, Pubmed
Arispe, Giant multilevel cation channels formed by Alzheimer disease amyloid beta-protein [A beta P-(1-40)] in bilayer membranes. 1993, Pubmed
Arispe, Zn2+ interaction with Alzheimer amyloid beta protein calcium channels. 1996, Pubmed
Bode, Tarantula peptide inhibits atrial fibrillation. 2001, Pubmed
Bourinet, Endogenous Xenopus-oocyte Ca-channels are regulated by protein kinases A and C. 1992, Pubmed , Xenbase
Bucciantini, Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases. 2002, Pubmed
Cahalan, STIMulating store-operated Ca(2+) entry. 2009, Pubmed
Camandola, Aberrant subcellular neuronal calcium regulation in aging and Alzheimer's disease. 2011, Pubmed
Capone, Amyloid-beta-induced ion flux in artificial lipid bilayers and neuronal cells: resolving a controversy. 2009, Pubmed
Cras, Senile plaque neurites in Alzheimer disease accumulate amyloid precursor protein. 1991, Pubmed
De Felice, Abeta oligomers induce neuronal oxidative stress through an N-methyl-D-aspartate receptor-dependent mechanism that is blocked by the Alzheimer drug memantine. 2007, Pubmed
Demuro, "Optical patch-clamping": single-channel recording by imaging Ca2+ flux through individual muscle acetylcholine receptor channels. 2005, Pubmed , Xenbase
Demuro, Optical single-channel recording: imaging Ca2+ flux through individual ion channels with high temporal and spatial resolution. 2005, Pubmed
Demuro, Imaging single-channel calcium microdomains. 2006, Pubmed , Xenbase
Demuro, Optical single-channel recording: imaging Ca2+ flux through individual N-type voltage-gated channels expressed in Xenopus oocytes. 2003, Pubmed , Xenbase
Demuro, Imaging the activity and localization of single voltage-gated Ca(2+) channels by total internal reflection fluorescence microscopy. 2004, Pubmed , Xenbase
Demuro, Calcium dysregulation and membrane disruption as a ubiquitous neurotoxic mechanism of soluble amyloid oligomers. 2005, Pubmed
Demuro, Imaging single-channel calcium microdomains by total internal reflection microscopy. 2004, Pubmed , Xenbase
Demuro, Calcium signaling and amyloid toxicity in Alzheimer disease. 2010, Pubmed
Deshpande, Different conformations of amyloid beta induce neurotoxicity by distinct mechanisms in human cortical neurons. 2006, Pubmed
Durell, Theoretical models of the ion channel structure of amyloid beta-protein. 1994, Pubmed
Glabe, Conformation-dependent antibodies target diseases of protein misfolding. 2004, Pubmed
Hardy, Alzheimer's disease: the amyloid cascade hypothesis. 1992, Pubmed
Hertel, Inhibition of the electrostatic interaction between beta-amyloid peptide and membranes prevents beta-amyloid-induced toxicity. 1997, Pubmed
Inoue, In situ Abeta pores in AD brain are cylindrical assembly of Abeta protofilaments. 2008, Pubmed
Jang, New structures help the modeling of toxic amyloidbeta ion channels. 2008, Pubmed
Jarrett, Seeding "one-dimensional crystallization" of amyloid: a pathogenic mechanism in Alzheimer's disease and scrapie? 1993, Pubmed
Jensen, Quantification of Alzheimer amyloid beta peptides ending at residues 40 and 42 by novel ELISA systems. 2000, Pubmed
Kagan, Amyloid peptide channels. 2004, Pubmed
Kawahara, Neurotoxicity of β-amyloid protein: oligomerization, channel formation, and calcium dyshomeostasis. 2010, Pubmed
Kawahara, Alzheimer's disease amyloid beta-protein forms Zn(2+)-sensitive, cation-selective channels across excised membrane patches from hypothalamic neurons. 1997, Pubmed
Kayed, Conformation dependent monoclonal antibodies distinguish different replicating strains or conformers of prefibrillar Aβ oligomers. 2010, Pubmed
Kayed, Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis. 2003, Pubmed
Kim, Sequence determinants of enhanced amyloidogenicity of Alzheimer A{beta}42 peptide relative to A{beta}40. 2005, Pubmed
Lal, Amyloid beta ion channel: 3D structure and relevance to amyloid channel paradigm. 2007, Pubmed
Lashuel, Neurodegenerative disease: amyloid pores from pathogenic mutations. 2002, Pubmed
Lemere, Sequence of deposition of heterogeneous amyloid beta-peptides and APO E in Down syndrome: implications for initial events in amyloid plaque formation. 1996, Pubmed
Lin, Amyloid beta protein forms ion channels: implications for Alzheimer's disease pathophysiology. 2001, Pubmed
Mashanov, Automatic detection of single fluorophores in live cells. 2007, Pubmed
Mason, Distribution and fluidizing action of soluble and aggregated amyloid beta-peptide in rat synaptic plasma membranes. 1999, Pubmed
Mattson, beta-Amyloid peptides destabilize calcium homeostasis and render human cortical neurons vulnerable to excitotoxicity. 1992, Pubmed
Miledi, Chloride current induced by injection of calcium into Xenopus oocytes. 1984, Pubmed , Xenbase
Picht, SparkMaster: automated calcium spark analysis with ImageJ. 2007, Pubmed
Pollard, A new hypothesis for the mechanism of amyloid toxicity, based on the calcium channel activity of amyloid beta protein (A beta P) in phospholipid bilayer membranes. 1993, Pubmed
Quist, Amyloid ion channels: a common structural link for protein-misfolding disease. 2005, Pubmed
Rhee, Amyloid beta protein-(1-42) forms calcium-permeable, Zn2+-sensitive channel. 1998, Pubmed
Rovira, Abeta(25-35) and Abeta(1-40) act on different calcium channels in CA1 hippocampal neurons. 2002, Pubmed
Schauerte, Simultaneous single-molecule fluorescence and conductivity studies reveal distinct classes of Abeta species on lipid bilayers. 2010, Pubmed
Shankar, Amyloid-beta protein dimers isolated directly from Alzheimer's brains impair synaptic plasticity and memory. 2008, Pubmed
Smith, Imaging the quantal substructure of single IP3R channel activity during Ca2+ puffs in intact mammalian cells. 2009, Pubmed
Sokolov, Soluble amyloid oligomers increase bilayer conductance by altering dielectric structure. 2006, Pubmed
Walsh, Naturally secreted oligomers of amyloid beta protein potently inhibit hippocampal long-term potentiation in vivo. 2002, Pubmed
Wang, beta-Amyloid(1-42) binds to alpha7 nicotinic acetylcholine receptor with high affinity. Implications for Alzheimer's disease pathology. 2000, Pubmed
Weber, Endogenous ion channels in oocytes of xenopus laevis: recent developments. 1999, Pubmed , Xenbase