The Rockefeller University Press $30.00
J. Cell Biol. Vol. 195 No. 3 515–524
Correspondence to Angelo Demuro: firstname.lastname@example.org
Abbreviations used in this paper: AD, Alzheimer’s disease; APP, amyloid pre-
cursor protein; SCCaFT, single-channel Ca2+ fluorescence transient; TIRF, total
internal reflection fluorescence.
A seminal event in the pathogenesis of Alzheimer’s disease
(AD) is the abnormal proteolytic processing of amyloid precur-
sor protein (APP), resulting in increased production of a self-
aggregating form of amyloid (A). Mutations in APP and in
presenilins (secretase enzymes involved in APP processing)
that are linked to early-onset familial forms of AD cause exces-
sive production of A, resulting in massive accumulation of
fibrillar A in the amyloid plaques that are a hallmark of brains
from AD patients (Cras et al., 1991). For many years, the
plaques were thought to be the primary culprit of AD pathol-
ogy, as formulated by the amyloid hypothesis (Hardy and
Higgins, 1992). However, that hypothesis fails to explain several
important pathological and clinical characteristics of AD. Mark-
edly, there is little correlation between the amounts of fibrillar
A deposit at autopsy and the clinical severity of AD (Lemere
et al., 1996). In contrast, a good correlation has been found be-
tween early cognitive dysfunction and levels of soluble forms
of A in the brain (Jensen et al., 2000; Shankar et al., 2008).
Aggregation of A proceeds through several steps, includ-
ing the formation of soluble low molecular weight spherical
oligomers, before assuming a final and stable conformation
as the insoluble fibrils from which plaques are constituted
(Bucciantini et al., 2002; Kayed et al., 2003; Glabe, 2004).
Growing evidence suggests that the neurotoxicity is associated
with soluble aggregates of As rather than with the plaques
themselves (Walsh et al., 2002; Kayed et al., 2003; Demuro
et al., 2005; Deshpande et al., 2006). Moreover, soluble oligo-
meric forms of amyloidogenic peptides associated with dis-
eases including Huntington’s and Parkinson’s have been shown
to be similarly toxic, and this toxicity is directly related to the
morphological structure of the aggregates rather than to their
protein sequence (Kayed et al., 2003; Demuro et al., 2005). Con-
sistent with this, antibodies raised against A peptides in their
oligomeric form recognize oligomeric species of other amy-
loidogenic proteins (-synuclein, polyglutamin, and lysozime)
but not the monomeric or the fibrillar forms of A and other
peptides (Kayed et al., 2003).
Amyloid oligomers, but not monomers, exert a common
pathophysiological action in disrupting the integrity of cell
membranes, resulting in uncontrolled influx of extracellular Ca2+
(Ca2+) elevation. Proposed mechanisms by which A me-
diates its effects include lipid destabilization, activation
of native membrane channels, and aggregation of A
into Ca2+-permeable pores. We distinguished between
these using total internal reflection fluorescence (TIRF)
microscopy to image Ca2+ influx in Xenopus laevis
oocytes. A1–42 oligomers evoked single-channel
Ca2+ fluorescence transients (SCCaFTs), which resembled
those from classical ion channels but which were not
ligomeric forms of A peptides are implicated
in Alzheimer’s disease (AD) and disrupt mem-
brane integrity, leading to cytosolic calcium
attributable to endogenous oocyte channels. SCCaFTs
displayed widely variable open probabilities (Po) and
stepwise transitions among multiple amplitude levels
reminiscent of subconductance levels of ion channels.
The proportion of high Po, large amplitude SCCaFTs
grew with time, suggesting that continued oligomer ag-
gregation results in the formation of highly toxic pores.
We conclude that formation of intrinsic Ca2+-permeable
membrane pores is a major pathological mechanism in
AD and introduce TIRF imaging for massively parallel
single-channel studies of the incorporation, assembly,
and properties of amyloidogenic oligomers.
Single-channel Ca2+ imaging implicates A1–42
amyloid pores in Alzheimer’s disease pathology
Angelo Demuro,1 Martin Smith,1 and Ian Parker1,2
1Department of Neurobiology and Behavior and 2Department of Physiology and Biophysics, University of California, Irvine, Irvine, CA 92697
© 2011 Demuro et al. This article is distributed under the terms of an Attribution–
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T H E J O U R N A L O F C E L L B I O L O G Y
JCB • VOLUME 195 • NUMBER 3 • 2011 516
support a pore-forming mechanism of A toxicity and pro-
vide insights into the formation and functional characteristics
of A pores.
A oligomers induce Ca2+ influx across the
We had previously shown that application of soluble A oligo-
mers (but not monomers or fibrils) to cultured neuroblastoma
cells evoked large increases in cytosolic (Ca2+) that arise largely
through Ca2+ influx across the plasma membrane (Demuro et al.,
2005). Our object here was to study this plasmalemmal Ca2+ flux
at the single-channel level using total internal reflection fluores-
cence (TIRF) microscopy of near-membrane Ca2+ signals, which
we had developed as a technique to resolve the gating of indi-
vidual ion channels in the membrane of Xenopus oocytes
(Demuro and Parker, 2005a). The oocyte offers several advan-
tages for study of the actions of A oligomers, including a pau-
city of native ligand– and voltage-gated channels (Bourinet et al.,
1992) that have been proposed to be targets of A modulation in
neuronal cells (Wang et al., 2000; De Felice et al., 2007; Alberdi
et al., 2010). We chose to investigate the molecular mechanisms
of toxicity of the A1–42 peptide, rather than the A1–40 form,
in light of the reported correlation between the early onset of
familial AD and the increased level of A1–42 in the AD brain
(Jarrett and Lansbury, 1993; Kim and Hecht, 2005).
A1–42 oligomers were prepared by incubating aque-
ous solutions of peptide monomer for up to 48 h. Western blots
(Fig. 1, A and B) showed a wide range of oligomeric species
with molecular masses ranging between 35 and 300 kD, cor-
responding to roughly 5–40 peptide multimers, which were
recognized by 6E10 and OC, sequence- and fibrillar-specific
A antibodies, respectively (Kayed et al., 2010). A oligomer
preparations were assayed for their ability to induce macro-
scopic Ca2+ influx in oocytes. Fig. 1 C shows representative
membrane currents and fluorescence signals in an oocyte loaded
with the Ca2+ indicator fluo-4 generated in response to voltage-
clamped hyperpolarizing pulses from 0 to 100 mV to tran-
siently increase the electrical driving force for Ca2+ entry. The
pulses initially evoked only a small inward leakage current and
a small rise in fluorescence, but both signals increased over
with devastating consequences for cellular Ca2+ homeostasis
(Mattson et al., 1992; Demuro et al., 2005; Deshpande et al.,
2006). Major questions remain as to how amyloid oligomers
exert their action on the cell membrane, and three distinct mech-
anisms have been proposed: (1) the fluidizing action hypoth-
esis postulates that A destabilizes membrane integrity via a
nonreceptor-mediated mechanism that alters the physicochemical
properties of membrane lipids and proteins (Hertel et al., 1997;
Mason et al., 1999; Sokolov et al., 2006); (2) A oligomers have
been proposed to directly activate endogenous Ca2+-permeable
receptor/channels such as neuronal nicotinic and glutamate re-
ceptors (Wang et al., 2000; De Felice et al., 2007; Alberdi et al.,
2010); and (3) A and other amyloidogenic oligomers are pro-
posed to incorporate into the cell membrane to form pores with
high cation conductivity (Arispe et al., 1993; Pollard et al., 1993;
Lin et al., 2001; Quist et al., 2005). Evidence in support of the
pore-forming mechanism derives from electrophysiological re-
cordings from both artificial and biological membranes (Lin
et al., 2001; Lashuel et al., 2002; Quist et al., 2005; Capone et al.,
2009) and from theoretical modeling (Durell et al., 1994; Jang
et al., 2008). Moreover, EM reveals porelike A structures in
cell membranes of postmortem brains from AD but not control
patients (Inoue, 2008), and similar A globular structures have
been visualized by atomic force microscopy in lipid membranes
treated with synthetic A peptides (Lin et al., 2001; Lashuel
et al., 2002).
Studies of the molecular mechanisms by which amyloid
oligomers induce membrane permeability have largely been
confined to electrophysiological recordings of currents across
artificial lipid bilayers (Kagan et al., 2004) or excised patches
of neuronal membranes (Kawahara et al., 1997). Here, we
used a technique (optical patch-clamping) we had previously
developed for high-resolution imaging of Ca2+ flux through
single ligand– and voltage-gated channels (Demuro and
Parker, 2004a, 2005a) to study the functioning of individual
A142 oligomer pores in the plasma membrane of Xenopus
laevis oocytes, which substantially lack endogenous Ca2+
channels (Weber, 1999). We find that extracellular applica-
tion of A142 oligomers induces punctate Ca2+ transients
that closely resemble the single-channel Ca2+ fluorescence
transients (SCCaFTs) generated by classical protein mem-
brane channels. The results obtained with this novel approach
Figure 1. Western blot analysis of A142 oligomer prepa-
rations and assay of potency to evoke Ca2+ influx in Xenopus
oocytes. (A and B) Western blots showing aggregation profiles
of A1–42 at 30 min after initially dissolving A peptide mono-
mer (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 A, 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 A1–42 oligomer prepara-
tions 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 A142 oligomers to a nonpeeled oocyte loaded with fluo-4 dextran. (D) Mea-
surements of inward currents (without leak subtraction) evoked by voltage steps from 0 to 100 mV taken at 5-min intervals after application of A142
(final bath concentration of 1 µg/ml). Data show means ± 1 SEM from four oocytes using two different A142 oligomer preparations.
517Imaging single A amyloid pore activity • Demuro et al.
(Fig. 3, A and B), showing a voltage dependence and extra-
polated suppression potential (Fig. 3 A) mirroring that of SCCaFTs
from Ca2+-permeable nicotinic acetylcholine receptors expressed
in the oocyte (Demuro and Parker, 2005a), as expected from the
electrical driving force for Ca2+ influx.
Zinc ions inhibit the Ca2+ conductance induced by A
oligomers (Arispe et al., 1996, 2007; Kawahara et al., 1997;
Rhee et al., 1998). Application of 300 µM extracellular Zn2+
strongly attenuated the appearance of A SCCaFTs (Fig. 3 C),
and the mean fluorescence change across 10 × 10–µm mem-
brane regions in response to hyperpolarization reduced 10-fold
from F/Fo 0.14 ± 0.03 (n = 7 trials) to 0.016 ± 0.002 (n = 4),
which is close to the mean value seen in the absence of A
(0.013 ± 0.002; n = 6).
40 min after adding A142 oligomers to the bathing solution
(Fig. 1 D), reflecting an increase in membrane Ca2+ permeabil-
ity and consequent activation of a Ca2+-activated Cl conduc-
tance (Miledi and Parker, 1984). This action was specific to the
oligomeric species of A that aggregated after long-term incu-
bation (12–48 h) because application of solutions of A pep-
tide that had been prepared shortly beforehand evoked little or
Imaging Ca2+ signals from individual
To resolve Ca2+ influx through individual A pores, oocytes
were stripped of the vitelline envelope, allowing the cell mem-
brane to appose closely to the cleaned cover glass of the imag-
ing chamber so that TIRF microscopy of fluo-4 could be used to
image Ca2+ signals restricted within the 100-nm-deep evanes-
cent field immediately adjacent to the plasma membrane. We
selected regions with uniform background-resting fluores-
cence close to the edge of the membrane footprint, so as to
maximize diffusional access of A. A142 oligomers dis-
solved at 1 µg/ml in Ringer’s solution were delivered by super-
perfusion from a micropipette (30–50-µm tip) within <200 µm
of the edge of the imaging field. A total volume of 3 ml was
delivered at 0.3–0.5 ml/min to a chamber initially containing
0.5 ml Ringer’s solution.
Before application of A, hyperpolarizing steps to 100 mV
usually evoked little or no change in resting fluorescence,
and we discarded oocytes showing sustained or transient local
Ca2+ elevations. In contrast, hyperpolarizing pulses evoked
numerous localized, flickering bright spots when examined
>20 min after A application (Fig. 2 A). We used a custom
software routine (CellSpecks) to locate the positions of these
Ca2+ hotspots and to automatically generate plots of the fluores-
cence time course from localized regions of interest. For exam-
ple, Fig. 2 B presents a representative map of all sites (n = 785)
identified within the 40 × 40–µm imaging field during a 20-s
hyperpolarization. The inhomogeneous distribution across Fig. 2 B
is representative of eight similar experiments and probably re-
flects gradients of A oligomer concentration away from the
application pipette, which was located near the bottom right
corner of the imaged region.
Fluorescence signals at individual sites were restricted
within 1 µm and flickered on and off at millisecond timescales
(Fig. 2 C), closely resembling both patch-clamp recordings
from ion channels and optical recordings of Ca2+ flux through
Ca2+-permeable voltage- and ligand-gated ion channels (Demuro
and Parker, 2003, 2004b, 2005a). In light of this similarity,
together with other properties described later, we refer to them
as SCCaFTs (Demuro and Parker, 2005a).
A SCCaFTs arise through local influx of
Ca2+ across the plasma membrane
SCCaFTs induced by A142 oligomers disappeared after re-
moval of external Ca2+ from the bathing solution (Ringer’s so-
lution without added Ca2+ plus 3 mM EGTA), consistent with
an extracellular source of Ca2+. Moreover, the mean ampli-
tudes of SCCaFTs reduced at more positive membrane potentials
Figure 2. A1–42 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 re-
gion was close to the edge of the footprint of the oocyte on the cover
glass, and 1 µg/ml A142 oligomers was applied to the bathing solu-
tion 18 min beforehand from a pipette positioned near the bottom right
corner of the image frame. No activity was observed before application
of A142. The grayscale image of fluorescence ratio changes (F/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 (A 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) Repre-
sentative fluorescence profiles from regions of interest (1 µm2) centered on
two pore locations.
JCB • VOLUME 195 • NUMBER 3 • 2011 518
before and after gsmtx-4, respectively). Moreover, during acti-
vation of store-operated influx in oocytes, we observed only a
generalized rise in fluorescence Ca2+ signal without evidence of
discrete SCCaFTs, suggesting that, like Ca2+ release–activated
Ca2+ channels in other cells (Cahalan, 2009), these channels in
the oocyte have an extremely low Ca2+ conductance, incom-
patible with our observation of the large SCCaFTs generated
Collectively, these results indicate that TIRF imaging of
SCCaFTs provides a means to study the properties of pores
directly constituted from A oligomers, an approach with im-
portant advantages over electrophysiological techniques such
as lipid bilayer reconstitution, in that measurements can be ob-
tained simultaneously and independently from hundreds of
pores within the membrane of intact cells.
Single A142 pores exhibit multiple Ca2+
A characteristic feature of A SCCaFTs was the appearance
of stepwise transitions between multiple fluorescence levels
(Fig. 4 A). The fluorescence signal recorded by TIRF micros-
copy closely reflects instantaneous Ca2+ flux (Ca2+ current)
through single channels (Demuro and Parker, 2005a; Smith and
Parker, 2009), and the stepwise changes are reminiscent of cur-
rent recordings from lipid bilayer systems describing inter-
conversion of A140 pores between multiple conductance
levels (Arispe et al., 2007). To further analyze this behavior, we
measured dwell-state fluorescence amplitudes by visual inspec-
tion from 76 A pore sites. The resulting multimodal amplitude
distribution (Fig. 4 B) revealed several (five or more) peaks,
which do not appear to recur at precise integer multiples.
Variability in calcium permeability and
gating kinetics among A142 pores
In addition to the variability within and between individual
SCCaFTs at a given site (e.g., Fig. 4 A), further variation in
fluorescence amplitudes was apparent between different pores.
We characterized this by using the CellSpecks program to de-
rive the distribution of the maximal event amplitude detected
at each of 2,820 sites (Fig. 4 C). A few sites gave SCCaFTs >2
F/F0, whereas at others, no events larger than 0.4 F/F0
were observed. Moreover, the latter value likely represents an
upper bound, owing to the inability of the automated routine to
reliably detect yet smaller events.
An even greater variability among A142 pores was
apparent in their kinetic properties. This is illustrated in Fig. 5 A,
showing a channel chip representation (Demuro and Parker,
2005a) of 172 pores presented in order of decreasing open
probability (Po; top to bottom), and in Fig. 5 B, showing repre-
sentative traces from pores that showed frequent large ampli-
tude SCCaFTs and those that gave only infrequent smaller
events. The overall distribution of mean SCCaFT durations
among 2,820 pores within a single 40 × 40–µm membrane re-
gion was well fitted by a double exponential function with time
constants of 5 and 16 ms (Fig. 6 A); the distribution of mean
closed times (intervals between successive SCCaFTs) for the
same pores fitted a double exponential decay function with time
SCCaFTs do not involve A activation
of endogenous ion channels in the
Several studies indicate that A may increase membrane con-
ductance by activating diverse endogenous channels, including
voltage-gated Ca2+ channels and nicotinic, AMPA, N-methyl-d-
aspartic acid, and serotonin receptors (Rovira et al., 2002;
De Felice et al., 2007; Alberdi et al., 2010). However, it is im-
probable that the SCCaFTs we observed arose from actions on
any of these channels, as they are absent or present only occa-
sionally and at very low density in oocytes (Weber, 1999). The
only known Ca2+-permeable channels present at high density in
the oocyte membrane are stretch-activated channels and store-
operated channels that mediate Ca2+ influx after depletion of ER
Ca2+ (Weber, 1999). Neither is likely to account for our results.
Application of 5 µM of the specific stretch-activated channel
blocker gsmtx-4 (Bode et al., 2001) failed to significantly inhibit
whole-cell currents induced 40 min after application of 1 µg/ml
A1–42 oligomers (n = 3 oocytes; 2.1 ± 0.51 and 1.85 ± 0.7 µA
Figure 3. Ca2+ influx through A142 pores is voltage dependent and
blocked by Zn2+. (A) Amplitudes of SCCaFTs from A142 pores plotted
as function of membrane potential. Points show mean amplitudes (F/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 A pore sites at the voltages indicated. (C) Representa-
tive traces from three regions of interest showing control records of SCCaFTs
induced by A142 (top) and records from the same regions 5 min after
adding 300 µM Zn2+ to the bathing solution.
519Imaging single A amyloid pore activity • Demuro et al.
Respective Ca2+ load contributed by
A142 pores with differing Po
The toxicity of A pores will presumably vary in proportion to
the amount of Ca2+ that passes through them into the cytosol
within a given time. Given the wide variation in permeability
and gating properties among different pores, we were thus
interested to determine which might represent the major toxic
species: for example, whether a relatively small number of high
Po pores might contribute a greater fraction of the total cellular
Ca2+ load than the more numerous low Po pores. To obtain a rel-
ative estimate of the amount of Ca2+ entering the cell through a
given pore in a fixed time, we analyzed fluorescence traces such
as those in Fig. 5 B by integrating the local fluorescence signal over
the time during which the pore was open (F/Fo × seconds).
The blue bars in Fig. 7 A show the distribution of fluorescence
constants of 3.9 and 55 s (Fig. 6 B). The corresponding dis-
tribution of Po showed a very wide range (Fig. 6 C), which
could again be fitted by two exponential components. A major-
ity of sites had low (<0.005) Po values, but a few showed
Po values as high as 0.2. This spread in Po primarily reflects dif-
ferences in mean closed times among different pores and not
differences in mean open times. After segregating the data into
relatively low (Po < 0.02) and high (Po ≥ 0.02) populations, we
found only a roughly twofold difference in mean open time
between these groups (respective values derived from single-
exponential fits of 4.33 ± 0.20 and 19 ± 0.36 ms), whereas the
mean closed times differed more than 10-fold (respective time
constants 521 ± 55 and 4,271 ± 333 ms). Interestingly, the Po
of a pore correlated strongly with the amplitude of the largest
event observed from that pore (Fig. 6 D).
Figure 4. SCCaFTs generated by A142 display multiple Ca2+
flux levels. (A) The trace at the left shows a representative record
of SCCaFTs induced by A142 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 (F/F0) cor-
responding to different amplitude dwell states during SCCaFTs.
(B) Distribution of dwell-state amplitude levels during A142 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. A142 pores display wide vari-
ability in kinetics and Po. (A) Channel-chip rep-
resentation of the activity of A142 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 de-
tected by CellSpecks within a single imaging
frame (40 × 40 µm2). Pores are depicted top
to bottom in order of decreasing Po. The bot-
tom 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.
JCB • VOLUME 195 • NUMBER 3 • 2011 520
Fig. 7 (D–F), showing corresponding data from the same mem-
brane region as in Fig. 7 (A–C) but obtained 10 min later (i.e.,
30 min after beginning application of A). Although the num-
ber of pores (SCCaFT sites) in the imaging field increased only
slightly (from 768 to 805), the relative contributions among dif-
ferent pores to the cumulative Ca2+ load changed dramatically
(Fig. 7 D). The proportion of pores with high fluorescence inte-
grals (Fig. 7 D, blue bars) and high Po (Fig. 7 E) was greater
than at the earlier time point, such that the top 5% of pores now
contributed almost 80% of the total Ca2+ load (Fig. 7 D, red
curve), as compared with 50% 10 min earlier. Again, the Po of
a pore was the main factor in determining its contribution to the
Ca2+ load (Fig. 7 E), whereas maximal SCCaFT amplitudes var-
ied over only about a threefold range between pores (Fig. 7 F).
Similar results were obtained in a total of four oocytes.
Disruption of membrane integrity by soluble A amyloids and
consequent dysregulation of intracellular Ca2+ homeostasis are
implicated as important pathogenic factors in AD (Demuro et al.,
2005, 2010; Arispe et al., 2007; Kawahara, 2010; Camandola and
Mattson, 2011). Although the specific molecular mechanisms re-
main controversial (Demuro et al., 2010), there is good evidence
that A oligomers create Ca2+-permeable pores in the cell mem-
brane (Arispe et al., 2007). The structure (Lal et al., 2007) and
electrophysiological properties of these pores have been studied
in artificial lipid membrane systems (Arispe et al., 2007) and in
excised patches of neuronal membranes (Kawahara et al., 1997).
Here, we used TIRF microscopy (Demuro and Parker, 2004b,
2005a) to resolve the permeation of Ca2+ through individual A
pores in the membrane of Xenopus oocytes. After bath applica-
tion of A1–42 oligomers, we observed that numerous localized
transient hotspots of Ca2+ influx, which were dependent on the
electrochemical driving force for influx of extracellular Ca2+, were
blocked by Zn2+ and closely resembled Ca2+ signals previously
seen from single classical Ca2+-permeable channels (Demuro
and Parker, 2006). Moreover, we did not observe any spatially
diffuse Zn2+-insensitive Ca2+ elevations as expected if amyloid
oligomers were to cause membrane thinning and reduction of the
dielectric barrier to Ca2+ ion translocation (Sokolov et al., 2006);
Xenopus oocytes lack endogenous channels reported to interact
with A amyloids (De Felice et al., 2007; Alberdi et al., 2010).
Therefore, we conclude that the local Ca2+ transients (SCCaFTs)
arise through openings of individual A oligomer pores.
Thus, TIRF Ca2+ imaging provides a novel and powerful
means to study the permeation and gating properties of amyloid
membrane pores. Specific advantages include the incorporation
of pores in a native cell membrane rather than an artificial lipid
environment and the ability to simultaneously and independently
monitor the activity of hundreds of pores within a small region
of the cell membrane. This combination of high-throughput and
single-molecule resolution is of particular importance because
preparations of amyloid oligomers typically contain a mix of
widely differing aggregation states (Demuro et al., 2010). Pop-
ulation measurements (e.g., whole-cell assays) yield no infor-
mation regarding this diversity, and existing single-pore assays
integrals for 768 pores within a representative imaging field,
presented in rank order from those with the lowest integral at
the left to those with the greatest integral at the right. To better
visualize the fraction of the total Ca2+ load carried by the pores
in relation to their respective fluorescence integrals, we further
showed (Fig. 7 A, red curve) the cumulative contribution to-
ward the normalized total Ca2+ load among the rank-ordered
pores. This reveals a disproportionate contribution to the Ca2+
load among the population of A pores. Analogous to the highly
unequal distribution of incomes among US citizens, the top 5%
of pores with greatest fluorescence integrals contribute about
one half the total Ca2+ load. Fig. 7 shows the corresponding
mean open probabilities (Po; Fig. 7 B) and Ca2+ permeabilities
(peak SCCaFT amplitude; Fig. 7 C) of the pores, ordered again
by their fluorescence integrals to match Fig. 7 A. From these
data, it is clear that the pores carrying a high toxic Ca2+ load are
characterized primarily by their high Po and that, although they
also display on average a higher Ca2+ permeability, this factor
plays a lesser role.
Time-dependent changes in A142
Visual inspection of image records indicated that the amplitudes
of fluorescence signals at given sites increased progressively
over several minutes after A application. This is quantified in
Figure 6. Gating kinetics of A142 pores. (A) Distribution of mean
open durations (SCCaFT durations) among the 2,820 pores. (B) Corre-
sponding 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.
521Imaging single A amyloid pore activity • Demuro et al.
peptides that have aggregated to form the pore. A recent study
combining imaging of individual fluorescently labeled A pep-
tides into lipid bilayers together with conductance measure-
ments led to a similar conclusion (Schauerte et al., 2010).
Monomers and dimers were found to be nonconducting, whereas
small oligomers (up to 14 mers) induced conductances of a
few tens of picosiemens, and large aggregates (which formed
only at high A concentrations) gave conductances of hundreds
of picosiemens. Schauerte et al. (2010) was unable to directly
associate conductance measurements to individual fluorescent
aggregates and instead segregated aggregation states into just
three classes. Because the values from electrophysiological
measurements are based on monovalent ion currents, it is not
possible to make a direct comparison with our estimates of the
specific Ca2+ conductance of A pores. Nevertheless, our find-
ings of a wide spectrum of permeability and gating properties
point to a more nuanced continuum of properties within a popu-
lation of A pores that likely correspond to the class II morpho-
logical aggregates of Schauerte et al. (2010).
A related question is whether the A oligomers that create
Ca2+-permeable pores are preformed in solution or aggregate
after interaction of smaller precursors within the membrane.
The available evidence suggests involvement of both processes.
Consistent with rapid membrane incorporation of preestab-
lished and functional oligomeric complexes, we had found that
extracellular application of oligomeric A resulted in rapid
induction of membrane Ca2+ permeability, whereas monomers
were ineffective (Demuro et al., 2005). On the other hand,
Schauerte et al. (2010) described binding of individual fluores-
cently labeled A monomers to lipid bilayers and subsequent
appearance of ion-conducting oligomers at a rate much faster
than the oligomerization rate (hours) in aqueous solution.
Our finding that individual A pores show progressive increases
(conductance measurements from bilayer systems) are labori-
ous and provide only a very small sample size.
Concordant with a spectrum of oligomeric compositions,
we observed enormous variation in functional properties be-
tween A1–42 pores, even at the same time and within the same
membrane region. Most dramatically, mean open probabilities
spanned a nearly 1,000-fold range, and permeabilities (ampli-
tudes of the largest Ca2+ fluorescence signal observed at each
given site) varied as much as 10-fold (F/Fo 0.2–2.0). Based on
comparison with fluorescence signals recorded under compara-
ble conditions from single acetylcholine receptor/channels of
known Ca2+ conductance (Ca2+ current of 0.25 pA equivalent
to F/Fo 1.0; Demuro and Parker, 2005a), we estimate that Ca2+
currents through A1–42 pores thus ranged between 0.05 and
0.5 pA at 100 mV, corresponding to Ca2+ conductances of
0.4–4 picosiemens assuming a reversal potential of 30 mV.
The maximal Ca2+ permeability of a pore correlated strongly
with its Po, and, similar to the transitions between conductance
levels reported in electrophysiological recordings from A pores
in bilayers (Arispe et al., 1993, 2007), we observed frequent
transitions between Ca2+ fluorescence dwell states during
SCCaFTs. Several observations indicate that the multistep
Ca2+ permeability levels did not arise because multiple A1–42
pores colocalized within a region smaller than our limit of reso-
lution; the stepwise behavior was seen even when the overall
density of A pores was very low, the amplitude levels were
not distributed as integer multiples as expected if they represent
the summated Ca2+ flux through multiple identical and indepen-
dent pores, and we frequently observed apparently synchronous
transitions across as many as four amplitude levels. Instead, we
favor a mechanism wherein the multiple permeability levels
represent different open states of a single pore, and the number
of levels exhibited by a pore is determined by the number of A
Figure 7. A142 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 F/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 nor-
malized to 100%. (A–C) Data obtained 20 min after application
of A142 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 inte-
gral of the fluorescence signals during all SCCaFTs detected at
that site. The curve (left ordinate; red) plots the cumulative contri-
bution of pores toward the normalized total Ca2+ load.
JCB • VOLUME 195 • NUMBER 3 • 2011 522
current, indicating that the monomeric peptide was ineffective at inducing
membrane Ca2+ permeability. Preparations that evoked currents ≥1 µA
were used immediately for TIRF imaging or were stored at 20°C before
use (Demuro et al., 2005). A1–42 samples were further characterized by
Western blot analysis (Kayed et al., 2010). Samples containing 4 µg
A1–42 were diluted in SDS treatment buffer, boiled for 5 min, and sepa-
rated on a 4–20% Tris-HCl gel (Bio-Rad Laboratories) at 4°C for 2 h at 85 V.
Proteins were transferred onto a nitrocellulose membrane, which was then
blocked for 1 h in 5% nonfat dry milk in TBS/Tween 20 buffer. The mem-
branes were probed with the mouse monoclonal anti-A antibody 6E10
(0.1 µg/ml; Sigma-Aldrich), and the conformation-specific polyclonal anti-
body OC (0.4 µg/ml; a gift from C. Glabe, University of California, Irvine,
Irvine, CA) overnight at 4°C. The membranes were then incubated with
anti–rabbit IgG conjugated with HRP (1:10,000) for 1 h at room tempera-
ture. Blots were developed with a chemiluminescence kit (SuperSignal
West Pico; Thermo Fisher Scientific).
Oocyte preparation and electrophysiology
Experiments were performed on defolliculated stage VI oocytes obtained
from Xenopus. Oocytes were injected 1 h before imaging with fluo-4
dextran (molecular mass of 10 kD and Ca2+ affinity of 3 µM) to a final
intracellular concentration of 40 µM. For TIRF microscopy experiments,
oocytes were placed in a hypertonic solution (200 mM K aspartate,
20 mM KCl, 1 mM MgCl2, 10 mM EGTA, and 10 mM Hepes, pH 7.2) at
4°C to shrink them so that the vitelline envelope could be manually torn
apart and removed using fine forceps. Oocytes were then placed animal
hemisphere down in a chamber whose bottom is formed by a fresh ethanol-
washed microscope cover glass (type-545-M; Thermo Fisher Scientific) and
were bathed in Ringer’s solution (110 mM NaCl, 1.8 mM CaCl2, 2 mM
KCl, and 5 mM Hepes, pH 7.2) at room temperature (23°C) continually
exchanged at a rate of 0.5 ml/min1 by a gravity-fed superfusion sys-
tem. The membrane potential was clamped at a holding potential of 0 mV
using a two-electrode voltage clamp (Gene Clamp 500; Molecular De-
vices) and was stepped to more negative potentials (100 mV unless
otherwise indicated) when imaging Ca2+ flux through amyloid pores to in-
crease the driving force for Ca2+ entry in to the cytosol. Solutions contain-
ing A42 oligomers were delivered from a glass pipette with a tip diame-
ter of 30 µm positioned near the edge of the membrane footprint of the
oocyte membrane on the cover glass.
TIRF microscopy, image acquisition, and processing
Imaging was accomplished by using a custom-built TIRF microscope
system based around a microscope (IX70; Olympus) equipped with a 60×
TIRF microscopy objective (1.45 NA; Olympus; Demuro and Parker,
2005b). Fluorescence excited by a 488-nm laser was imaged using an
electron-multiplied charge-coupled device camera (Cascade 128+; Roper
Scientific) at full resolution (128 × 128 pixel; 1 pixel = 0.33 µm at the
specimen) at a rate of 500 s1. Image data were acquired using the Meta-
Morph software package (Universal Imaging) and were black-level cor-
rected by subtracting the camera offset. To compensate for differential
time-dependent changes in basal fluorescence across different locations in
the image field, a strongly smoothed (10 × 10–pixel Gaussian blur) copy
of each frame was calculated to create a running baseline (F0) image. The
raw image stack was then divided, frame by frame, by the smoothed copy
to create a baseline-corrected pseudoratio stack in which each pixel repre-
sents localized differences in fluorescence relative to the spatially aver-
aged baseline fluorescence (F/F0). In initial experiments, data were
analyzed manually using MetaMorph to visually identify sites of pore activ-
ity, and traces of fluorescence versus time such as those in Figs. 2–5 were
obtained as the maximum pixel intensity within fixed 3 × 3–pixel (1 × 1 µm)
regions of interest centered on putative pore locations. Fluorescence mea-
surements were not confounded by movement of A pores, as tracking the
centroid locations of SCCaFTs with subpixel resolution revealed only slight
random deviations throughout the imaging record (SD typically <0.3
pixel), which are negligible in comparison with the size of the regions of
interest used to measure fluorescence intensities. The maximum observed
fluorescence signals were small (maximum F/Fo <2.0) in comparison with
the full dynamic range of fluo-4 (F/Fo >30 in saturating Ca2+) and are
thus expected to be linearly proportional to Ca2+ flux.
Event detection algorithm and automated data analysis (CellSpecks)
TIRF microscopy recordings produce large (128 × 128 pixel × 15,000 frame)
image stacks, which can represent the activity of >1,000 discrete A pores.
Thus, it was extremely laborious to compile and analyze population data by
visual inspection, and we did not find existing software, including GMimPro
and SparkMaster (Mashanov and Molloy, 2007; Picht et al., 2007),
in Po and permeability over several minutes further supports
the idea that pores already active in the membrane may grow
and change their properties by continued accretion of mono-
mers or small oligomers.
SCCaFTs arising from A pores remained at fixed loca-
tions over several minutes. This greatly simplified analysis but is
surprising because free diffusion of proteins in the lipid mem-
brane would be expected to result in clearly detectable move-
ments of several micrometers (Demuro and Parker, 2005a). We
do not believe that the immotility of pores arose through mechan-
ical interaction with the cover glass because a similar lack of
motility was evident in oocytes with intact vitelline envelope.
Instead, A may bind to some static cytoskeletal structure.
Our results were obtained using Xenopus oocytes, and it
remains to be determined how closely the actions of A on this
model cell system replicate those on neuronal membranes. More-
over, it is not clear how the progressive changes in gating and
permeability that we observe over a timescale of minutes may
relate to the progression of AD over years, during which time
neurons are continually exposed to low but rising concentrations
of soluble A in the cerebrospinal fluid. Nevertheless, we specu-
late that the initial evolution of the disease may involve formation
of small-conductance, low Po pores that, although not immedi-
ately triggering cell death, produce chronic pathological cell
stress. A tipping point may then be reached when a sustainable
level of Ca2+ influx through these pores is abruptly exceeded by
the appearance of even a few high-conductance, high Po pores
that contribute a disproportionately large Ca2+ load.
In summary, our application of high-resolution TIRF imag-
ing provides strong support for a mechanism whereby A amy-
loid oligomers form intrinsic Ca2+-permeable pores in the cell
membrane and enables massively parallel characterization of the
permeation and gating properties of thousands of pores. Our
results reveal an enormous range in activities that may reflect
concentration—and time dependent—differences in stoichiom-
etries of A pores and suggest a mechanism wherein continued
aggregation of oligomers leads to the formation of highly active
high-permeability pores that may play a disproportionate cyto-
toxic role in the pathogenesis of AD. Moreover, our single-channel
Ca2+ imaging approach would be equally applicable to studies of
numerous other amyloid-related diseases, including Huntington’s,
Parkinson’s, and prion disease, in which increased membrane
Ca2+ permeability is implicated as a major pathological mecha-
nism (Demuro et al., 2005).
Materials and methods
Preparation and characterization of A1–42 oligomers
Soluble oligomers were prepared by dissolving 0.5 mg of human recom-
binant A1–42 peptide (hexafluoroisopropanol pretreated; rPeptide) in
20 µl of freshly prepared DMSO and were quickly diluted with 480 µl
of double-distilled water in a siliconized Eppendorf tube. After a 10-min
sonication, samples were incubated at room temperature for 10 min and
then centrifuged for 15 min at 14,000 g. The supernatant fraction was
transferred to a new siliconized tube and stirred at 500 rpm using a Teflon-
coated microstir bar for 8–48 h at room temperature. Aliquots were taken
at intervals and were assayed by pipette application to voltage-clamped
oocytes at a final bath concentration of 1 µg/ml. Membrane currents were
recorded 30 min after application in response to hyperpolarization from
0 to 100 mV. A preparations incubated for <6 h evoked little or no
Imaging single A amyloid pore activity • Demuro et al.
brings up plots as these values as functions of time, and sampling can be
modified to select a mean or maximum of pixel values over a user-specified
region of interest. Data (including individual measurements of event dura-
tions, intervals, and fluorescence amplitudes together with population mea-
surements of mean pore open/closed times, Po, and mean fluorescence)
are exported as ASCII text files.
Fluo-4 dextran was purchased from Invitrogen, and human A142 pep-
tide was purchased from Millipore and rPeptide. gsmtx-4 was a gift from
F. Sachs (University at Buffalo, Buffalo, NY). All other reagents and anti-
bodies were purchased from Sigma-Aldrich.
We are grateful to Anna Pensalfini for assistance in performing Western blot
analyses of oligomer preparations. We thank Dr. Frederick Sachs for kindly
providing gsmtx-4 and Dr. Charles Glabe for the OC antibody.
This work was supported by National Institutes of Health grants
GM48071 (to I. Parker) and P50-AG16573 (to A. Demuro).
Submitted: 26 April 2011
Accepted: 26 September 2011
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adequate for our purposes. Accordingly, we developed a Java-based pro-
gram (CellSpecks) to identify active sites, generate idealized traces of pore
gating, and derive parameters, including mean open and closed times and
open probabilities as presented in Figs. 6 and 7. CellSpecks is coded in
Java and accepts image files in MetaMorph stack format or using Sun Java
standard libraries for opening directories of sequential images. Each frame
is converted into a new 2D array and stored as a stack object in memory.
The steps described in the next sections are then performed to identify
fluorescence signals, associate these with pore locations, and output final
Fluorescence thresholding. For each pixel, a 1D array of intensity val-
ues over the time is created and is corrected for baseline drift by subtrac-
tion of a heavily smoothed copy formed by filtering with a boxcar mean
51 frames in length. The modal value (most commonly observed value) of
the resulting array is then taken as the baseline, and an estimate of base-
line noise is calculated as the SD of only those source values that fall below
the mode. A value of 2.5 times the negative SDs above the mode was se-
lected as the noise threshold to minimize false positives, and a new stack
is created in which all pixel values below the threshold are set to 0, and
those above threshold are set to their original values relative to the mode.
Event detection. The event detection process is based on an object-
oriented programming paradigm. For each pixel, a new 3D array (x,y,t) of
event-part object pointers is generated to represent the pixel in space and
time. Each event-part object contains the intensity value at that location in
time as well as pointers to event-parts above, below, left, right, and before
and after (in time) the current pixel. When these event-parts are loaded into
each other’s pointers, contiguous signal pixels are then linked to each
other and bounded by null pointers to nonsignal pixels. Events are built
from these 3D groups of contiguous signal pixels by scanning the stack and
recursively linking all contiguous event parts to a single event object
(SCCaFT). At the end of this process, a list of all event locations in the stack
is generated. After constructing a list of events, CellSpecks performs a
series of steps to generate a list of pore locations.
Channel attribution. The events are then analyzed one by one and
assigned to a designated pore location. To facilitate the location of
pores, a weight array, g(x,y), is generated as a 2D array containing the
sum of pixel values from the original stack over time. Local maxima are
interpreted as likely pore locations and are used to create an initial list of
putative pore objects. Events are then linked to pores one by one, either
to the closest known pore or to a new pore location. Because the coordi-
nates of a pore are dynamically generated from their constituent events,
events early in the stack could bias the location of pores to which subse-
quent nearby larger events may be assigned. The algorithm avoids this
problem by seeding the list of pores with the locations of larger events
(whose fluorescence spread over a larger area and could therefore
be more flexibly associated) regardless of their order, so that they are
assigned their own putative pore locations at the outset. Whether or not
an event is added to the existing pore list depends on two criteria. The
first is binary overlap verification that checks whether or not the coordi-
nates of the event and pore in consideration when rounded to the nearest
pixel in space are equal. Event locations are calculated from their constit-
uent event parts using a mean of the event-part coordinates weighted by
intensity. The coordinate for the location of the pore is than calculated using
a mean of the coordinates of the pore’s constituent events weighted by
each event’s total intensity, calculated as the sum of the intensity mea-
sured in all the pixels involved in space and time. The second criterion for
event-pore association takes into consideration the relative intensity, Ir, of
the event’s pixels at the assigned pore location over the duration of the
event. Ir is calculated as the ratio between the sums of the intensity of
each pixel over the duration of the events versus the maximum intensity
measured at this location. The relative intensity of an event at the center
of the pore multiplied by the inverse of distance between the event and
pore centers must be >0.2, a threshold value empirically determined for
our experimental conditions.
Events that do not fulfil either of these criteria are assigned to new
pore locations. Finally, putative pore locations from the initial list that are
not associated with any events are discarded. By the end of the detection
and attribution processes, a final list of designated pore locations has been
produced, and each location is associated with a list of linked events.
Data visualization, analysis, and export. The list of events associated
with each pore location is used to derive distributions of pore open and
closed times (i.e., event durations and inter-event intervals), from which
population statistics are then calculated. A graphical user interface allows
on-screen viewing of raw and processed data (e.g., intermediate stack,
baseline, signal, and noise information). Clicking on a map of locations
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