The “Aβ ion channel
hypothesis” suggests that
Aβ forms ion channel-like pores in lipid membranes causing transmembrane
currents via Aβ ion channels.
10,15,22,24−27,31,56−58 Upon addition of the Aβ peptide to one side
of the PLB chamber, Aβ must first bind to the bilayer and then
likely undergo a conformational change that helps the Aβ peptide
overcome the barrier to insert or slip into the bilayer. Once in the
bilayer, monomers or oligomers need to interact with one another to
form a porelike structure. There are three different types of ion
channel activity that have been described for Aβ channels.
22 The first type of channel activity is the “bursting”
fast cation channel that, as its name implies, is a short burst of
activity that gives a nonlinear current–voltage relationship.
The second type of channel activity is the so-called “spiky”
fast cation channel that is similar to a burst of activity; however,
the spikes are short-lived compared to bursting activity. Lastly,
the third type of channel activity is the “step” or
“steplike” activity. With the steplike behavior, a clear,
defined jump in current is seen as a channel opens and closes. We
observed these types of current activity for wild-type Aβ
1–42 in the DOPS/DOPE lipid bilayers. Figure A illustrates bursting and spiky behavior of the
wild-type Aβ
1–42 channels. At −80 mV,
these channels began to exhibit the so-called “bursting fast
cation channel”. The only truly apparent difference between
bursting and spiky behavior is that the bursting channel activity
is characterized by the absence of the long closures of channels.
22 As we increased the applied potential from −80
to −50 mV in 10 mV steps, the magnitude of the current decreased,
as well. The inset of Figure A shows a higher
time resolution of the steplike activity seen with Aβ
1–42 channels. These steps are much less frequent in folded bilayers
than in painted bilayers.
Figure B depicts 30 min
of recording and
the ability of zinc to block a portion of the Aβ
1–42 activity as previously shown.
27,31,32,60 Zn
2+ appears to inhibit
Aβ
1–42 in two sites; it binds to the N-terminal
histidines (His6, His13, and His14) on the peptide,
51 inducing a conformational change, and it has been shown
also to inhibit the current activity induced by the Aβ
17–42 fragment (p3), a fragment without histidines.
28 The arrow in Figure B points to
the time at which zinc was added to the same chamber. The channels
are not immediately blocked by zinc (Figure B), yet as the experiment progressed, we observed decreased conductances.
The electrical recordings show the ionic current with multiple
conductances; this heterogeneous nature is typical of Aβ ion
channels and other channel-forming amyloids. One possible explanation
for this behavior is that the channels consist of distinct oligomeric
species, forming channel-like structures.
10,28,45,61 Given the
different conductances of the Aβ channels, this could explain
the variance in conductance measured in the electrical recordings.
In 13 experiments with wild-type Aβ
1–42, we
measured channel activity in six cases, making the frequency of channel
activity 46%, which is comparable to reports from previous work with
Aβ
1–42.
27,42,62,63 Interestingly, we also found
that different lots of Aβ
1–42 differentially
affected the percentage of channel activity observed. This suggests
that the aggregation state of Aβ
1–42 affects
channel activity. Our results show that Aβ
1–42 activity occurred at concentrations as low as 0.5–1 μM.
We did not exceed concentrations of 4.5 μM with Aβ
1–42. Channel-like behavior was never observed in bilayers
prior to the addition of the peptide, or without its addition.
Although work showing channel activity of Aβ
1–42 has previously been done,
23,27,31,42,62,63 one prerequisite for this work was to characterize
Aβ
1–42 activity in our membrane/buffer system.
We also needed a reference to which we could compare our results with
those of the Aβ
1–42 mutants. To the best of
our knowledge, this is the second report showing Aβ channel-like
activity with folded bilayers,
31,42 and only one report
exists using the tip-dip method.
20 Most
groups studying Aβ ion channel formation in model membranes
used painted bilayers.
10,22,23,26,27,63 Generally, solvent free bilayers (folded and tip-dip)
are considered better models of bilayers, because the bilayers are
thinner and contain smaller amounts of nonbiological solvents in the
hydrophobic core. We found that Aβ
1–42 in
folded bilayers generally exhibited more spiky and bursting types
of activity (short-lived) than steplike activity. The combined results
of the membrane-only experiments and Aβ
1–42 activity in DOPS/DOPE folded bilayers validate a system for testing
the activity of Aβ
1–42 mutants. To gather
structural information about Aβ ion channels, we studied amino
acid substitutions of Aβ
1–42. By studying
these mutants, we aim to infer structural features of Aβ and
its function in membranes. This functional approach provides structural
information that cannot be elucidated by other techniques.
The F19P Mutant Does Not Exhibit Channel-like Activity
The amino acid proline (Pro or P) is under-represented in β-sheets
of proteins with known structure.
64 Thermodynamic
studies of amino acid replacements identify proline as the amino acid
least compatible with β-sheet structure.
64−66 Consequently,
proline mutagenesis of Aβ has been intensively studied, specifically
the ability of these types of mutants to form fibrils.
64−66 Proline is energetically unfavorable in the extended cross-β-sheet
structure and, as a result, inhibits amyloid aggregation.
64−66 The substitution with proline introduces a “kink”
into the strands of the U-shaped peptide. In previous work, we proposed
that the Aβ
17–42 fragment (p3) with the F19P
substitution (p3-F19P) exhibited no channel activity.
28 We show here that Aβ
1–42 F19P exhibits
no channel activity, suggesting that the Pro substitution at this
position hinders the formation of Aβ-conducting structures.
Figure shows a current versus time trace
of the nonconductive F19P mutant. At voltages as high as ±150
mV, there was still no visible conductance from channel formation
(see the inset of Figure ).
We conducted these experiments for up to 4 h and
then verified
again the integrity of bilayers by adding gramicidin A, a well-established
pore former.
49 F19P was tested in folded
bilayers (
n = 10) at concentrations ranging from
4.5 to 13.5 μM. After data had been recorded for more than 40
h (
n = 10, 4 h each) with F19P, we observed only
100 s of channel-like activity with very low conductance. The activities
are presented in Figure B,C. The pores shown
in Figure B,C have conductances of 4.6 and
2.2 pS (below the level for gA in this membrane/electrolyte system),
compared to the wide range of higher conductances for wild-type Aβ
1–42, generally between 50 pS and 1 nS (Figure ). F19P was also tested in DOPS/POPE painted bilayers
[
n = 7 (data not shown)] and showed no sign of instability
for extended periods of time, with an average of 104 ± 40 min.
In these painted bilayers, the lowest recorded point of bilayer instability
was 75 min and the highest 190 min. This is comparable to the normal
life span of these bilayers without any addition of peptide. However,
the unusually low and unique conductance measurements (Figure ) might suggest that either F19P forms collapsed
pores or this mutant is impaired in its binding and/or insertion into
bilayers. The most rigorous interpretation of the F19P results is
that F19P does not form conducting structures in the bilayers, which
suggests that the β-sheet structure of the U-turn is necessary
for Aβ
1–42 pore formation (see below).
The F20C Mutant Behaves Like the Wild Type in Lipid Bilayers
For the Aβ
1–42 F20C mutant, we substituted
Phe20 with Cys. To the best of our knowledge, the membrane behavior
of the F20C mutant has not been described previously. We found that
the F20C mutant conducts in a manner somewhat comparable to that of
the wild type, in good agreement with the model presented here (see
below). We performed seven experiments with F20C and observed activity
in four (57%). In all experiments, we added F20C directly to one side
of the PLB chamber. We initially added the mutant to a final concentration
of 4.5 μM and if needed added additional 4.5 μM mutant
every 45 min until a final concentration of 13.5 μM was reached.
Under these conditions, F20C activity appears mostly as spikes and
bursts and occasionally as short-lived steplike activity. Spiky and
bursting activities are shown in the
Supporting
Information.
Figure presents
a 1 h current versus time trace recording of a continuous activity
by F20C. During this time, we added Zn
2+ ions and observed
inhibition of this activity. For the sake of simplicity, the figure
is presented in 15 min panels. Figure A presents
a current trace that can be described in four smaller parts that closely
follow the voltage plot under the trace. Initially, (i) the applied
voltage is −50 mV showing channel-like activity, followed by
(ii) a gradual decrease in the applied potential. Once there is no
current (at −1.5 mV), (iii) the voltage is set to zero and
sustained at this level. Finally, (iv) the applied voltage is increased
to 30 mV. In Figure B, we added 0.5 mM Zn
2+ to the same side of F20C. Following the addition of Zn
2+, the activity begins to subside, although not immediately.
In the next 30 min (Figure C,D), the amplitude
and frequency of membrane conductance decrease and ultimately appear
to be inhibited by Zn
2+. This result shows that F20C activity
is sensitive to Zn
2+ ions. The replacement of Phe20 with
Cys in Aβ
1–42 does not preclude channel formation.
This region of the Aβ peptide is central to its ability to form
fibrils. We observed precipitates in Aβ
1–42 F20C residual aliquots. In fact, a scanning cysteine mutagenesis
study of Aβ
1–40 found that the F20C mutant
was accessible to alkylation in the fibril state, indicating that
F20 is exposed to the solvent in fibers.
67Both F19P and F20C Mutants Form Channel-like Structures but
Have Different Pore Morphologies
For two Aβ
1–42 conformers, we performed 100 ns explicit MD simulations on the wild-type,
F19P, and F20C barrels embedded in an anionic lipid bilayer composed
of DOPS and POPE (1:2 molar ratio). Conformer 1 barrels have a turn
at Ser26–Ile31, and conformer 2 barrels have a turn at Asp23–Gly29.
Both Aβ conformers can be divided into four domains: the N-terminal
chain (residues 1–16 and 1–8 for conformers 1 and 2,
respectively), pore-lining β-stand (residues 17–25 and
9–22 for conformers 1 and 2, respectively), turn (residues
26–31 and 23–29 for conformers 1 and 2, respectively),
and C-terminal β-strand (residues 32–42 and 30–42
for conformers 1 and 2, respectively). Both point mutations were in
the pore-lining β-stand. All Aβ barrels were initially
designed as a perfect annular shape. However, the initial annular
conformation is gradually lost via relaxation of the lipid bilayer,
and environmentally relaxed peptides can be observed after 30 ns.
The membrane-embedded U-shaped portions of the Aβ barrels reach
equilibration after the initial transient state, while the extramembranous
N-termini of the peptides are disordered. Heterogeneous channel conformations
as observed in the average Aβ barrels structure (Figure ) are similar to the structure of Aβ channels
with various sequences in our previous simulations.
13,14,28,42−44,52,53 However, although the outer shapes of Aβ barrels are similar
to each other, the morphology of the solvated pore is quite different.
For the wild-type Aβ barrels, both conformers preserve the solvated
pore, wide enough for ions to enter and exit at the same time. The
averaged pore diameters are ~1.83 and ~1.86 nm for the
conformer 1 and 2 Aβ barrels, respectively. However, both conformers
of each mutant significantly decrease the size of the solvated pore.
For F19P, the averaged pore diameters are ~1.48 and ~1.69
nm for the conformer 1 and 2 Aβ barrels, respectively, and for
F20C, they are ~1.67 and ~1.69 nm for the conformer
1 and 2 Aβ barrels, respectively. In particular, the conformer
1 F19P mutant completely blocks the channel mouth in the lower bilayer
leaflet (Figure C).
The pore diameter was calculated using HOLE.
68 Figure shows the pore
diameter
measured along the pore axis for the averaged barrel conformations.
The averaged pore heights calculated from the program are ~4.1,
~5.6, and ~5.2 nm for conformer 1 and ~4.5, ~5.9,
and ~5.3 nm for conformer 2 wild-type, F19P, and F20C barrels,
respectively. The pore heights for the wild-type Aβ barrels
match the bilayer thickness, while both mutant barrels have longer
and narrower pores than the wild type. Especially, both F19P barrels
have a collapsed pore due to interacting N-terminal chains at the
lower channel mouth blocking the entry into the pore (Figure A,B). The N-terminal chains contain several charged
residues; they normally stretch toward the lipid headgroups, and only
a few chains can fluctuate toward the channel mouth in wild-type barrels.
However, in the F19P barrels, the N-terminal chains easily stick together
because of kinks produced by the Pro19 residues in the pore-lining
β-stands. No kinks can be observed in the F20C barrels, but
still the F20C mutants provide a less stable and smaller pore than
wild-type Aβ
1–42.
In the wild-type Aβ barrels, we observe that
few ions cross
through the water pore, but most ions spend time at the binding sites
and are frequently near the channel mouth during the simulation. However,
in F19P barrels, we found that no ions cross through the water pore
during the simulations, although ions can spend time at the binding
sites located in the upper channel mouth. The behavior of ions in
the F20C pores is similar to those in the wild-type pores. To observe
the fluctuation of the ion across the pore, we calculated the change
in the total charge in the pore as a function of simulation time (Figure C,D). For the conformer 1 Aβ barrels, a pore
height cutoff along the pore axis is |z| < 10
nm, while for the conformer 2 Aβ barrels, the cutoff is |z| < 15 nm. With these cutoffs, charge fluctuations involve
only contributions of ions fluctuating in the middle of the pore.
Apparently, the wild-type pores have larger charge fluctuations than
any mutant pore, because the wild-type Aβ barrels have the wider
pore. The electrical charge fluctuation due to movement of a number
of ions across the pore appears to resemble the single-channel conductance,
although the measured time frame is significantly limited.
We
provide experimental evidence that amino acid substitutions
can have a direct and profound impact on Aβ
1–42 membrane activity measurements. This functional tool can be coupled
with molecular dynamics (MD) models to gain valuable insights into
structural aspects of the Aβ
1–42 channel structure.
The initial results presented in this work suggest that (i) the nature
of the bilayer affects the type of Aβ
1–42 activity.
In folded DOPS/DOPE bilayers, we rarely observed steplike activity
with wild-type Aβ
1–42. To the best of our
knowledge, this is the second report showing Aβ
1–42 channel-like activity with folded bilayers.
31,42 (ii) Results with the Aβ
1–42 F19P mutant
may imply that the β-sheet U turn is needed for activity in
bilayers. The fact that F19P has been previously shown not to form
fibers
64,66 provides further information about the structural
significance of this residue. The lack of membrane activity for F19P
suggests that a β-sheet in the U turn is necessary for Aβ
1–42 pore formation. Generally, Pro substitutions form
a kink hindering either β-sheets or α-helices. Additional
amino acid substitutions at position Phe19 would be needed to exclude
other Aβ
1–42 secondary structures,
69 which could be embedded in the bilayer. The
F19P peptide could be used as a structure-impaired negative control
in experiments aiming to address toxicity or other structural features
of Aβ
1–42. In model bilayers, the heterogeneous
activity of Aβ
1–42 has been at times interpreted
as nonspecific membrane perturbations by the peptide. The lack of
F19P activity (
n = 17; 10 folded and 7 painted) lends
support to the electrical activity most likely occurring through a
defined structure(s), i.e., pore formation. While this conclusion
is strictly applicable for only the folded and painted bilayers with
the electrolyte used in this study, we may expect similar behavior
in other lipid compositions where Aβ
1–42 exhibits
bilayer activity.
These results led us to suggest that, like
p3-F19P, the Aβ1–42 F19P mutant might form
a collapsed pore that is
nonconductive. Further support for this suggestion was obtained by
the MD simulations in which we observed that the pore is blocked by
the N-terminal chains due to kinks at Pro residues in the pore-lining
β-strands. We observed F19P minor channel conductances in one
experiment. This F19P activity is inconclusive, because this activity
was seen in 100 s out of 40 h of recording, yet we did not see such
activity in 28 h in identical bilayers without peptide. The MD model
proposed in this study, together with the PLB results, suggests a
collapsed pore. Results with F19P are negative (no bilayer activity);
therefore, its implications are inferential, and other experimental
approaches are needed to exclude the possibility that the F19P peptide
might simply not insert or bind the membrane or that the peptide binds
and inserts itself into the membrane and forms a collapsed pore.
Here, we present evidence showing that Aβ
1–42 F20C forms ion channels with membrane activity comparable to that
of the wild type, and this activity can be inhibited by low millimolar
concentrations of Zn
2+ like the wild type. Cysteine residues
have a reactive sulfhydryl group that if in the proximity of other
monomers could form −S–S– bridges with neighboring,
in-register monomers, somewhat stabilizing the pore structure. This
possibility remains unaddressed in this study. In future work, we
would like to react the F20C residue with MTS reagents.
70 These experiments are often difficult without
prior knowledge of the pore structure.
71,72 If we succeed,
we will be able to experimentally determine if the Phe20 residue lines
the pore of the ion channel as suggested by the MD models presented
here.
and Ratnesh Lal*†
This article has been 
