Effect of calcium and cadmium ions on amyloid beta
peptide 1-42 channel activity
Notarachille G., Meleleo D., Gallucci E., Micelli S.
Dept. Farmaco-Biologico, Università degli Studi di Bari, via Orabona 4, 70126 Bari; Italy
Summary
Heavy metals are known to pollute the environment and can be taken up by the
organism in food. They can accumulate within organs and tissues, with sometimes
dramatic effects. Heavy-metal accumulation in the brain seems to be involved in neurodegenerative conditions, such as Alzheimer’s disease (AD). Recent reports have shown
the presence of heavy metals in senile plaques, hallmarks of AD, constituted of AβP140 and AβP1–42 peptide deposits. There is increasing evidence that heavy metals can
interact with amyloid β peptides, contributing to the neurodegenerative events of AD.
In this study, we analyse AβP1-42 incorporation and channel formation in planar
lipid membranes (PLMs) and the effects of calcium and cadmium ions on AβP1-42
channel activity.
Introduction
Environmental factors, such as heavy metals, appear to play an important role in
pathological processes, such as Alzheimer’s disease (AD) [1, 2]. AD is a progressive
neurodegenerative disorder, associated with memory loss and cognitive decline that
gradually leads to neuronal cell death. Amyloid β peptides (AβP), consisting of 40-42
amino acids (AβP1-40 and AβP1-42), are the major components of senile plaques,
one of the main pathological features in Alzheimer’s. Several studies have shown
that AβP1-42 is more neurotoxic than AβP1-40, owing to its greater aggregation
propensity [3, 4]. It has been proposed that the neurotoxicity of AβP1-42 is highly
dependent on its conformation, aggregation state and ability to form transmembrane
ion channels [5, 6, 7].
The aim of this study was to use the voltage-clamp technique to investigate the
propensity of AβP1-42 to incorporate and form ion channels in planar lipid membranes
(PLMs), made up of phosphatidylcholine:cholesterol (70:30,w:w). Further, as metal
ions are involved in the pathophysiology of disease, we investigate the role of Ca++
and Cd++ in AβP1-42 incorporation, channel formation and channel activity.
©2009 by MEDIMOND s.r.l.
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Fig. 1: Examples of chart recordings of AβP1-42 channel activity incorporated into PLMs made up of
POPC:Chol.
AβP1-42 alone (control) (a); AβP1-42 with CaCl2 added to the cis side (b); AβP1-42 with CaCl2 added to
the trans side (c); AβP1-42 preincubated with CaCl2 for 1h (d), for 1h and 30’(e), for 6h (f) and for 24h
(g); AβP1-42 with CdCl2 added to the cis side (h); AβP1-42 with CdCl2 added to the trans side (i); AβP1-42
preincubated with CdCl2 for 1h (l), for 1h and 30’(m), for 6h (n) and for 24h (o).
Experimental conditions: AβP1-42=5·10-8M; KCl 0.1M; pH=7; CaCl2 or CdCl2=2.5·10-4M; applied voltage
(Vs) 80mV and 100mV with CaCl2 or CdCl2, respectively.
Each trace represents a fragment of activity recorded in individual experiments.
Material and Methods
Channel activity was recorded in PLMs made up of phosphatidylcholine:cholesterol
(POPC:Ch) 70:30,w:w in 1% n-decane. Bilayers were formed across a 300µm-diameter
circular hole in a teflon partition separating two teflon chambers that hold symmetrical
KCl 0.1M (pH=7, temperature 23±1°C). The concentration of AβP1-42 and of CaCl2
or CdCl2 chosen in all series of experiments were 5⋅10-8M and 2.5⋅10-4M, respectively;
three different experimental procedures were performed:
1. in the first series of experiments, the AβP1-42 was added to the cis side of
the membrane. Subsequently, in the open channel state, CaCl2 or CdCl2 was added
to the same side as the AβP;
2. in the second series of experiments, after AβP1-42 channel formation, in the
open channel state, CaCl2 or CdCl2 was added to the trans side of the membrane.
3. in the third series of experiments, AβP1-42 was preincubated with CaCl2 or
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CdCl2 for different times (1h, 1h and 30’, 6h, 24h); subsequently the AβP1-42-cation
mixture was added to the cis side of the membrane.
In single-channel experiments, the membrane current was monitored with an oscilloscope and recorded on a chart recorder for data analysis by hand. The cis and trans
chambers were connected to the amplifier head stage by Ag/AgCl electrodes in series
with a voltage source and a highly sensitive current amplifier. The single-channel
instrumentation had a time resolution of 1-10msec depending on the magnitude of
the single-channel conductance. The polarity of the voltage was defined according to
the side where AβP was added (the cis side).
The single-channel data were obtained from at least three experiments. The current mediated by each channel is proportional to the applied voltage; dividing the
current flowing through each channel by the applied voltage gives the conductance
of the channel.
Results
AβP1-42 incorporation into PLMs causes discrete current jumps, compatible with
channel-type opening and closure. The traces present fluctuations between opening
(conducting) and closing (non-conducting) states, with different conductance levels,
occurrence frequency and lifetime. Fig. 1 shows examples of chart recordings of AβP142 channel formation in PLMs, in the absence and in the presence of cations.
The channel activity of AβP1-42 is evidently intense, characterized by different
conductance levels and high channel turnover. The presence of Ca++ or Cd++ leads to
a gradual decrease in channel activity and turnover as compared to AβP1-42 alone.
In particular, Cd++ has a more drastic effect than Ca++, because it “blocks” channel
activity in a time-dependent manner, both when Cd++ is added to the cis or trans
chamber and when preincubated with peptide.
For each voltage applied to the membrane, all single-channel events were used to
construct a histogram of conductance distribution. The conductance fluctuations are
not homogeneous in size, but distributed over a certain range. Fig. 2 reports a conductance fluctuation histogram at an applied voltage of 80mV in control conditions
(AβP1-42 alone).
For each applied voltage, the average conductance (Λ ) was determined by recording
not less than 100 single-events and averaging over the distribution of conductance
values [8]. For all series of experiments, we studied the voltage-conductance relationship in the applied voltage range of Vs= ±100mV. We found that:
a) theΛ values decrease as the applied voltage increases in all tested conditions;
b) where the events could be monitored, theΛ values are not modified by the
presence of cations for each applied voltage;
c) it is worth noting that in the preincubation experiments at 24h, the limited number
of events makes it impossible to construct a histogram of conductance distribution.
The occurrence frequency values (numbers of events in 60 sec) ± SD, for each
voltage applied and in all tested conditions, indicate that the addition of Ca++ or
Cd++ determines a decrease in channel turnover that culminates in the case of Cd++
in a drastic blockage of channel activity. As an example, Fig. 3 reports the values
of AβP1-42 occurrence in all different experimental conditions at an applied voltage
of 80mV.
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Fig. 2: Probability P(Λ) as % of occurrence of a conductance step of magnitude Λ (nS) at an applied voltage
of 80mV in control conditions (AβP1-42 alone).
P(Λ) is the number of steps observed within an interval of width (∆Λ=0.0125nS) divided by the total
number of steps.
Fig. 3: The occurrence (events/60sec) ±SD of AβP1-42 channels in POPC:Chol PLMs at applied potentials
of 80mV, in control conditions and in the presence of Ca++ or Cd++ added to the cis or trans side of
the PLM or in preincubation experiments.
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The analysis of channel lifetime was performed in experiments in which a total
of not less than 100 channels were present, with the known equation with a singleor two-exponential function [9], but this analysis, owing to the reduced number of
channels, was difficult in the presence of the two cations .
AβP1-42, at an applied voltage of 100mV, exhibits a lifetime in the range of
0.25-19.75 sec. Neither calcium nor cadmium, no matter how they added, modify
the channel lifetime range.
Conclusions
Our results indicate that AβP1-42 incorporates into PLMs made up of POPC:Ch
and forms voltage-dependent ion channels.
Calcium and cadmium are able to modulate AβP1-42 channel activity - in fact
they decrease channel activity and turnover.
The inhibition of channel activity takes place in the case of channels already
incorporated into the membrane, as well as when Ca++ or Cd++ are preincubated with
peptide. This effect is more drastic in the case of Cd++.
Ca++ and Cd++ preincubated with AβP are suggestive for a possible conformational
variation of peptide that renders it incompatible for membrane incorporation. Alternatively, it could be hypothesized that cations could interact with specific groups of
AβP1-42 that alter the folding of the peptide.
The most intensively studied examples of conformational diseases are Alzheimer’s
disease, Parkinson’s disease, Huntington’s disease and spongiform encephalopathies. As
AD is associated with protein misfolding, we can speculate that the presence of cations
such as Ca++ or Cd++ may be an important predisposing factor for the formation of
small and toxic aggregates of AβP, directly or indirectly involved in neurodegenerative
mechanisms [10, 11, 12]. This is consistent with recent studies in vitro and in vivo
that demonstrated that metal ions, such as zinc, copper, iron and aluminium, play a
crucial role in AD, acting as mediators of neurotoxicity by favouring protein aggregation or oxidative damage [13]. Owing to the complexity of the interplay between
peptide and Ca++ or Cd++, further studies are in progress to clarify this aspect.
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