Cu2+-induced modification of the kinetics of A{beta}(1-42) channels

Randa Bahadi,1 Peter V. Farrelly,1 Bronwyn L. Kenna,1 Cyril C. Curtain,2 Colin L. Masters,2 Roberto Cappai,2 Kevin J. Barnham,2,3 and Joseph I. Kourie1

1Membrane Transport Group, Department of Chemistry, The Faculties, The Australian National University, Canberra, Australian Capital Territory 0200; 2Department of Pathology, The University of Melbourne, Victoria 3010; and 3The Mental Health Research Institute, Parkville, Victoria 3052, Australia

Submitted 14 April 2003 ; accepted in final form 21 May 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We found that the amyloid {beta} peptide A{beta}(1-42) is capable of interacting with membrane and forming heterogeneous ion channels in the absence of any added Cu2+ or biological redox agents that have been reported to mediate A{beta}(1-42) toxicity. The A{beta}(1-42)-formed cation channel was inhibited by Cu2+ in cis solution ([Cu2+]cis) in a voltage- and concentration-dependent manner between 0 and 250 µM. The [Cu2+]cis-induced channel inhibition is fully reversible at low concentrations between 50 and 100 µM [Cu2+]cis and partially reversible at 250 µM [Cu2+]cis. The inhibitory effects of [Cu2+]cis between 50 and 250 µM on the channel could not be reversed with addition of Cu2+-chelating agent clioquinol (CQ) at concentrations between 64 and 384 µM applied to the cis chamber. The effects of 200-250 µM [Cu2+]cis on the burst and intraburst kinetic parameters were not fully reversible with either wash or 128 µM [CQ]cis. The kinetic analysis of the data indicate that Cu2+-induced inhibition was mediated via both desensitization and an open channel block mechanism and that Cu2+ binds to the histidine residues located at the mouth of the channel. It is proposed that the Cu2+-binding site of the A{beta}(1-42)-formed channels is modulated with Cu2+ in a similar way to those of channels formed with the prion protein fragment PrP(106-126), suggesting a possible common mechanism for Cu2+ modulation of A{beta} and PrP channel proteins linked to neurodegenerative diseases.

neurodegenerative diseases; transitional metals; ion channel pathologies; membrane injuries; calcium homeostasis


ALZHEIMER'S DISEASE (AD) is a neurodegenerative disorder that affects the cognitive function of the brain. Pathological changes in AD are characterized by the formation of amyloid plaques and neurofibrillary tangles as well as extensive neuronal loss. The plaques, which accumulate extracellularly in the brain, are composed of aggregates and cause direct neurotoxic effects and/or increase neuronal vulnerability to excitotoxic insults. The major components of the extracellular neurofibrillar bundles are polymerized amyloid {beta}(A{beta}) peptides A{beta}(1-40), A{beta}(1-42), and A{beta}(1-43). It has been shown that A{beta} familial AD-linked mutations of the amyloid protein precursors presenilin-1 and presenilin-2 increase the concentration of A{beta}(1-42) (53), which has been shown to be toxic in primary neuronal culture at micromolar concentrations (56). The major mechanisms proposed for A{beta}-induced cytotoxicity involve the loss of Ca2+ homeostasis (see Refs. 46 and 47) and the generation of reactive oxygen species (see Refs. 9, 11, 12, and 25). The changes in Ca2+ homeostasis could be the result of 1) alterations in endogenous ion transport systems and 2) formation of heterogeneous ion channels (see Refs. 32, 33, and 35). Several laboratories have found that A{beta}(1-40) and other fragments of amyloid precursor protein that contain A{beta} also possess the ability to form ion channels in both artificial and biological membranes. Electrophysiological studies have shown that A{beta} fragments, e.g., A{beta}(25-35), A{beta}(1-40), and A{beta}(1-42), elicit cation-selective currents when reconstituted into lipid bilayers (1-4, 23, 24, 30, 35, 37, 41, 55) and in the plasma membrane of neurons (28, 29, 48) as well as in Xenopus oocytes (21). A{beta}(1-42) and A{beta}(1-40) increase Ca2+ uptake in liposomes in a dose-dependent manner (38, 49), and soluble A{beta}s induce Ca2+ influx in neurons and nonneuronal cells (10, 50, 51, 57).

In addition to the A{beta} being linked to AD, a role for transition metals has also been recognized. Cu2+ and Zn2+ have been implicated in AD (11, 12, 42), Parkinson's disease (54), prion protein (PrP) (26), and immunoglobulin light chain amyloidosis (17). The mechanisms underlying the interaction between A{beta} and these metals may mediate their role in neurotoxicity. There is also evidence to show that A{beta}, and also PrP, binds Cu2+ (5, 6) to a site resembling that of superoxide dismutase (16) and metalloenzyme-like activity (44). The A{beta}-formed channels are redox sensitive and modulated by Zn2+ (3, 4, 35). In this study we have examined the Cu2+ dependency of A{beta}(1-42)-formed channels.


    MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Solutions. Solutions contained KCl (250 mM cis/50 mM trans) plus 1 mM CaCl2 and 10 mM HEPES (pHcis 7.4, adjusted with KOH). The Cu2+ concentration of the cis solution ([Cu2+]cis) was adjusted by adding aliquots of concentrated Cu2+ in cis solution.

Lipid bilayer technique. Bilayers were formed across a 150-µm hole in the wall of a 1-ml Delrin T cup using a mixture of palmitoyl-oleoyl-phosphatidylethanolamine, palmitoyl-oleoyl-phosphatidylserine, and palmitoyl-oleoyl-phosphatidylcholine (5:3:2, by volume) and palmitoyl-oleoyl-phosphatidylethanolamine, palmitoyl-oleoyl-phosphatidylserine (7:3 by volume) (36, 40), obtained in chloroform from Avanti Polar Lipids (Alabaster, AL). The lipid mixture was dried under a stream of N2 and redissolved in n-decane at a final concentration of 50 mg/ml. A{beta}(1-42) was synthesized by the W. Keck Laboratory (Yale University, New Haven, CT). A{beta} stock solutions were prepared in experimental cis solutions and kept at -80°C till use. A{beta}(1-42) was then incorporated into the negatively charged lipid bilayer by addition to the cis chamber of A{beta}(1-42) liposomes (2) or aliquots of A{beta} stock solution at a final peptide concentration of 0.1-1 µg/ml. The side of the bilayer to which the peptide or liposomes were added is defined as cis, and the other side as trans. Ion channels were also recorded from a peptide-lipid mixture of 1:50. Unless stated otherwise, the initial experimental solution for incorporating synthetic A{beta}(1-42) into the bilayers contained KCl (250 mM cis/50 mM trans). Other reagents were obtained from Sigma unless otherwise noted. The experiments were conducted at 20-25°C.

Preparation of liposomes. In some experiments, liposomes of A{beta}(1-42) were used. The method for the preparation of these liposomes was described by Arispe et al. (3). A 20-µl aliquot of palmitoyl-oleoyl-phosphatidylserine dissolved in chloroform (10 mg/ml) was placed in a glass tube. After evaporation of the chloroform (by blowing filtered N2 gas), a 30-µl aliquot of 1 M potassium aspartate (pH adjusted to 7.2) was added and the resulting mixture was sonicated for 5 min. Next, a 20 µl (2 mg/ml) stock solution of the A{beta}(1-42) in water was added, and the adduct was sonicated for 2 min.

Ion channel recording. The pCLAMP6 program (Axon Instruments) was used for voltage command and acquisition of ionic current families with an Axopatch 200 amplifier (Axon Instruments). The current was monitored with an oscilloscope, and the data were stored on a computer. The cis and trans chambers were connected to the amplifier head stage by Ag-AgCl electrodes in agar salt bridges containing the solutions present in each chamber. Voltages and currents are expressed relative to the trans chamber. An outward current is defined as a cation moving from the cis chamber to the trans chamber or an anion moving from the trans chamber to the cis chamber. Data were filtered at 1 kHz (4-pole Bessel, -3 dB) and digitized via a TL-1 DMA interface (Axon Instruments) at 2 kHz.

Data analysis. Modifications in the bilayer thickness, mediated via lipid solvents, e.g., n-decane, contribute to changes in channel gating kinetics of ion channels (31, 36) and particularly those formed with short peptides, e.g., the 15-amino acid gramicidin-formed channel (43). Therefore, standardizing the specific membrane capacitance (Cb) and its time independence is important in comparative and detailed investigations of ion channel characteristics. Kinetic analysis for the A{beta}(1-42)-formed channels was conducted only for optimal bilayers having a Cb of >0.42 µF/cm2 and containing a single active channel. The criteria for defining ion currents as belonging to a "single channel" have been described elsewhere (14a). Single-channel activity was analyzed for overall characteristics using the program CHANNEL 2 (developed by Gage PW and Smith M; see Ref. 36). The following kinetic parameters of single-channel activity (32- to 128-s-long records) were determined: mean open time, To (i.e., the average of the open times of all intervals where the current exceeded the baseline noise for 0.5 ms); frequency of opening to all conductance levels, Fo; and open probability, Po (i.e., the sum of all open times as a fraction of the total time). CHANNEL 2 also allows online analysis of the entire current record for computation of the maximal current (I) and mean current (I'). I' is defined as the integral of the current passing through the channel divided by the total time. The integral current is determined by computation of the area between a line set on the noise of the closed state and channel opening to various levels. The threshold level for the detection of single-channel events was set at 50% of the maximum current (45). The maximal current is the current amplitude of a fully open channel. The maximal current was obtained by measuring the distance (in pA) between two lines, one set on the noise of the closed level where the current amplitude is 0 pA and the other set on the noise of the majority of distinct events that were in the open state. The maximal current was also obtained by measuring the distance (in pA) between the peak at 0 pA (representing the closed state) and the extreme peak on the right (representing the open state) in the all-point histogram generated using CHANNEL 2 (see Ref. 36). Both methods were used, and the results were generally in agreement. The reversal potential Erev was corrected for ionic mobility and liquid junction potential (8). SigmaPlot (Jandel Scientific Software) was used to plot and calculate curve fit of the data. Data are reported as means ± SE, and the difference in means was analyzed by using Student's t-test. Data were considered statistically significant when P values were <=0.05.


    RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cu2+ effects on A{beta}(1-42) channel activity. Experiments conducted on bilayers before the addition of A{beta}(1-42) revealed that the biophysical properties of the phospholipids forming the bilayer (n = 12) were not affected by [Cu2+]cis between 50 and 200 µM or clioquinol ([CQ]cis) between 8 and 256 µM (n = 16). The bilayers maintained a specific bilayer capacitance value of ~0.42 µF/cm2 and a cord conductance value for the leak of ~12.5 pS in the presence of different [Cu2+]cis or [CQ]cis. The effects of Cu2+ and [CQ]cis on A{beta}(1-42) channels were investigated after incorporation of the peptide into the lipid bilayer membranes. Excess peptide, 0.1-1 µg/ml, in the cis chamber was removed by perfusion with fresh control cis solution. The lack of any significant effects of CQ on the cation channel is shown in Fig. 1, where 1) 192 µM [CQ]cis did not affect the cation channel in control cis solution, before the addition of any Cu2+, and 2) 50 µM Cu2+ affected the kinetics of this channel in the presence of 192 µM [CQ]cis. These experiments suggested that CQ had no effects on this channel and that any background traces of Cu2+ had no significant effects on the channel activity.



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Fig. 1. Inhibitory effects of Cu2+ concentrations in the cis solution ([Cu2+]cis) on the activity of an amyloid {beta}-peptide [A{beta}(1-42)]-formed channel, in the burst mode, activated at membrane potentials (Vm)of +140 mV in KCl (250 mM cis/50 mM trans) containing clioquinol (CQ). Current traces are shown for control (A), 192 µM [CQ]cis (B), and 192 µM [CQ]cis + 50 µM [Cu2+]cis (C). Following convention, the upward deflections denote activation of outward ion current. For a better display, the data have been filtered at 1 kHz, digitized at 2 kHz, and reduced by a factor of 5. Current traces are separated by a 12-pA offset.

 

Figure 2, A-F, shows groups of four representative current traces recorded from the same bilayer containing A{beta}(1-42) channels exposed to different [Cu2+]cis between 0 and 250 µM and clamped at +140 mV. Several seconds after the addition of 50 µM Cu2+, changes in channel activity were observed (Fig. 2B). The most apparent effects were the lengthening of the long durations of channel inactivity, i.e., intrabursts, and changes in the activity of the channel within the burst. The channel activity was modified further after subsequent additions to 100, 150, 200, and 250 µM [Cu2+]cis (Fig. 2, C-F). These findings are in agreement with Cu2+-induced inhibition of the deamidated-type of PrP(106-126)-formed channels (34), where Cu2+ induced long-duration channel closures at positive voltages. The effects of 250 µM [Cu2+]cis on A{beta}(1-42) channels could not be reversed with the addition of 112 µM CQ, which selectively binds Cu2+, to the cis solution (Fig. 2G). The channel activity recovered partially after wash with control cis solution containing 250 mM KCl (Fig. 2H). Furthermore, the addition of 128 µM [CQ]cis produced only a little additional recovery to the channel activity (Fig. 2I). Similarly, 384 µM [CQ]cis did not prevent channel inhibition induced with 100 µM [Cu2+]cis (data not shown).



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Fig. 2. Effects of different [Cu2+]cis on the activity of an A{beta}(1-42)-formed channel activated at Vm of +140 mV in KCl (250 mM cis/50 mM trans). Current traces are shown for control (A), 50 µM [Cu2+]cis (B), 100 µM [Cu2+]cis (C), 150 µM [Cu2+]cis (D), 200 µM [Cu2+]cis (E), 250 µM [Cu2+]cis (F), 250 µM [Cu2+]cis + 112 µM [CQ]cis (G), wash with control cis solution (H), and 128 µM [CQ]cis (I). Following convention, the upward deflections denote activation of outward ion current. For clarity, only 4 of 16 typical current traces are shown at each treatment. For a better display, the data have been filtered at 1 kHz, digitized at 2 kHz, and reduced by a factor of 5. Current traces are separated by a 12-pA offset.

 

Effects on A{beta}(1-42) channel conductance. Data analysis with CHANNEL 2 (36) revealed that the Cu2+-induced decrease in I and I' was exponential (Fig. 3). Cu2+ reduced the channel current amplitude by 38.34%; the current amplitudes were 8.9 ± 0.4 and 6.3 ± 0.6 pA in the presence and absence of 100 µM Cu2+ at a membrane potential (Vm) of +140 mV, respectively (n = 16 traces). However, current amplitude was further reduced to 5.5 ± 0.4 pA in 200 µM Cu2+ and a Vm of +140 mV. I' was reduced from 4.5 to 1.22 pA (27.11%) as [Cu2+]cis increased to 200 µM. No channel activity was observed in the presence of 250 µM [Cu2+]cis.



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Fig. 3. Dose-dependent effects of [Cu2+]cis on the A{beta}(1-42)-formed channel parameters maximal current (I) and mean current (I') are shown in A and B, respectively. Solid lines are drawn to a third-order polynomial.

 

Effects on voltage dependence of A{beta}(1-42) channel conductance. To examine the voltage dependency of [Cu2+]cis-induced effects on the channel activity, we used a voltage protocol to obtain single-channel currents. From an initial holding potential of +60 mV, the bilayer potential was stepped to voltages ranging from -160 to +140 mV, in steps of +20 mV. After the first few milliseconds of the clamp, during which a capacitive transient current occurred, a single-channel current was activated following each voltage step. The current-voltage (I-V) relationships show the Cu2+-induced changes in the voltage dependence of the maximal current (I) (Fig. 4A), where the maximal conductance (maximal slope of I-V) was reduced nonsignificantly from 62 pS in control to 60 pS in 100 µM [Cu2+]cis. On the other hand, the mean current (I') was reduced significantly at positive voltages (Fig. 4B), being 4.5 ± 0.5 and 1.35 ± 0.2 pA in the absence and presence of 100 µM [Cu2+]cis, respectively. The current's Erev (approximately -22 mV; Fig. 4A) shifted nonsignificantly (~2.5 mV), indicating that there were no changes in the nature of the [Cu2+]cis-reduced current, which remained a K+ current. The effects of 100 µM [Cu2+]cis on I and I' were reversible (Fig. 4, A and B).



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Fig. 4. Effects of 100 µM [Cu2+]cis on A{beta}(1-42)-formed cation channel recorded from voltage-clamped optimal bilayers at voltages between -160 and +140 mV in KCl (250 mM cis/50 mM trans). Current-voltage relationships were constructed for I and I' in A and B, respectively: {circ}, control; {bullet}, 100 µM [Cu2+]cis; and {square}, wash with control cis solution.

 

Effects on A{beta}(1-42) channel burst kinetics. The changes in the kinetic parameters of the channel bursts were obtained at different [Cu2+]cis. Figure 5, A-D, shows the concentration dependence of the mean burst and intraburst durations and frequencies for a channel in a bilayer that is clamped to +140 mV. The intraburst duration and burst duration increase as [Cu2+]cis is incremented. The burst frequency and intraburst frequency decrease as a function of increasing [Cu2+]cis. The [Cu2+]cis-induced changes in these parameters were fitted with two or three exponentials.



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Fig. 5. [Cu2+]cis dependence of the kinetic parameters of the burst of A{beta}(1-42)-formed channel, in the burst mode, activated at different voltages in KCl (250 mM cis/50 mM trans). A: burst duration. B: intraburst duration. C: burst frequency. D: intraburst frequency. Solid lines are drawn to a third-order polynomial. The threshold for channel burst detection was set at 0.2 s before channel activation and 0.2 s after channel inactivation; i.e., a cluster of activity separated by at least 0.2-s closure durations on each side.

 

Effects on A{beta}(1-42) channel kinetics within the burst. The changes in the kinetic parameters of the channel within the burst were also calculated at different [Cu2+]cis. Figure 6, A-D, shows the concentration dependence of the Po, Fo, To, and closed time (Tc) for a channel in a bilayer that is clamped to +140 mV. The parameters Po, Fo, and To decreased while Tc increased as a function of increasing [Cu2+]cis. The values of Po, Fo, To, and Tc were 0.49, 106 s-1, 2.8 ms, and 3.01 ms for control and 0.06, 91 s-1, 0.81 ms, and 14.2 ms. The [Cu2+]cis-induced changes in these parameters were fitted with two or three exponentials. These findings are in agreement with those fits where drugs typically induce open channel block (18).



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Fig. 6. [Cu2+]cis dependence of the kinetic parameters of A{beta}(1-42)-formed channel, in the burst mode, activated at +140 mV in KCl (250 mM cis/50 mM trans). A: open probability (Po). B: frequency (Fo). C: mean open time (To). D: mean closed time (Tc). Solid lines are drawn to a third-order polynomial.

 

Effects on A{beta}(1-42) channel desensitization. Figure 7 shows the [Cu2+]cis dependence of the desensitization parameter (burst duration x Po) of an A{beta}(1-42)-formed channel, in the burst mode, activated at +140 mV in KCl (250 mM cis/50 mM trans). The [Cu2+]cis-induced changes in the A{beta}(1-42) channel desensitization are biphasic, an inverted bell-shaped curve with a minimum at 50 µM [Cu2+]cis and a bell-shaped curve with a peak at between 100 and 150 µM [Cu2+]cis. The third-order polynomial fit of the data shows a decline in the channel desensitization as a function of increasing [Cu2+]cis. The value decreases from 0.83 s-1 at 50 µM [Cu2+]cis to 0.17 s-1 at 200 µM [Cu2+]cis.



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Fig. 7. [Cu2+]cis dependence of the desensitization parameter (burst duration x Po) of A{beta}(1-42)-formed channel, in the burst mode, activated at +140 mV in KCl (250 mM cis/50 mM trans). Solid line is drawn to a third-order polynomial. The threshold for channel detection was set at 50% of the current amplitude.

 

Voltage dependence of effects of Cu2+ on A{beta}(1-42) channel bursts kinetics. The voltage dependence of the burst (Fig. 8A) and intraburst durations (Fig. 8B) could be described by an inverted bell-shaped third-order polynomial fit with a peak at approximately +80 mV. In 100 µM [Cu2+]cis, mean burst duration was reduced and mean intraburst duration increased at all voltages. The data show that [Cu2+]cis affected the burst (Fig. 8A) and intraburst durations (Fig. 8B) in a voltage-dependent manner. The mean values of the burst and intraburst durations in the control solution were 1.22 and 0.54 at +80 mV and 2.63 and 1.77 at +140 mV, respectively. In 100 µM [Cu2+]cis, these mean values for the burst and intraburst durations were 0.60 and 5.92 s at +80 mV and 0.16 and 6.79 s at +140 mV, respectively.



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Fig. 8. Effects of [Cu2+]cis on the voltage dependence of the burst kinetics of A{beta}(1-42)-formed channel, in the burst mode, activated at different voltages in KCl (250 mM cis/50 mM trans). A: burst duration. B: intraburst duration. C: burst frequency. D: intraburst frequency. Data are means of 1-3 channels. Solid lines are drawn to a third-order polynomial for control ({circ}) and 100 µM [Cu2+]cis ({bullet}).

 

The voltage dependence of the burst frequency (Fig. 8C) and intraburst frequency (Fig. 8D) could also be described by a bell-shaped third-order polynomial fit with a peak between +80 and 120 mV. In 100 µM [Cu2+]cis, burst frequency was reduced and intraburst frequency increased. The data show that [Cu2+]cis shifted the peak of the burst frequency (Fig. 8C) and the peak of the intraburst (Fig. 8D) shifted to more positive voltages.


    DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The main finding of this study is that Cu2+ modulates ion current activity of A{beta}(1-42) channel recorded in KCl solutions (250 mM cis/50 mM trans). Cu2+ inhibited the A{beta}(1-42)-formed channels in the absence and presence of CQ, which has been reported recently to have in vivo efficacy in reducing brain A{beta} accumulation in amyloid-bearing transgenic mice (14) and H2O2 generation by A{beta} in vitro (25, 44). It is assumed that CQ action is mediated by chelation of A{beta}-associated Cu2+ and Zn2+ (14). However, CQ did not reverse the Cu2+-induced inhibition of the A{beta}(1-42) channel formed in artificial membranes in the absence of Cu2+ and Zn2+. These facts indicate that the inhibitory effects of CQ on the A{beta}-induced toxicity in neuronal cultures as well as the reported beneficial effects of CQ (14, 25, 44) might be mediated via CQ action on other neuronal signaling mechanisms affected by A{beta}.

The effect of Cu2+ on the kinetics of A{beta}(1-42)-formed channels is observed within 1-10 s, and the Cu2+-induced changes in the kinetics of A{beta}(1-42)-formed channels become evident at concentrations as low as 50 µM (see, e.g., Figs. 2 and 6). The physiological concentration of Cu2+ in the synapse has been reported at 250 µM (27). However, the in vivo Cu2+ concentration threshold necessary to induce changes in the A{beta}-formed ion channels that may underlie neuronal dysfunction remains to be quantified.

A{beta}(1-42) is more lipophilic than A{beta}(1-40), and therefore it is expected that A{beta}(1-42) and its intermediate aggregates should be more stable in lipids (see Ref. 15). The findings reported in this study on the ability of A{beta}(1-42) to form channels together with those reported for A{beta}(1-40)-formed channels (35) suggest that the two additional residues in A{beta}(1-42) are not essential in channel formation. However, differences in the distribution of A{beta}(1-40) and A{beta}(1-42) channel types and in their stabilities in lipid membranes cannot be ruled out.

Kinetic of the burst and intraburst. At the single-channel level, the effects of Cu2+ are characterized by changes in the kinetics of the burst and the kinetics of the events within the burst (Figs. 1 and 2). In agreement with previous findings, the kinetic parameters Po, Fo, and To decrease while Tc increases as [Cu2+]cis increased (Figs. 5 and 6). The Cu2+-induced changes in the kinetics of the bursts and the events within the burst are described at least by two exponentials. This behavior appears to be in agreement with that of open channel blocking drugs, which exhibit a two-phase change in current kinetics (18), suggesting that Cu2+ may act on both open and closed states of the channel. Cu2+ also affects desensitization of the A{beta}(1-42) channels (Fig. 7). According to Auerbach and Akk (7) and Spitzmaul et al. (52), the value of ({tau}bPo)-1 (where {tau}b is the mean cluster duration and Po is the probability of being open within a cluster) is a direct measure of the rate constant of desensitization. Under control conditions, these values are as follows: Po = 0.49 and {tau}b = 4 s, so that ({tau}bPo)-1 = 1.96 s-1, whereas at 100 µM [Cu2+]cis, Po = 0.16 and {tau}b = 0.12 s, so that ({tau}bPo)-1 = 0.019 s-1. The duration of the burst and intraburst increases, whereas the number of bursts and intrabursts decreases at Cu2+ concentrations between 0 and 200 µM. At 250 µM [Cu2+]cis the channel is fully inhibited and no channel activity is observed. The lengthening of the burst and intraburst durations suggests that Cu2+ decreases both activation and inactivation rates of the channel. Between 50 and 200 µM [Cu2+]cis, Po declines rapidly (Fig. 6A) because of the decline in To and increase in Tc (Figs. 6C and 5D). The reversible effects of Cu2+ at low concentrations (50-100 µM) on the channel kinetics are consistent with its binding to a site at the mouth of the channel.

Binding site and "fast" open channel block. Consistent with the hydrophilic and structural properties of A{beta}(1-42), the Cu2+-induced changes in the kinetic parameters of this channel suggest that the Cu2+ binding site could be located at the mouth of the channel. This is consistent with findings of Curtain et al. (15, 16), which showed that Cu2+ bound to the histidine residues His6, His13, and His14 near the NH2 terminus of the peptide, and that although the metal ions induced a channel-like structure, the metal binding site resided above the plane of the lipid bilayer. The findings reported here show that transition metals modulate the activity of A{beta}(1-42)-formed channels. This channel modulation by transition metals could be common to other channel-forming amyloidogenic peptides. Indeed, {beta}2-microglobulin, another amyloid channel-forming protein, formed channels that are blocked by zinc (22). We also have reported that PrP(106-126) is sensitive to Cu2+ and that the metal binding site is localized at the NH2-terminal amino group, His111, and Met109 (26, 34). We also reported that millimolar concentrations of Zn2+ were needed to induce a reduction in the current amplitude of the "bursting" channel and the slow mode of the large-conductance A{beta} channel (35). Similarly, Lin et al. (38) showed that Zn2+ inhibited A{beta} channel activity. The findings in this study suggest that these A{beta} channels have higher affinity to Cu2+ than Zn2+, consistent with findings of Curtain et al. (16). Furthermore, Cu2+ induces a fast open channel block, whereas Zn2+ induces slow block of A{beta} channels, and the binding site for Zn2+ could be deep in the channel's conductive pathway (35).

One proposed molecular channel model suggests that the A{beta}(1-40)-formed channel is located asymmetrically within the membrane (19). This asymmetry is reflected at the putative entrances to the aqueous pore, which has three histidine residues (His6, His13, and His14) and several anionic residues including Asp7 and Glu11. It is thought that metalloproteases bind Zn2+ and Cu2+ to these sites (3, 19). This is consistent with the primary structure of A{beta}(1-42), which contains two separate local sequences containing histidine (13). The local sequence FRHDS contains His6, whereas the other local sequence, EVHHQ, contains His13 and His14. The A{beta}(1-40)-formed channel has rings of His6 and Asp7 surrounding one pore entrance. Successive rings of Glu11, His13, and His14 encircle the other entrance (3). There is evidence to show that His13 is crucial in the zinc ion-induced aggregation of A{beta} (39). It is interesting to note that His at position 13 is also involved in mediating the binding of Cu2+ and Zn2+ in nonnative states of {beta}2-microglobulin, which is deposited as amyloid plaques in the joint space that underlies the debilitating complications of long-term hemodialysis (20). Additionally, the oligomeric {beta}2-microglobulin channels are also blocked with Zn2+ (22).

We propose that in vivo membrane damage results from the interaction of age- and mutation-induced malfunctioned proteins with lipid membranes. A{beta}(1-42) is capable of inducing membrane damage, as deduced from formation of channels, in the absence of an increase in the background traces of Cu2+. The formation of a Cu2+-A{beta}(1-42) channel complex may act as an additional mechanism affecting Cu2+ homeostasis, which normally prevents the production of aberrant reactive oxygen species, and thus accentuating the neurotoxic effects of A{beta}(1-42).


    DISCLOSURES
 
This research work is supported by National Health and Medical Research Council Project Grants 970122 and 120808 and Australian Research Council Small Research Grants F99123 and F0047.


    ACKNOWLEDGMENTS
 
We thank Dr W. L. F. Armarego and R. McCart for suggestions and critical reading of the manuscript.


    FOOTNOTES
 

Address for reprint requests and other correspondence: J. I. Kourie, Membrane Transport Group, Dept. of Chemistry, The Faculties, Science Road Bldg. 33, The Australian National Univ., Canberra, Australian Capital Territory 0200, Australia (E-mail: joseph.kourie{at}anu.edu.au).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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