COMMUNICATION
Amyloid beta  Protein-(1-42) Forms Calcium-permeable, Zn2+-sensitive Channel*

Seung Keun RheeDagger §, Arjan Pieter QuistDagger , and Ratneshwar LalDagger

From the Dagger  Neuroscience Research Institute, University of California, Santa Barbara, California 93106 and the § Biochemistry Department, Yeungnam University, Kyongsan 712-749 Korea

    ABSTRACT
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Abstract
Introduction
Procedures
Results & Discussion
References

Amyloid beta  protein (Abeta P) forms senile plaques in the brain of the patients with Alzheimer's disease. The early-onset AD has been correlated with an increased level of 42-residue Abeta P (Abeta P1-42). However, very little is known about the role of Abeta P1-42 in such pathology. We have examined the activity of Abeta P1-42 reconstituted in phospholipid vesicles. Vesicles reconstituted with Abeta P show strong immunofluorescence labeling with an antibody raised against an extracellular domain of Abeta P suggesting the incorporation of Abeta P peptide in the vesicular membrane. Vesicles reconstituted with Abeta P showed a significant level of 45Ca2+ uptake. The 45Ca2+ uptake was inhibited by (i) a monoclonal antibody raised against the N-terminal region of Abeta P, (ii) Tris, and (iii) Zn2+. However, reducing agents Trolox and dithiothreitol did not inhibit the 45Ca2+ uptake, indicating that the oxidation of Abeta P or its surrounding lipid molecules is not directly involved in the Abeta P-mediated Ca2+ uptake. An atomic force microscope was used to image the structure and physical properties of these vesicles. Vesicles ranged from 0.5 to 1 µm in diameter. The stiffness of the Abeta P-containing vesicles was significantly higher in the presence of calcium. The stiffness change was prevented in the presence of zinc, Tris, and anti-Abeta P antibody but not in the presence of Trolox and dithiothreitol. Thus the stiffness change is consistent with the vesicular uptake of Ca2+. These findings provide biochemical and structural evidence that Abeta P1-42 forms calcium-permeable channels and thus may induce cellular toxicity by regulating the calcium homeostasis in Alzheimer's disease.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results & Discussion
References

A pathological hallmark in brain tissue from patients with Alzheimer's disease (AD)1 is the accumulation of amyloid beta  protein (Abeta P), a 39-43-amino acid-long polypeptide, as morphologically heterogeneous neuritic plaques and cerebrovascular deposits (1, 2). Abeta P is derived primarily from a proteolytic cleavage of the beta -amyloid precursor protein (Abeta PP), a highly conserved and widely expressed integral membrane protein with a single membrane-spanning polypeptide. The amount and the nature of polypeptides vary considerably among various forms of ADs: Abeta P1-40 and Abeta P1-42 are differentially accumulated in sporadic Alzheimer's disease and non-demented brain samples (3) and a mutation in presenilins is linked with an increased ratio of Abeta P1-42/Abeta P1-40 in familial Alzheimer's disease (4-7). The early-onset familial AD has been correlated with an increased level of Abeta P1-42. However, very little is known about the role of Abeta P1-42 in such pathology and about the mechanism(s) of its action.

Accumulating evidence suggests an early and causative role of Abeta Ps in the pathogenic cascade (8-11). Postulated mechanisms of Abeta P toxicity include, by its interaction with the tachykinin neuropeptide system, a surface membrane effect (12); by changing cellular ionic concentration via formation of plasma membrane channels (13-15); and by activating oxidative pathways and making cells more responsive to oxidative stress (for review see Refs. 16 and 17). Reactive oxygen species and the antioxidant defenses work probably by altering the lipid peroxidation and membrane composition. However, Abeta P polypeptides associated with the reactive oxygen hypothesis have produced conflicting effects on cytoskeletal organization and cell lysis (18-23).

The commonly observed change in the cellular ion concentration involves increased calcium level (24-26) either indirectly via modulating the existing Ca2+ channel or directly via cation-selective channels formed by Abeta Ps. Support for the cation-selective Abeta P channels are accumulating. Arispe and his collaborators (13-15, 27) have reported cation-selective channels formed by Abeta P1-40 when reconstituted into lipid bilayers and in the membrane patches excised from hypothalamic gonadotropin-releasing hormone neurons. Kagan and his collaborators (28) have also recorded channel-like activity when Abeta P25-35 was reconstituted in lipid bilayers as well as for both Abeta P1-40 and Abeta P1-42 reconstituted in lipid bilayer,2 though, with less reliability and reproducibility than the Abeta P25-35 current (28). Whether Abeta P1-42 toxicity is also mediated via Abeta P1-42 forming calcium-permeable ion channel is unclear.

The molecular structure of Abeta P oligomers, especially as an ion channel, is unknown. Durell et al. (29) have developed theoretical models for the structure of ion channel formed by the membrane-bound Abeta P1-40. However, no direct structural data from EM, NMR, x-ray diffraction, or other microscopic techniques are available to support the presence of the Abeta P channel.

We have used an atomic force microscope (AFM) (30) integrated with a light and fluorescence microscope (31) to examine the mechanism(s) of Abeta P1-42 toxicity. Abeta P1-42 was reconstituted in phospholipid vesicles and were imaged in buffer to reveal the Abeta P-membrane complex and a channel-like structure. Consistent with the possibility of fresh Abeta Ps forming ion-permeable channels (i) fluorescently labeled anti-Abeta P antibodies were localized in Abeta P-reconstituted vesicles, (ii) vesicles reconstituted with Abeta P show a significant level of 45Ca2+ uptake which was blocked by anti-Abeta P-antibody, Zn2+, and tromethamine, but not blocked by antioxidants, and (iii) Abeta P-reconstituted vesicles have considerably higher stiffness in the presence of calcium.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results & Discussion
References

Reconstitution of Abeta P1-42 into Liposomes-- Human Abeta P1-42 and phospholipids were purchased from Bachem (Torrance, CA) and Avanti Polar (Birmingham, AL), respectively. Liposomes were prepared from both palmitoyloleoylphosphatidylethanolamine and palmitoyloleoylphosphatidylserine. 10 µl of phospholipids (10 mg/ml palmitoyloleoylphosphatidylethanolamine: palmitoyloleoylphosphatidylserine::1:1) in chloroform was dried under argon gas, and then 2 ml of buffer (10 mM HEPES, pH 7.4) was added. The mixture was then bath sonicated for 20 min. For the incorporation of Abeta P1-42 into liposomes, the phospholipids were first dried under argon gas, then 1.5 ml of buffer was added, followed by 20 min of bath sonication. The Abeta P stock solution (5 mg/ml) was added, and the mixture was sonicated for another 20 min. The final concentration of phospholipids and Abeta P is 1 mg/ml.

Immunolabeling with Anti-Abeta P Antibody-- A mouse monoclonal antibody raised against the N' terminus of Abeta P (named 3D6 antibody [anti-Abeta P 1-5 (DAEFR)]) was obtained from Dr. Russell Rydel at Athena Neurosciences (San Francisco, CA). Goat anti-mouse IgG conjugated with Cy3 (1 mg/ml) was purchased from Chemicon International (Temecula, CA). Liposomes were adsorbed on glass coverslips and fixed with 4% paraformaldehyde for 10 min, then washed with phosphate-buffered saline (PBS), and then blocked with PBS containing 3% BSA and 1% goat serum to minimize nonspecific binding. Primary antibody (diluted 500-fold) was added in the presence of 3% BSA and 1% goat serum and incubated for 1 h at room temperature. After washing with PBS, the sample was incubated with secondary antibody (500-fold dilution) at the same condition as primary incubation. Fluorescent images were obtained using 40× high numerical aperture objective lens with an inverted Olympus inverted fluorescence microscope.

Measurement of 45Ca2+ Uptake into Liposomes-- 45Ca2+ uptake was measured by the modified method of Nakade et al. (32). 25 µl of liposomes reconstituted with/without Abeta P1-42 was incubated with 75 µl of HEPES buffer (10 mM, pH 7.4) containing 2 µCi of 45Ca2+. Calcium influx was measured separately for each perturbation: anti-Abeta P-antibody, Zn2+, Tris, Trolox, and DTT, respectively. After incubation for 1 min at 30 °C, in the presence of a perturbation, the calcium influx reaction was stopped with a blocking solution containing 300 µl HEPES buffer, 0.3 mM CaCl2, 5 mM ZnCl2, 10 mM Tris, and 15 µg/ml 3D6 antibody. The 400-µl mixture of liposomes and the blocking solution was immediately loaded onto a Chelex 100 column (bead volume: 3 ml) (Bio-Rad) which was pre-equilibrated with a buffer containing 200 mM sucrose, 20 mM Tris-HCl (pH 7.4 at room temperature), and 0.3% BSA. The column was then washed immediately with 5 ml of a buffer containing 200 mM sucrose and 20 mM Tris-HCl (pH 7.4) to take the liposomes. The 45Ca2+ content of the liposomes was measured with a Beckman liquid scintillation counter.

Imaging Liposomes with Atomic Force Microscopy-- AFM images were obtained as described (33),3 using a prototype of Bioscope AFM and a Multimode AFM (Digital Instruments, Santa Barbara, CA). Contact mode AFM was used for most of the images. Oxide-sharpened silicon nitride tips with a nominal spring constant of about 0.06 newton/m (Digital Instruments) were used for most experiments. All imaging was conducted on wet and hydrated liposomes. For liposomes reconstituted with/without Abeta P1-42, 20-50 µl of sample was deposited on a clean glass Petri dish and left for 30 min. The surface of the Petri dish was then rinsed with a buffer (10 mM HEPES, pH 7.4) and imaged in the buffer. The imaging force was regularly monitored and kept to a minimum. The imaging force varied from subnanonewton to tens of nanonewtons. All imaging was performed at room temperature (22-24 °C).

Measuring Viscoelastic Properties-- We measured stiffness of Abeta P vesicles by the AFM force-mapping technique as described (34) using the Nanoscope III software (Version 4.23R2; Digital Instruments, Santa Barbara). Force maps were taken by raster scanning the tip over the sample with 64 × 64 measuring points with a pixel resolution of 95 nm. Each force map thus consists of a topographical image of 64 × 64 pixels, and a force curve is stored for every pixel. Force maps were obtained alternately with the regular height and error modes of imaging the surface topography, using the same cantilever.

Vesicle stiffness was calculated from the force-volume imaging data. Force maps were exported to and analyzed with IGOR Pro software (Wavemetrics, Lake Oswego, OR) and macros written by Dan Laney (34). To calculate the elastic modulus of vesicles from the force curves, the Hertz model was used and the vesicle-tip system was equated to a sphere-sphere interaction (35, 36). First, the indentation of the tip into the sample was determined by comparing force curves on the vesicles with those on the hard glass substrate. Then, the elastic modulus was extracted from the portion of the force curve where the indentation was still very small (~10 nm).

    RESULTS AND DISCUSSION
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Abstract
Introduction
Procedures
Results & Discussion
References

Immunolocalization of Abeta P1-42 on the Reconstituted Liposomes-- Liposomes reconstituted with fresh Abeta P1-42 show strong immunolabeling with an anti-Abeta P antibody. Fig. 1D shows a fluorescence labeling image of liposomes reconstituted with Abeta P1-42. For comparison, no immunofluorescence labeling was observed in the liposomes prepared without Abeta P1-42 (Fig. 1B). Also, there was no immunofluorescence labeling observed in vesicles reconstituted with aged Abeta P1-42 (Abeta P stored for 24 h or longer). All immunolabeled liposomes have a well defined vesicular structure as revealed by AFM imaging (Fig. 1, A and C). Vesicles with strong immunofluorescence signals appear to have double-layered (two membranes) disc-shaped structures in AFM images. Vesicle size ranged from 0.5 to 1 µm. Vesicles without Abeta Ps are on average half in diameter but more spherical compared with the vesicles with Abeta Ps. The average height of vesicles without Abeta P is 40 nm and 31 nm for vesicles with Abeta P. The vesicular flattening and larger size are probably due to the protein-lipid interactions (37, 38) as is the case with other membrane proteins such as gap junctions and acetylcholine receptors.


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Fig. 1.   Panels A and B, AFM and fluorescence images, respectively, of liposomes prepared in the absence of Abeta P1-42. Panels C and D, AFM and fluorescence images, respectively, of liposomes prepared in the presence of Abeta P1-42. The inserted frames in A and C are the higher resolution images of liposomes.

Since Abeta PP is a membrane protein, it was believed that the membrane-bound Abeta P portion would not be found as a free peptide except in case of membrane injury leading to a proteolytic cleavage of Abeta PP and also that the hydrophobic and self-aggregating nature of Abeta P would prohibit it from existing as a soluble, circulating peptide in normal biological fluids. Subsequent studies, however, revealed secreted soluble Abeta P in the conditioned media of a variety of primary or transfected Abeta PP-expressing cells under normal metabolic conditions (39-41). Our result is consistent with the presence of soluble Abeta Ps that could interact with lipid membrane and form channels.

Abeta P Channel-specific Calcium Uptake-- Liposomes reconstituted with Abeta P1-42 show a significantly larger (>4-fold) increase in the influx of 45Ca2+ compared with the liposomes without Abeta P1-42 (compare A and B in the top panel of Fig. 2). In the presence of an antibody raised against the amino-terminal domain of Abeta P, there was no significant influx of 45Ca2+ in the liposomes reconstituted with Abeta P (compare A, B, and C in the top panel of Fig. 2). Such inhibition of calcium uptake by the anti-Abeta P antibody shows the specificity of Abeta P1-42-induced calcium uptake. The level of inhibition by anti-Abeta P antibody would have been greater if all epitopes of the reconstituted Abeta Ps were oriented outside of the liposomes.


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Fig. 2.   Top panel, influx of 45Ca2+ into liposomes. A, influx of 45Ca2+ into liposomes containing no Abeta P1-42. B, influx of 45Ca2+ into liposomes containing Abeta P at a concentration of 1 mg/ml. C, influx of 45Ca2+ into liposomes containing Abeta P1-42 in the presence of antibody raised against the amino-terminal domain of Abeta P. Bottom panel, influx of 45Ca2+ into liposomes containing Abeta P with various perturbations: control (A), 5 mM Zn2+ (B), 10 mM Tris (C), 100 mM Trolox (D), and 2 mM dithiothreitol (E).

We examined the mechanisms of Abeta P1-42-specific calcium uptake. Recent studies suggest that Abeta Ps form cation-selective ion channels which can be inhibited by zinc, Tris, and other related compounds. Consistent with such a possibility, calcium uptake was prevented when the liposomes reconstituted with Abeta P1-42 were incubated with Tris or zinc (compare A, B, and C, in the lower panel of Fig. 2). Whether this is also true at the cellular level needs to be examined. Abeta P1-40-specific cationic channels are reported in AD-free fibroblasts and neuronal patches(24-27, 47).3

Abeta P is reported to bind specifically and saturably with zinc in a biphase mode: at high affinity (KD = 107 nM) or at low affinity (KD = 5.2 µM) (42). The zinc-binding site was mapped to a stretch of contiguous residues between positions 6 and 28 of the Abeta P sequence. Zinc, at more than 1 µM concentration in the Abeta P-solubilized solution, is thought to facilitate the precipitation of Abeta P (43, 44). This property is important because zinc is abundant in the same neocortical regions where Abeta P deposits are most commonly found, and high micromolar concentration of zinc is achieved during glutamatergic neurotransmission (45, 46), providing one possible explanation for the propensity of Abeta P to deposit close to the neocortical synaptic vicinity.

The influx of 45Ca2+ into liposomes containing Abeta P1-42 was not prevented by anti-oxidants Trolox (100 mM) and dithiothreitol (2 mM) (compare A, D, and E in the lower panel of Fig. 2). Rather, these anti-oxidants often appear to increase the calcium uptake. Anti-oxidants have been proposed to interact with the membrane lipid components and change the membrane permeability (for review see Refs. 16-18). However, in our study, there was no significant change in calcium uptake in the liposomes prepared without Abeta Ps. Our results thus strongly suggest that the anti-oxidants have little to no effect on Abeta P1-42 channel-mediated ionic exchange in vitro. Whether this is also true at the cellular level needs to be determined.

Calcium Uptake Induced Change in Vesicular Elasticity-- In cells, calcium uptake would lead to altered metabolic load and an imbalance in the ionic homeostasis. In reconstituted vesicles, calcium uptake should change the physicochemical properties, especially vesicle stiffness and charge-charge interactions. We examined the change in vesicle stiffness using the force-mapping feature of the AFM imaging on vesicles reconstituted with/without Abeta P1-42 (Fig. 3) with a well defined morphology as revealed in AFM images. The shape and size of the vesicles appear to vary, but the apparent height of the unilammellar vesicles was similar.


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Fig. 3.   Height images obtained during force-mapping of liposomes in non-calcium medium before adding any perturbation. For liposomes shown in panels A and B, no perturbation was used. Liposomes shown in panel B contained no Abeta P. Liposomes shown in panels C, D, E, F, and G were incubated with anti-Abeta P antibodies, zinc, Tris, Trolox, and DTT, respectively, before adding calcium. Height images after adding perturbations and calcium have not changed from these pre-perturbation images.

Change in stiffness was examined for each treatment, e.g. anti-Abeta P antibody, Zn2+, Tris, Trolox, and DTT. Stiffness was measured on the same vesicles before and after the application of a perturbation, thus each vesicle serving as its own control. Stiffness change was normalized with respect to the average stiffness in the calcium-free medium. In parallel with the change in the calcium uptake, stiffness of the vesicles reconstituted with Abeta P1-42 increased significantly compared with that for the vesicles without Abeta Ps (compare A, B, and C in the top panel of Fig. 4). This stiffness change was inhibited by the anti-Abeta P antibody suggesting the presence of Abeta P. The calcium uptake-induced increased stiffness was inhibited by the presence of zinc or Tris (compare A, B, and C in the bottom panel in Fig. 4). On the other hand, anti-oxidants, such as Trolox and DTT, had diverse effects on the calcium uptake-induced stiffness increase. DTT inhibited the stiffness change whereas Trolox enhanced the stiffness change. The inhibitory effect of DTT, however, is weaker than the inhibitory effect of zinc, Tris, and antibody. Such a complex effect of these antioxidants and the channel blockers may be reflected in the modes of action of these perturbations.


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Fig. 4.   Normalized change in elastic modulus of the vesicles after adding perturbation and calcium, measured using AFM force-mapping. Top panel: A, liposomes containing no Abeta P and with no perturbation; B, liposomes containing Abeta P1-42 and with no perturbation; C, liposomes containing Abeta P1-42 and with anti-Abeta P antibodies. Bottom panel: A, liposomes containing Abeta P and without perturbation (control); B, liposomes containing Abeta P1-42 and incubated with zinc; C, liposomes containing Abeta P1-42 and incubated with Tris; D, liposomes containing Abeta P1-42 and incubated with Trolox; and E, liposomes containing Abeta P1-42 and incubated with DTT.

The change in stiffness is expected to result from (i) calcium ion-induced charge-charge repulsion energy inside liposomes, (ii) the binding of calcium to the lipids and proteins, and also (iii) the increased efficacy of lipid-protein interactions. However, the binding between calcium and the lipids or peptides on the external surface of the liposomes is unlikely the main driving force to increase in stiffness of liposomes because the same divalent cation zinc blocked the increase in stiffness of liposomes (Fig. 4, lower panel). It is possible that antioxidants do not prevent calcium binding to lipids on the internal face of the vesicle whereas zinc, Tris, and antibody (perhaps by steric effect) all prevent the binding of calcium to the lipids and proteins on the external face of the vesicles. Further study is required to examine this issue. In summary, our study strongly suggests that Abeta P1-42 forms calcium-permeable, Zn2+-sensitive channels in vitro and allows calcium transport. Such calcium exchange could destabilize cellular calcium homeostasis and lead to the cell toxicity.

    ACKNOWLEDGEMENTS

We thank Drs. Bruce Kagan and Dennis Clegg for useful advice and critical evaluation of the manuscript, Dr. Russell Rydel of Athena Neurosciences for kindly providing us the antibodies used in our study, and Dr. Paul Hansma for encouragement with the AFM study of Alzheimer's disease.

    FOOTNOTES

* This work was supported by grants from the State of California, Department of Health Services, Alzheimer's Disease Program, 95-23336, and Yeungnam University in Korea. Portions of this work have been published as an abstract from the Biophysical Society annual meeting, 1998.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.

To whom correspondence should be addressed. Tel.: 805-893-2350; Fax : 805-893-2005; E-mail: rlal{at}physics.ucsb.edu.

1 The abbreviations used are: AD, Alzheimer's disease; Abeta P, amyloid beta  protein; APP, amyloid precursor protein; AFM, atomic force microscope; PBS, phosphate-buffered saline; BSA, bovine serum albumin; DTT, dithiothreitol.

2 T. Mirzabekov, M. C. Lin, W. L. Yuan, P. J. Marshall, M. Carman, K. Tomaselli, I. Lieverburg, and B. L. Kagan, personal communication.

3 Y. J. Zhu, Y. Zhang, and R. Lal, submitted for publication.

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Abstract
Introduction
Procedures
Results & Discussion
References

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