Alzheimer's beta-amyloid, human islet amylin, and prion protein fragment evoke intracellular free calcium elevations by a common mechanism in a hypothalamic GnRH neuronal cell line

A growing number of reports suggest that elevated levels of extracellular Alzheimer's beta-amyloid protein alter the homeostasis of free [Ca(2+)](i) in different cell types of the mammalian brain. In line with these results, we have previously shown that AbetaP[1-40] forms cation-selective channels (Ca(2+) included) across artificial planar bilayers formed from acidic phospholipids and across excised membrane patches from immortalized hypothalamic GnRH neurons (GT1-7 cells), suggesting that the nonregulated Ca(2+)-influx through these spontaneously formed "amyloid channels" may provide a mechanism to explain its toxicity (1). We have now found and report here that the application of AbetaP[1-40] to GT1-7 neurons consistently elevates [Ca(2+)](i) levels. We also found that human islet amylin and the prion protein fragment (PrP106-126), peptides that acquire beta-pleated sheet conformation in water solutions and have been reported to form ion channels across planar bilayer membranes, also increase cytosolic free calcium in GT1-7 neurons. Searching for protective agents, we found that soluble cholesterol, known to decrease the fluidity of the cell membrane, inhibits AbetaP[1-40]-evoked [Ca(2+)](i) rise. These results suggest that unregulated Ca(2+) entry across amyloid channels may be a common mechanism causing cell death, not only in diseases of the third age, including Alzheimer's disease and type 2 diabetes mellitus, but also in prion-induced diseases.     

Displacement currents associated with the insertion of Alzheimer disease amyloid beta-peptide into planar bilayer membranes

The role of endogenous amyloid beta-peptides as causal factors of neurodegenerative diseases is largely unknown. We have previously reported that interactions between Alzheimer's disease A beta P[1-40] peptide in solution and planar bilayer membranes made from anionic phospholipids lead to the formation of cation-selective channels. We now find and report here that the spontaneous insertion of free A beta P[1-40] across the bilayer can be detected as an increase in bilayer capacity. To this end we recorded the displacement currents across planar bilayers (50 mM KCl on both sides) in response to sudden displacements of the membrane potential, from -300 to 300 mV in 20-mV increments. To monitor the A beta P[1-40]-specific displacement currents, we added A beta P[1-40] (1-5 microM) to the solution on either side of the membrane and noted that the direction of the displacement current depended on the side with A beta P[1-40]. The size of the A beta P[1-40]-specific charge displaced during a pulse was always equal to the charge returning to the original configuration after the pulse, suggesting that the dipole molecules are confined to the membrane. As a rule, the steady-state distribution of the A beta P[1-40]-specific charges within the bilayer could be fit by a Boltzmann distribution. The potential at which the charges were found to be equally distributed (V(o)) were approximately -135 mV (peptide added to the solution in the compartment electrically connected to earth) and 135 mV (peptide added to the solution connected to the input of the amplifier). The A beta P[1-40]-specific transfer of charge reached a maximum value (Q(max)) when the electrical potential of the side containing the amyloid beta-protein was taken to either -300 or 300 mV. For a circular membrane of 25-microm radius ( approximately 2000 microm(2)), the total A beta P[1-40]-specific charge Q(max) was estimated as 55 fC, corresponding to some 170 e.c./microm(2). Regardless of the side selected for the addition of A beta P[1-40], at V(o) the charge displaced underwent an e-fold change for a approximately 27-mV change in potential. The effective valence (a) of the A beta P[1-40] dipole (i.e., the actual valence Z multiplied by the fraction of the electric field chi acting on the dipole) varied from 1 to 2 electronic charges. We also tested, with negative results, the amyloid peptide with the reverse sequence (A beta P[40-1]). These data demonstrate that A beta P[1-40] molecules can span the low dielectric domain of the bilayer, exposing charged residues (D(1), E(3), R(5), H(6), D(7), E(11), H(13), and H(14)) to the electric field. Thus the A beta P[1-40] molecules in solution must spontaneously acquire suitable conformations (beta-pleated sheet) allowing specific interactions with charged phospholipids. Interestingly, the domain from residues 676 to 704 in the APP(751) is homologous with the consensus sequence for lipid binding found in other membrane proteins regulated by anionic phospholipids.     

Synthetic mammalian C-type natriuretic peptide forms large cation channels

We report the first evidence that synthetic human C-type natriuretic peptide-22 and the OaC-type natriuretic peptide-39(18-39), a 22 amino acid fragment of the OaC-type natriuretic peptide-39 from platypus venom, can function directly by forming a novel voltage-gated weakly cation-selective channel in negatively charged artificial lipid bilayer membranes. The channel activity is characterized by a tendency for inactivation at negative voltages, e.g. -60 and -70 mV, whereas at positive voltages the channel is fully open. The channel has a maximal cord conductance of 546+/-23 pS (n = 16) and shows weak outward rectification. The sequence and the permeability ratios were P(K)+: P(Cs)+: P(Na)+: P(choline)+ 1:0.88:0.76:0.13, respectively. The addition of 50 mM TEA+ cis (a blocker of outwardly rectifying K+ channels), 20 mM Cs+ cis (a blocker of inwardly rectifying K+ channels) or 0.5 mM glibenclamide cis (a blocker of ATP-sensitive K+ channels) to the cis chamber did not affect the conductance or the kinetics of the OaC-type natriuretic peptide-39(18-39)-formed channels (n = 2-5). It is concluded that the weak cation selectivity, large conductance and high open probability as well as their voltage dependency are consistent with the ability of these peptides to cause that loss of compartmentation of the membrane, which is a characteristic feature of adverse conditions that cause C-type natriuretic peptide-related pathologies.     

β-amyloid Ca2+-channel hypothesis for neuronal death in Alzheimer Disease

The Alzheimer's Disease (AD) amyloid protein (AßP[1-40]) forms cation selective channels when incorporated into planar lipid bilayers by fusion with liposomes containing the peptide. Since the peptide has been proposed to occurin vivo in both membrane-bound and soluble forms, we also tested the possibility of direct incorporation of the soluble AßP[1-40] into the membrane. We found the peptide can also form similar channels in acidic phospholipid bilayers formed at the tip of a patch pipet, as well as in the planar lipid bilayer system. As in the case of liposome mediated incorporation, the AßP[1-40]-channel in the solvent-free membrane patch exhibits multiple cation selectivity (Cs⁺>Li⁺>Ca²⁺K⁺) and sensitivity to tromethamine. The fact that equivalentAßP[1-40] amyloid channels can be detected by two different methods thus provides additional validation of our original observation. Further studies with aßP-channels incorporated into planar lipid bilayers from the liposome complex have also revealed that the channel activity can express spontaneous transitions to a much higher range of conductances between 400 and 4000 pS. Under these conditions, the amyloid channel continues to be cation selective but loses its tromethamine sensitivity. By contrast, amyloid channels were insensitive to nitrendipine at either conductance range. We calculate that if such channels were expressed in cells, the ensuing ion fluxes down their electrochemical potential gradients would disrupt cellular homeostasis. We therefore interpret these data as providing further support for our ß-amyloid Ca²⁺-channel hypothesis for neuronal death in Alzheimer's Disease.     

A comparison of calcium-activated potassium channel currents in cell-attached and excised patches

Single channel currents from Ca-activated K channels were recorded from cell-attached patches, which were then excised from 1321N1 human astrocytoma cells. Cells were depolarized with K (110 mM) so that the membrane potential was known in both patch configurations, and the Ca ionophore A23187 or ionomycin (20-100 microM) was used to equilibrate intracellular and extracellular [Ca] (0.3 or 1 microM). Measurements of intracellular [Ca] with the fluorescent Ca indicator quin2 verified that [Ca] equilibration apparently occurred in our experiments. Under these conditions, where both membrane potential and intracellular [Ca] were known, we found that the dependence of the channel percent open time on membrane potential and [Ca] was similar in both the cell-attached and excised patch configuration for several minutes after excision. Current-voltage relations were also similar, and autocorrelation functions constructed from the single channel currents revealed no obvious change in channel gating upon patch excision. These findings suggest that the results of studies that use excised membrane patches can be extrapolated to the K-depolarized cell-attached configuration, and that the relation between [Ca] and channel activity can be used to obtain a quantitative measure of [Ca] near the membrane intracellular surface.     

Effect of high hydrostatic pressure on the BK channel in bovine chromaffin cells.

The activity of the BK channel of bovine chromaffin cells was studied at high hydrostatic pressure, using inside-out patches in symmetrical KCl solution, Ca2+-free and at V(H) = -60 to -40 mV. Pressure increased the probability of channels being open (900 atm increasing the probability 30-fold), and it increased the minimum number of channels apparent in the patches. The pressure activation of the channel was reversed on decompression. Channel conductance was unaffected. It was shown that pressure did not act by raising the temperature, or by affecting [Ca] or pH, or the order of the membrane bilayer, and it was concluded that pressure most likely acted directly on the channel proteins and/or their modulating reactions.     

Mass spectrometry of purified amyloid beta protein in Alzheimer's disease.

The amyloid beta-protein (A beta) that is progressively deposited in Alzheimer's disease (AD) arises from proteolysis of the integral membrane protein, beta-amyloid precursor protein (beta APP). Although A beta formation appears to play a seminal role in AD, only a few studies have examined the chemical structure of A beta purified from brain, and there are discrepancies among the findings. We describe a new method for the rapid extraction and purification of A beta that minimizes artifactual proteolysis. A beta purified by two-dimensional reverse-phase HPLC was analyzed by combined amino acid sequencing and mass spectrometry after digestion with a lysylendopeptidase. The major A beta peptide in the cerebral cortex of all five AD brains examined was aspartic acid 1 to valine 40. A minor species beginning at glutamic acid 3 but blocked by conversion to pyroglutamate was also found in all cases. A species ending at threonine 43 was detected, varying from approximately 5 to 25% of total A beta COOH-terminal fragments. Peptides ending with valine 39, isoleucine 41, or alanine 42 were not detected, except for one brain with a minor peptide ending at valine 39. Our findings suggest that A beta 1-40 is the major species of beta-protein in AD cerebral cortex. A beta 1-40 and A beta 1-43 peptides could arise independently from beta APP, or A beta 1-43 could be the initial excised fragment, followed by digestion to yield A beta 1-40. These analyses of native A beta in AD brain recommend the use of synthetic A beta 1-40 peptide to model amyloid fibrillogenesis and toxicity in vitro.     

A9 Fibroblasts Transfected with the m3 Muscarinic Receptor Clone Express a Ca2+ Channel Activated by Carbachol,GTP and GDP

Muscarinic m3 receptor-mediated changes in cytosolic Ca²⁺ concentration ([Ca²⁺]_l) occur by activation of Ca²⁺ release channels present in the endoplasmic reticulum membrane and Ca²⁺ entry pathways across the plasma membrane. In this report we demonstrate the coupling of m3 muscarinic receptors to the activation of a voltage-insensitive, cation-selective channel of low conductance (3.2 ± 0.6 pS; 25 mm Ca²⁺ as charge carrier) in a fibroblast cell line expressing m3 muscarinic receptor clone (A9m3 cells). Carbachol (CCh)-induced activation of the cation-selective channel occurred both in whole cell and excised membrane patches (CCh on the external side), suggesting that the underlying mechanism involves receptor-channel coupling independent of intracellular messengers. In excised inside-out membrane patches from nonstimulated A9m3 cells GTP (10 μm) and GDP (10 μm) activated cation-selective channels with conductances of approximately 4.3 and 3.3 pS, (25 mm Ca²⁺ as charge carrier) respectively. In contrast, ATP (10 μm), UTP (10 μm) or CTP (10 μm) failed to activate the channel. Taken together, these results suggest that carbachol and guanine nucleotides regulate the activation of a cation channel that conducts calcium. Received: 14 November 1996/Revised: 4 April 1997     

Theoretical models of the ion channel structure of amyloid beta-protein.

Theoretical methods are used to develop models for the ion channel structure of the membrane-bound amyloid beta-protein. This follows recent observations that the beta-protein forms cation-selective channels in lipid bilayers in vitro. Amyloid beta-protein is the main component of the extracellular plaques in the brain that are characteristic of Alzheimer's disease. Based on the amino acid sequence and the unique environment of the membrane, the secondary structure of the 40-residue beta-protein is predicted to form a beta-hairpin followed by a helix-turn-helix motif. The channel structures were-designed as aggregates of peptide subunits in identical conformations. Three types of models were developed that are distinguished by whether the pore is formed by the beta-hairpins, the middle helices, or by the more hydrophobic C-terminal helices. The latter two types can be converted back and forth by a simple conformational change, which would explain the variable conduction states observed for a single channel. It is also demonstrated how lipid headgroups could be incorporated into the pore lining, and thus affect the ion selectivity. The atomic-scale detail of the models make them useful for designing experiments to determine the real structure of the channel, and thus further the understanding of peptide channels in general. In addition, if beta-protein-induced channel activity is found to be the cause of cell death in Alzheimer's disease, then the models may be helpful in designing counteracting drugs.     

Poly-L-glutamine forms cation channels: relevance to the pathogenesis of the polyglutamine diseases

We report that long-chain poly-L-glutamine forms cation-selective channels when incorporated into artificial planar lipid bilayer membranes. The channel was permeable to alkali cations and H(+) ions and virtually impermeable to anions; the selectivity sequence based on the single-channel conductance was H(+) > Cs(+) > K(+) > Na(+). The cation channel was characterized by long-lived open states (often lasting for several minutes to tens of minutes) interrupted by brief closings. The appearance of the channel depended critically on the length of polyglutamine chains; ion channels were observed with 40-residue stretches, whereas no significant conductance changes were detected with 29-residue tracts. The channel-forming threshold length of poly-L-glutamine was thus between 29 and 40 residues. A molecular mechanics calculation suggests a mu-helix (. Biophys. J. 69:1130-1141) as a candidate molecular structure of the channel. The channel-forming nature of long-chain poly-L-glutamine may provide a clue to the elucidation of the pathogenetic mechanism of the polyglutamine diseases, a group of inherited neurodegenerative disorders including Huntington's disease.     

A Ca2-activated cation-selective channel in the basolateral membrane of the cortical thick ascending limb of Henle's loop of the mouse

The patch-clamp technique was used to investigate the properties of a cation-selective channel in the basolateral membrane of microdissected collagenase-treated fragments of cortical thick ascending limbs of Henle's loop from mouse kidney. The channel activity was seldom observed in cell-attached patches (2 out 15 studied cases). In inside-out excised patches immersed in symmetrical NaCl Ringer's solutions, the unit channel conductance was ohmic and ranged from 22 to 33 pS (mean, 26.8 +/- 0.6 pS, n = 24). When NaCl was replaced by KCl (n = 8) or sodium gluconate (n = 2) on the cytoplasmic side of the membrane, single-channel currents still reversed at 0 mV and the conductance was unchanged. The reversal potential was +28.8 +/- 0.4 mV (n = 8) when a NaCl concentration (140 vs. 42 mmol/l) gradient was applied, close to the expected value (approx. 30 mV) for a cation selective channel. The channel was found to discriminate poorly between Na+, K+, Cs+, and Li+ ions. The activity of the channel was not clearly voltage-dependent but was dependent upon the free Ca2+ concentration on the cytoplasmic side of the membrane. We conclude that the channel resembles the non-selective cation channel which has been previously described in several tissues.     

Ion selectivity of the nerve membrane sodium channel incorporated into liposomes

Tetrodotoxin-sensitive sodium channels of lobster nerve membranes were incorporated into soybean liposomes by the freeze-thaw-sonication procedure and their ionic selectivity was studied. Veratridine and grayanotoxin-I were used to activate the sodium channels and the increment of the ionic flux through them was specifically abolished by tetrodotoxin. The drug-sensitive 22Na+, 42K+, 86Rb+ and 137Cs+ influxes were measured. The permeability ratios calculated directly from ion fluxes showed that the channels preferably allow the passage of Na+. No anion influx ([32P]phosphate, [35S]sulfate, 36Cl) sensitive to the drugs was observed. The data reveal that the sodium channels incorporated into liposomes remain cation-selective and discriminate among different cations.     

Ion channel formation by Alzheimer's disease amyloid beta-peptide (Abeta40) in unilamellar liposomes is determined by anionic phospholipids

Incorporation of Alzheimer's disease amyloid beta-proteins (AbetaPs) across natural and artificial bilayer membranes leads to the formation of cation-selective channels. To study the peptide-membrane interactions involved in channel formation, we used cation reporter dyes to measure AbetaP-induced influx of Na+, Ca2+, and K+ into liposomes formed from phosphatidylserine (PS), phosphatidylinositol (PI) and phosphatidylcholine (PC). We found that Abeta40, but not Abeta40-1 or Abeta28, caused a dose-dependent increase in the concentration of each cation in the lumen of liposomes formed from the acidic phospholipids PS and PI. The Abeta40-induced changes in cation concentration, which we attribute to ion entry through Abeta40 channels, were not observed when using liposomes formed from the neutral phospholipid PC. Using mixtures of phospholipids, the magnitude of the AbetaP40-induced ion entry increased with the acidic phospholipid content of the liposomes, with entry being observed with as little as 5% PS or PI. Thus, while negatively charged phospholipids are required for formation of cation-permeable channels by Abeta40, a small amount is sufficient to support the process. These results have implications for the mechanisms of AbetaP cytotoxicity, suggesting that even a small amount of externalized negative charge could render cells susceptible to the deleterious effects of unregulated ion influx through AbetaP channels.     

Cholesterol and Clioquinol modulation of A beta(1-42) interaction with phospholipid bilayers and metals

The beta-sheet plaques that are the most obvious pathological feature of Alzheimer's disease are composed of amyloid-beta peptides and are highly enriched in the metal ions Zn, Fe and Cu. The interaction of the full-length amyloid peptide, A beta(1-42), with phospholipid lipid bilayers was studied in the presence of the metal-chelating drug, Clioquinol (CQ). The effect of cholesterol and metal ions was also determined using solid-state 31P and 2H NMR. CQ modulated the effect of metal ions on the integrity of the bilayer and although CQ perturbed the phospholipid membrane, the bilayer integrity was maintained. Model membranes enriched in cholesterol were studied under conditions of peptide association and incorporation. Solid-state NMR showed that the bilayer integrity was preserved in cholesterol-enriched membranes in comparison to phosphatidylcholine-phosphatidylserine bilayers. Changes in peptide structure, consistent with an increase in beta-sheet, were observed using specifically 13C-labelled A beta(1-42) by magic angle spinning NMR. Results using aligned phosphatidylcholine bilayers and completely 15N-labelled peptide indicated that the peptide aggregated. The results are consistent with oligomeric beta-sheet structured peptides only partially penetrating the bilayer and cholesterol reducing the membrane disruption.     

Cl- channels in intact human T lymphocytes.

We recently described a large, multiple-conductance Cl- channel in excised patches from normal T lymphocytes. The properties of this channel in excised patches are similar to maxi-Cl- channels found in a number of cell types. The voltage dependence in excised patches permitted opening only at nonphysiological voltages, and channel activity was rarely seen in cell-attached patches. In the present study, we show that Cl- channels can be activated in intact cells at physiological temperatures and voltages and that channel properties change after patch excision. Maxi-Cl- channels were reversibly activated in 69% of cell-attached patches when the temperature was above 32 degrees C, whereas fewer than 2% of patches showed activity at room temperature. Upon excision, the same patches displayed large, multiple-conductance Cl- channels with characteristics like those we previously reported for excised patches. After patch excision, warm temperatures were not essential to allow channel activity; 37% (114/308) of inside-out patches had active channels at room temperature. The voltage dependence of the channels was markedly different in cell-attached recordings compared with excised patches. In cell-attached patches, Cl- channels could be open at cell resting potentials in the normal range. Channel activation was not related to changes in intracellular Ca2+ since neither ionomycin nor mitogens activated the channels in cell-attached patches, Ca2+ did not rise in response to warming and the Cl- channel was independent of Ca2+ in inside-out patches. Single-channel currents were blocked by internal or external Zn2+ (100-200 microM), 4-acetamido-4' isothiocyanostilbene-2,2'-disulfonate (SITS, 100-500 microM) and 4,4'-diisothiocyanostilbene 2,2'-disulfonate (DIDS, 100 microM). NPPB (5-nitro-2-(3-phenylpropylamino)-benzoate) reversibly blocked the channels in inside-out patches.     

Calcium- and voltage-activated potassium channels in human macrophages.

Single calcium-activated potassium channel currents were recorded in intact and excised membrane patches from cultured human macrophages. Channel conductance was 240 pS in symmetrical 145 mM K+ and 130 pS in 5 mM external K+. Lower conductance current fluctuations (40% of the larger channels) with the same reversal potential as the higher conductance channels were noted in some patches. Ion substitution experiments indicated that the channel is permeable to potassium and relatively impermeable to sodium. The frequency of channel opening increased with depolarization and intracellular calcium concentration. At 10(-7) M (Ca++)i, channel activity was evident only at potentials of +40 mV or more depolarized, while at 10(-5) M, channels were open at all voltages tested (-40 to +60 mV). In intact patches, channels were seen at depolarized patch potentials of +50 mV or greater, indicating that the ionized calcium concentration in the macrophage is probably less than 10(-7) M.     

Patch clamp recordings from membranes which contain gap junction channels.

The septal membranes of the median and lateral giant axons of earthworm, which contain gap junctions, were exposed by cutting one segment of the cord. Patch recordings were obtained from the exposed cytoplasmic side of the septum. Seal resistances ranged from 2 to 15 G omega. The patch could be excised (detached) or left attached to the whole cell. Two types of channels were observed. One type was blocked by tetraethylammonium (TEA) or Cs+ and had a unitary conductance of 30-40 pS. It appears to be a K+ channel. The other channel type had a unitary conductance of 90-110 pS and was unaffected by TEA+ or Cs+. In the detached configuration the channel was shown to conduct Cs+, K+, Na+, TMA+, Cl- and TEA+ even in the presence of 2 mM Zn2+, 1 mM Ni2+, 1 mM Co2+, and 4 mM 4-aminopyridine. The conductance ratios relative to K+ were 1.0 for Cs+, 0.84 for Na+, 0.64 for TMA+, 0.52 for Cl- and 0.2 for TEA+. The channel appears to be voltage insensitive whether monitored in detached or attached recording mode. Both H+ and Ca2+ reduce the probability of opening. Thus, the 100 pS channel has many of the properties expected of a gap junction channel.     

Potassium channel activity recorded from the apical membrane of freshly isolated epithelial cells in rat caudal epididymis.

K+ channels were recorded in excised, inside-out patches from the apical membrane of the freshly isolated tubule of the caudal portion of the rat epididymis. With asymmetric K+ concentrations in bath and pipette (140 mM K+in/6 mM K+out), the channels had a slope conductance of 54.2 pS at 0 mV. The relative permeability of K+ over Na+ was about 171 to 1. The channels were activated by intracellular Ca2+ and by membrane depolarization. These channels belong to a class defined as "intermediate-conductance Ca2+-activated K+ channel. " External tetraethylammonium ions (TEA+) caused a flickery block of the channel with reduction in single-channel current amplitude measured at a range of holding membrane potentials (-40 to 60 mV). Activity of the K+ channels was inhibited by intracellular ATP (KD =1.188 mM). The channel activity was detected only occasionally in patches from the apical membrane (about 1 in 17 patches containing active channels). The presence of the intermediate-conductance Ca2+-activated K+ channels indicates that they could provide a route for K+ secretion in a Ca2+-dependent process responsible for a high luminal K+ concentration found in the epididymal duct of the rat.     

Amyloid beta protein (1-40) forms calcium-permeable, Zn2+-sensitive channel in reconstituted lipid vesicles.

Amyloid beta protein (A beta P) forms senile plaques in the cerebrocortical blood vessels and brain parenchyma of patients with Alzheimer's disease (AD). The nonfamilial or sporadic AD (SAD), the most prevalent form of AD, has been correlated with an increased level of 40-residue A beta P (A beta P1-40). However, very little is known about the role of A beta P1-40 in AD pathophysiology. We have examined the activity of A beta P1-40 reconstituted in phospholipid vesicles. A combined light fluorescence and atomic force microscope (AFM) was used to image the structure of reconstituted vesicles and 45Ca2+ uptake was used as an assay for calcium permeability across the vesicular membrane. Vesicles reconstituted with fresh and globular A beta P1-40 contain a significant amount of A0 beta P and exhibit strong immunofluorescence labeling with an antibody raised against the N-terminal domain of A beta P, suggesting the incorporation of A beta P1-40 peptide in the vesicular membrane. Vesicles reconstituted with A beta P1-40 exhibited a significant level of 45Ca2+ uptake. The vesicular calcium level saturated over time, showing an important ion channel characteristic. The 45Ca2+ uptake was inhibited by (i) a monoclonal antibody raised against the N-terminal region of A beta P and (ii) Zn2+. However, a reducing agent (DTT) did not inhibit the 45Ca2+ uptake, indicating that the oxidation of A beta P or its surrounding lipid molecules is not directly involved in A beta P-mediated Ca2+ uptake. These findings provide biochemical and structural evidence that fresh and globular A beta P1-40 forms calcium-permeable channels and thus may induce cellular toxicity by regulating the calcium homeostasis in nonfamilial or sporadic Alzheimer's disease.     

Heterogeneous amyloid-formed ion channels as a common cytotoxic mechanism: implications for therapeutic strategies against amyloidosis

The amyloidoses consist of human and animal chronic, progressive, and sometimes fatal diseases that are characterized by the deposition of insoluble proteinaceous amyloid fibrils in various tissues. Despite the biochemical diversity of amyloids, they share certain properties. The amphipathic and the charged nature of many amyloid-forming peptides point to their intrinsic ability to form diverse beta-sheet-based aggregates and channel types in negatively charged membranes. We hypothesize that the formation of heterogeneous channels represents a common cytotoxic mechanism that accentuates the changes in the signal transduction that underlie amyloid-induced cell malfunction. One group of amyloid-forming peptides that could mediate their action via the formation of heterogeneous channels includes the extensively examined prions and amyloid beta protein that are associated with conformational neurodegenerative diseases. The aim of this study is to examine heterogeneous channels formed in bilayers with amyloid-forming peptides as a common mechanism of malfunction of signal transduction. The observed amyloid-formed channel types include the following. (1) Natriuretic peptides: (i) 68-pS H2O2- and Ba2+-sensitive channel with fast kinetics. The fast channel had three modes (spike mode, burst mode, and open mode), which differ in their kinetics but not in their conductance properties; (ii) a 273-pS inactivating large conductance channel; and (iii) a 160-pS transiently activated channel. (2) Prions: (i) a 140-pS GSSG- and TEA-sensitive channel with fast kinetics; (ii) a 41-pS dithiothreitol (DTT)-sensitive channel with slow kinetics; (iii) a 900 to 1444-pS large channel. (3) Amyloid beta protein: (i) a 17 to 63-pS AbetaP[1-40]-formed "bursting" fast cation channel, (ii) the AbetaP[1-40]-formed "spiky" fast cation channel with a similar kinetics to the "bursting" fast channel except for the absence of the long intraburst closures, (iii) 275-pS AbetaP[1-40]-formed medium conductance channel, and (iv) 589- to 704-pS AbetaP[1-40]-formed inactivating large conductance channel. This heterogeneity is one of the most common features of these charged cytotoxic amyloid-formed channels, reflecting these channels' ability to modify multiple cellular functions. Although the diversity of these aggregated-peptide-formed channels may indicate that a stochastic mechanism governs their formation, the fact that certain channel types are often observed point to preferential channel protein conformations. In addition, the fact that other amyloids have similar structural properties (e.g. hydrophobicity, charged residues, and beta-structural linkages, suggests that, despite the intrinsic ability to form diverse conformations, certain conformations and, hence, certain channel types could be a common pathologic conformation among these amyloid-forming peptides. It is concluded that conformation-based channel diversity is an important mechanism for enhancing the toxicity of amyloid-forming peptides. The cytotoxic nature of these self-associated beta-based protein channels suggests that under normal physiological conditions cells employ well-evolved protective mechanisms against seeding and/or propagation of channel-forming peptides; for example, (a) compartmentalization of these peptides as membrane bound in internal vesicles and/or (b) degradation of these peptides by enzymes. The pharmacological diversity of the amyloid-forming channels implies that multiple therapeutic interventions may be necessary for blocking and reversing heterogeneous channel formations and preventing their associated diseases.