Amyloid fibrillization is multistep process involving soluble oligomeric intermediates, including spherical oligomers and protofibrils. Amyloid oligomers have a common, generic structure, and they are intrinsically toxic to cells, even when formed from non-disease related proteins, which implies they also share a common mechanism of pathogenesis and toxicity. Here we report that soluble oligomers from several types of amyloids specifically increase lipid bilayer conductance regardless of the sequence, while fibrils and soluble low molecular weight species have no effect. The increase in membrane conductance occurs without any evidence of discrete channel or pore formation or ion selectivity. The conductance is dependent on the concentration of oligomers and can be reversed by anti-oligomer antibody. These results indicate that soluble oligomers from many types of amyloidogenic proteins and peptides increase membrane conductance in a conformation-specific fashion and suggest that this may represent the common primary mechanism of pathogenesis in amyloid-related degenerative diseases.Soluble amyloid oligomers are a common intermediate in the pathway for amyloid fibril formation and have been implicated as the primary toxic species of amyloids related to neurodegenerative disease (1-6). More recent reports indicate that soluble amyloid oligomers are intrinsically toxic even when they are formed from proteins that are not normally related to degenerative disease (3), and the toxic activity of soluble oligomers may be related to a common generic structure that they share (6). Although the primary mechanism of amyloid toxicity is not clear, the fact that different amyloids reside in either the cytosolic or extracellular compartments and the observation that cytosolic amyloid aggregates are toxic when applied externally to cells (6, 7) points to the cell plasma membrane as a potential primary target of amyloid pathogenesis. Indeed, there are many reports of membrane perturbations caused by amyloids like A␤ (8), but it isn't clear whether these effects are specific to soluble oligomers nor whether they are common to other types of amyloids. Here we report that homogeneous populations of spherical amyloid oligomers and protofibrils increase the conductivity of membranes by a non-channel mechanism. This effect is observed for all soluble oligomers tested regardless of protein sequence and is not observed for amyloid fibrils or soluble low molecular weight species, suggesting that the increase in membrane conductivity may be a primary common mechanism of amyloid oligomer pathogenesis. MATERIALS AND METHODSPeptide Synthesis-Peptide synthesis: A␤ peptides, prion 106 -126, and IAPP 1 were synthesized by fluoren-9-ylmethoxy carbonyl chemistry using a continuous flow semiautomatic instrument as described previously (9). The purity was checked by analytical reverse phase-high performance liquid chromatography and by electrospray mass spectrometry. Polyglutamine KKQ40KK was a gift from Dr. Ronald Wetzel, and ␣-synuclein was a gif...

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Amyloid oligomers are believed to play causal roles in several types of amyloid-related neurodegenerative diseases. Several different types of amyloid oligomers have been reported that differ in morphology, size, or toxicity, raising the question of the pathological significance and structural relationships between different amyloid oligomers. Annular protofibrils (APFs) have been described in oligomer preparations of many different amyloidogenic proteins and peptides as ring-shaped or pore-like structures. They are interesting because their pore-like morphology is consistent with numerous reports of membrane-permeabilizing activity of amyloid oligomers. Here we report the preparation of relatively homogeneous preparations of APFs and an antiserum selective for APFs (␣APF) compared with prefibrillar oligomers (PFOs) and fibrils. PFOs appear to be precursors for APF formation, which form in high yield after exposure to a hydrophobic-hydrophilic interface. Surprisingly, preformed APFs do not permeabilize lipid bilayers, unlike the precursor PFOs. APFs display a conformation-dependent, generic epitope that is distinct from that of PFOs and amyloid fibrils. Incubation of PFOs with phospholipids vesicles results in a loss of PFO immunoreactivity with a corresponding increase in ␣APF immunoreactivity, suggesting that lipid vesicles catalyze the conversion of PFOs into APFs. The annular anti-protofibril antibody also recognizes heptameric ␣-hemolysin pores, but not monomers, suggesting that the antibody recognizes an epitope that is specific for a ␤ barrel structural motif.Many age-related neurodegenerative diseases are characterized by the accumulation of amyloid deposits derived from a variety of misfolded proteins (1). These diseases typically have both sporadic and inherited forms, and in many cases the mutations associated with the familial forms are in the gene encoding the protein that accumulates or in genes directly related to its production, processing, or accumulation (2). The genetic linkage between the mutant allele and disease is evidence of the causal relationship of amyloid accumulation to pathogenesis, and many of the mutations either destabilize the natively folded state, produce more amyloidogenic protein, or they increase its propensity to aggregate (3). Although fibrillar amyloid deposits are among the most obvious pathognomonic features of disease, their role in pathogenesis is not clear. The extent of fibrillar amyloid plaque deposition does not correlate well with Alzheimer's disease pathogenesis, and there are a significant number of non-demented individuals that have equivalent amounts of amyloid plaques as disease patients (4). Pathological changes are observed in transgenic animals before the onset of amyloid plaque accumulation (5, 6), and it has been reported that soluble A␤ oligomers correlate better with dementia than insoluble, fibrillar deposits (7, 8), suggesting that oligomeric forms of A␤ may represent the primary toxic species. Soluble oligomers have been implicated as the primary ...

[Brief letters to the Editor that make specific sciendfic reference to papers published previously in THE ,JOURNAL OF GENERAL PHYSIOLOGY are invited. Receipt of such letters will bc acknowledged, and those containing pcrtincnt scientific comments and scientific criticisms will be published.] Access Resistance of a Small Circular PoreDear Sir:The access resistance of a small circular pore is of some importance in estimating the conductance of pores in biological membranes (Hille, 1968(Hille, ,1970. This resistance is usually approximated as the convergence resistance to a hemisphere of the same radius as the pore. The approximation assumes that the contribution of the hemisphere itself is negligible. It turns out that this is not true and that the resistance of the hemisphere is in fact of the same order of magnitude as the resistance of the material in the halfspace outside the hemisphere.We can estimate the resistance of the hemisphere by assuming field lines inside the hemisphere are straight and perpendicular to the mouth of the pore. With this approximation the resistance of the hemisphere is Rh = p ,(1) 2~-a where a is the radius of the hemisphere and P resistivity of the medium. This is equal to the value obtained for the convergence resistance to a hemisphere (Hille, 1970, p. 21). Hence making the approximation that the convergence resistance to a circular pore is given by the convergence resistance to a hemisphere neglects a contribution possibly as large as the one calculated from the original approximation.Fortunately a more satisfying physical approximation can be solved exactly using a well-known result of electrostatics. If the mouth of the pore is an equipotential surface, the convergence resistance can be calculated exactly. This problem is equivalent to that of calculating the resistance between a conducting disk on an insulator and a half-spherical electrode very far from the disk.It can be shown that problems of resistance between electrodes in conducting media and problems of capacitance between electrodes in insulating media are related by a simple transformation. (See, for example, Smythe pp. 234 and 237.) For a given electrode geometry, resistance and capacitance are related by the equation where R is the resistance, C the capacitance, e the permitivity of the medium where the

The architecture of the pore-region of a voltage-gated K+ channel, Kv1.3, was probed using four high affinity scorpion toxins as molecular calipers. We established the structural relatedness of these toxins by solving the structures of kaliotoxin and margatoxin and comparing them with the published structure of charybdotoxin; a homology model of noxiustoxin was then developed. Complementary mutagenesis of Kv1.3 and these toxins, combined with electrostatic compliance and thermodynamic mutant cycle analyses, allowed us to identify multiple toxin-channel interactions. Our analyses reveal the existence of a shallow vestibule at the external entrance to the pore. This vestibule is approximately 28-32 A wide at its outer margin, approximately 28-34 A wide at its base, and approximately 4-8 A deep. The pore is 9-14 A wide at its external entrance and tapers to a width of 4-5 A at a depth of approximately 5-7 A from the vestibule. This structural information should directly aid in developing topological models of the pores of related ion channels and facilitate therapeutic drug design.

The Xenopus laevis oocyte is widely used to express exogenous channels and transporters and is well suited for functional measurements including currents, electrolyte and nonelectrolyte fluxes, water permeability and even enzymatic activity. It is difficult, however, to transform functional measurements recorded in whole oocytes into the capacity of a single channel or transporter because their number often cannot be estimated accurately. We describe here a method of estimating the number of exogenously expressed channels and transporters inserted in the plasma membrane of oocytes. The method is based on the facts that the P (protoplasmic) face in water-injected control oocytes exhibit an extremely low density of endogenous particles (212 +/- 48 particles/microns2, mean, SD) and that exogenously expressed channels and transporters increased the density of particles (up to 5,000/microns2) only on the P face. The utility and generality of the method were demonstrated by estimating the "gating charge" per particle of the Na+/glucose cotransporter (SGLT1) and a nonconducting mutant of the Shaker K+ channel proteins, and the single molecule water permeability of CHIP (Channel-like In-tramembrane Protein) and MIP (Major Intrinsic Protein). We estimated a "gating charge" of approximately 3.5 electronic charges for SGLT1 and approximately 9 for the mutant Shaker K+ channel from the ratio of Qmax to density of particles measured on the same oocytes. The "gating charges" were 3-fold larger than the "effective valences" calculated by fitting a Boltzmann equation to the same charge transfer data suggesting that the charge movement in the channel and cotransporter occur in several steps. Single molecule water permeabilities (pfs) of 1.4 x 10(-14) cm3/sec for CHIP and of 1.5 x 10(-16) cm3/sec for MIP were estimated from the ratio of the whole-oocyte water permeability (Pf) to the density of particles. Therefore, MIP is a water transporter in oocytes, albeit approximately 100-fold less effective than CHIP.

Summary. Alamethicin induces a conductance in black lipid films which increases exponentially with voltage. At low conductance the increase occurs in discrete steps which form a pattern of five levels, the second and third being most likely. The conductance of each level is directly proportional to salt concentration, inversely proportional to solution viscosity, and nearly independent of voltage.The probability distribution of the five steps is not a function of voltage, but as the voltage is increased, more levels begin to appear. These can be explained as superpositions of the original five, both in position and relative probability.This suggests that the five levels are associated with a physical entity which we call a pore. This point of view is confirmed by the following measurements. The kinetic response of the current to a voltage step is first order, and shows an exponential increase in rate of pore formation and an exponential decrease in rate of pore disappearance with voltage. If these rates are statistical, the number of pores should fluctuate about a voltage-dependent mean. High conductance current fluctuations are too large to be explained by fluctuation in the number of pores alone. But if fluctuations among the five levels are included, the magnitude of the fluctuations at high conductance is accurately predicted.Alamethicin adsorbs reversibly to the membrane surface, and the conductance at a fixed voltage depends on the ninth power of alamethicin concentration and on the fourth power of salt concentration, in the aqueous phase. In our bacterial phosphatidyl ethanolamine membranes, alamethicin added to one side of the membrane produces elevated conductance only when the voltage on that side is increased.

Aquaporins increase the water permeability in many cell types across many species. We investigated the effects of external pH and Ca 2؉ on water permeability of Xenopus oocytes injected with aquaporin cRNA by measuring the rate of swelling in hypotonic solutions. Lowering pH to 6.5 increased the water permeability of aquaporin (AQP0) 3.4 ؎ 0.4-fold. Diethylpyrocarbonate pretreatment increased water permeability 4.2 ؎ 0.5-fold and abolished pH sensitivity, suggesting that the pH regulation is mediated by an external histidine. Lowering Ca 2؉ increased water permeability 4.1 ؎ 0. acts at an internal site. Three different calmodulin inhibitors each increased AQP0 water permeability, suggesting that Ca 2؉ may act through calmodulin. None of the above altered the water permeability induced by AQP1 or AQP4. Because the greatest change in AQP0 water permeability is in the normal pH range found in the lens (7.2-6.5), this paper provides evidence for regulation of an aquaporin by pH under physiological conditions.The major intrinsic protein (MIP, now designated aquaporin 0 and abbreviated AQP0) 1 of the optical lens was the first sequenced member of the aquaporins, an ancient family of proteins found in bacteria, plants, and animals (1-4). Preston et al. (5) discovered that CHIP 28 (now called aquaporin 1 (AQP1)), a protein abundant in red blood cells, facilitates the diffusion of water across the plasma membrane when expressed in Xenopus oocytes. AQP4 (previously called MIWC for mercurial-insensitive water channel) is expressed strongly in the brain and kidney collecting duct (6, 7). Work in several laboratories subsequently demonstrated that many members of the aquaporin family facilitate the diffusion of water and other nonelectrolytes (3, 8 -12). Among the aquaporins, AQP0 forms a water channel with a relatively low water permeability (13,14); the water permeability per molecule is 40 times higher for AQP1 (13,15). The structural basis of this large difference in water permeability is unknown. AQP0 and AQP1 form tetrameric arrays in their native membranes and when reconstituted in lipid vesicles (6 -18). AQP0, AQP1, and AQP4 share ϳ40% sequence identity with each other. Attempts to increase AQP0 water permeability by exchanging parts of AQP0 for corresponding parts of AQP1 have been ineffective (19).Although low in water permeability per molecule, AQP0 comprises more than 60% of the membrane protein in the normal vertebrate lens and therefore provides the major permeability pathway for water movement across the membranes of lens fiber cells. If it is defective or missing from an otherwise normal lens, a cataract results (20, 21). In a chimeric mouse model, cataract can be prevented by the presence of 20% normal cells, which presumably supply the requisite AQP0 (22). The role of AQP0 in maintaining normal lens conditions is uncertain, but it likely facilitates the intrinsic circulation of fluid in the lens that maintains lens transparency and homeostasis in the absence of blood vessels (23). pH and Ca 2ϩ are likely ca...