Amyloid fiber formation is correlated with pathology in many diseases, including Alzheimer's, Parkinson's, and type II diabetes. Although β-sheet-rich fibrillar protein deposits define this class of disorder, increasing evidence points toward small oligomeric species as being responsible for cell dysfunction and death. The molecular mechanism by which this occurs is unknown, but likely involves the interaction of these species with biological membranes, with a subsequent loss of integrity. Here, we investigate islet amyloid polypeptide, which is implicated in the loss of insulin-secreting cells in type II diabetics. We report the discovery of oligomeric species that arise through stochastic nucleation on membranes and result in disruption of the lipid bilayer. These species are stable, result in all-or-none leakage, and represent a definable protein/lipid phase that equilibrates over time. We characterize the reaction pathway of assembly through the use of an experimental design that includes both ensemble and single-particle evaluations. Complexity in the reaction pathway could not be satisfied using a two-state description of membrane-bound monomer and oligomeric species. We therefore put forward a threestate kinetic framework, one of which we conjecture represents a non-amyloid, non-β-sheet intermediate previously shown to be a candidate therapeutic target.amylin | membrane pore | cytotoxicity | disordered protein A lzheimer's, Parkinson's, type II diabetes, and other epidemiologically important diseases are characterized, in part, by the deposition of proteinaceous plaques termed amyloid (1, 2). For each disease, a specific protein is involved in amyloid assembly via a process that is cytotoxic, ultimately leading to degeneration. Although a defining characteristic, it is now widely thought that these deposits are not the origin of cytotoxicity. Instead, it has been suggested that cell dysfunction and death are mediated by small oligomeric species that acquire the capacity to disrupt cellular membrane integrity (3, 4). The energetic and structural basis by which these states mediate toxicity, however, is unclear.Islet amyloid polypeptide (IAPP or amylin) is a 37-residue peptide hormone cosecreted with insulin by pancreatic β-cells. Unmodified, wild-type IAPP self-assembles to form amyloid in type II diabetic patients in a process that is associated with β-cell dysfunction (2, 5). Indeed, the capacity of species-based sequence variants to form amyloid is correlated with the observation of metabolic disease (6). Thus, whereas primates and cats readily form amyloid and acquire diabetes, rats and mice do not spontaneously develop diabetes, and rodent IAPP does not form amyloid or other β-sheet aggregates. Diabetic symptoms can be induced in model rodents either by using toxins or by using rodents transgenic for human IAPP (7).Previous in vitro studies have shown that the binding of human and rat IAPP to lipid bilayers is cooperative (8), an observation accounted for by the presence of oligomeric species. The pop...

Three families of membrane-active peptides are commonly found in nature and are classified according to their initial apparent activity. Antimicrobial peptides are ancient components of the innate immune system and typically act by disruption of microbial membranes leading to cell death. Amyloid peptides contribute to the pathology of diverse diseases from Alzheimer's to type II diabetes. Preamyloid states of these peptides can act as toxins by binding to and permeabilizing cellular membranes. Cell-penetrating peptides are natural or engineered short sequences that can spontaneously translocate across a membrane. Despite these differences in classification, many similarities in sequence, structure, and activity suggest that peptides from all three classes act through a small, common set of physical principles. Namely, these peptides alter the Brownian properties of phospholipid bilayers, enhancing the sampling of intrinsic fluctuations that include membrane defects. A complete energy landscape for such systems can be described by the innate membrane properties, differential partition, and the associated kinetics of peptides dividing between surface and defect regions of the bilayer. The goal of this review is to argue that the activities of these membrane-active families of peptides simply represent different facets of what is a shared energy landscape.

Invertebrate-specific gap junction proteins, termed innexins, form a large family of four-transmembrane proteins. These proteins oligomerize to constitute intercellular channels that allow for the passage of small signaling molecules associated with neural and muscular electrical activity. In contrast to the large number of structural and functional studies of vertebrate connexin gap junction channels, few structural studies of recombinant innexin channels have been published. Here we show a three-dimensional structure of two-dimensionally crystallized Caenorhabditis elegans innexin-6 (INX-6) gap junction channels. An N-terminal deletion INX-6 construct in which amino acids 2 through 19 were removed (INX-6-deltaN) was crystallized in lipid bilayers. The threedimensional reconstruction based on cryo-electron crystallography revealed that the two-dimensional crystals of INX-6-deltaN comprise two lipid bilayers, including fully docked gap junction channels. A single INX-6-deltaN gap junction channel comprises 16 subunits, a hexa-decamer, in contrast to vertebrate connexin channels, which comprise 12 subunits. Two bulb densities were observed in each hemichannel, one in the pore and the other at the cytoplasmic side of the hemichannel in the channel pore pathway. The former is reminiscent of the plug observed in the connexin26 mutant structure we previously reported. A fluorescent dye transfer assay revealed that INX-6-deltaN junction channels were essentially impermeable. These findings imply the structural diversity of gap junction channels among multicellular organisms, and provide insight into the functional properties characteristic of invertebrates. How connexin hemichannels open and close the pore in response to changes in voltage and extracellular calcium concentrations is still unknown. Previous work has shown that conformational changes at the extracellular entrance of the pore are critical for hemichannel gating by calcium and voltage. Recently, we found that negatively charged residues in this region could interact with calcium ions to produce occlusion of the pore. These residues form a ring, raising the possibility that a calcium-bound gating ring at the entrance of the pore forms a gate.To test whether such a gating ring serves as a physical gate that prevents the access of ions and small metabolites, we assessed the calcium state-dependent accessibility of Cd 2þ and MTSES to a substituted cysteine in Cx26 (Cx26G45C) located below the residues that form the ring. Extracellular application of MTSES to Xenopus oocytes expressing Cx26G45C mutants modified hemichannel currents, indicating accessibility to this residue. However, the estimated reaction rate was independent of the extracellular calcium concentration (i.e., the reaction rate was the same whether the channels were open or were closed by calcium). Similarly, Cd 2þ accessibility to residue G45C was nearly independent of extracellular Ca 2þ concentration. These results indicate that the calcium-gating ring does not prevent access of ions or metabo...

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