50 research outputs found
Insulated conducting cantilevered nanotips and two-chamber recording system for high resolution ion sensing AFM.
Biological membranes contain ion channels, which are nanoscale pores allowing controlled ionic transport and mediating key biological functions underlying normal/abnormal living. Synthetic membranes with defined pores are being developed to control various processes, including filtration of pollutants, charge transport for energy storage, and separation of fluids and molecules. Although ionic transport (currents) can be measured with single channel resolution, imaging their structure and ionic currents simultaneously is difficult. Atomic force microscopy enables high resolution imaging of nanoscale structures and can be modified to measure ionic currents simultaneously. Moreover, the ionic currents can also be used to image structures. A simple method for fabricating conducting AFM cantilevers to image pore structures at high resolution is reported. Tungsten microwires with nanoscale tips are insulated except at the apex. This allows simultaneous imaging via cantilever deflections in normal AFM force feedback mode as well as measuring localized ionic currents. These novel probes measure ionic currents as small as picoampere while providing nanoscale spatial resolution surface topography and is suitable for measuring ionic currents and conductance of biological ion channels
Structure and permeability of ion-channels by integrated AFM and waveguide TIRF microscopy.
Membrane ion channels regulate key cellular functions and their activity is dependent on their 3D structure. Atomic force microscopy (AFM) images 3D structure of membrane channels placed on a solid substrate. Solid substrate prevents molecular transport through ion channels thus hindering any direct structure-function relationship analysis. Here we designed a ~70 nm nanopore to suspend a membrane, allowing fluidic access to both sides. We used these nanopores with AFM and total internal reflection fluorescence microscopy (TIRFM) for high resolution imaging and molecular transport measurement. Significantly, membranes over the nanopore were stable for repeated AFM imaging. We studied structure-activity relationship of gap junction hemichannels reconstituted in lipid bilayers. Individual hemichannels in the membrane overlying the nanopore were resolved and transport of hemichannel-permeant LY dye was visualized when the hemichannel was opened by lowering calcium in the medium. This integrated technique will allow direct structure-permeability relationship of many ion channels and receptors
Role of the fast kinetics of pyroglutamate-modified amyloid-β oligomers in membrane binding and membrane permeability.
Membrane permeability to ions and small molecules is believed to be a critical step in the pathology of Alzheimer's disease (AD). Interactions of oligomers formed by amyloid-β (Aβ) peptides with the plasma cell membrane are believed to play a fundamental role in the processes leading to membrane permeability. Among the family of Aβs, pyroglutamate (pE)-modified Aβ peptides constitute the most abundant oligomeric species in the brains of AD patients. Although membrane permeability mechanisms have been studied for full-length Aβ1-40/42 peptides, these have not been sufficiently characterized for the more abundant AβpE3-42 fragment. Here we have compared the adsorbed and membrane-inserted oligomeric species of AβpE3-42 and Aβ1-42 peptides. We find lower concentrations and larger dimensions for both species of membrane-associated AβpE3-42 oligomers. The larger dimensions are attributed to the faster self-assembly kinetics of AβpE3-42, and the lower concentrations are attributed to weaker interactions with zwitterionic lipid headgroups. While adsorbed oligomers produced little or no significant membrane structural damage, increased membrane permeabilization to ionic species is understood in terms of enlarged membrane-inserted oligomers. Membrane-inserted AβpE3-42 oligomers were also found to modify the mechanical properties of the membrane. Taken together, our results suggest that membrane-inserted oligomers are the primary species responsible for membrane permeability
Structural Convergence Among Diverse, Toxic β-Sheet Ion Channels
Recent studies show that an array of -sheet peptides, including N-terminally truncated A peptides (A11-42/17-42), K3 (a 2-microglobulin fragment), and protegrin-1 (PG-1) peptides form ion channel-like structures and elicit single channel ion conductance when reconstituted in lipid bilayers and induce cell damage through cell calcium overload. Striking similarities are observed in the dimensions of these toxic channels irrespective of their amino acid sequences. However, the intriguing question of preferred channel sizes is still unresolved. Here, exploiting ssNMR-based, U-shaped, -strand-turn--strand coordinates, we modeled truncated A peptide (p3) channels with different sizes (12- to 36-mer). Molecular dynamics (MD) simulations show that optimal channel sizes of the ion channels presenting toxic ionic flux range between 16- and 24-mer. This observation is in good agreement with channel dimensions imaged by AFM for A9-42, K3 fragment, and PG-1 channels and highlights the bilayer-supported preferred toxic -channel sizes and organization, regardless of the peptide sequence
All-d-Enantiomer of β-Amyloid Peptide Forms Ion Channels in Lipid Bilayers
Alzheimer’s disease (AD) is the most common type
of senile
dementia in aging populations. Amyloid β (Aβ)-mediated
dysregulation of ionic homeostasis is the prevailing underlying mechanism
leading to synaptic degeneration and neuronal death. Aβ-dependent
ionic dysregulation most likely occurs either directly via unregulated
ionic transport through the membrane or indirectly via Aβ binding
to cell membrane receptors and subsequent opening of existing ion
channels or transporters. Receptor binding is expected to involve
a high degree of stereospecificity. Here, we investigated whether
an Aβ peptide enantiomer, whose entire sequence consists of d-amino acids, can form ion-conducting channels; these channels
can directly mediate Aβ effects even in the absence of receptor–peptide
interactions. Using complementary approaches of planar lipid bilayer
(PLB) electrophysiological recordings and molecular dynamics (MD)
simulations, we show that the d-Aβ isomer exhibits
ion conductance behavior in the bilayer indistinguishable from that
described earlier for the l-Aβ isomer. The d isomer forms channel-like pores with heterogeneous ionic conductance
similar to the l-Aβ isomer channels, and the d-isomer channel conductance is blocked by Zn2+, a known
blocker of l-Aβ isomer channels. MD simulations further
verify formation of β-barrel-like Aβ channels with d- and l-isomers, illustrating that both d- and l-Aβ barrels can conduct cations. The calculated
values of the single-channel conductance are approximately in the
range of the experimental values. These findings are in agreement
with amyloids forming Ca2+ leaking, unregulated channels
in AD, and suggest that Aβ toxicity is mediated through a receptor-independent,
nonstereoselective mechanism
Real-Time Afm Imaging of Surface-Induced Oligomerization of the Non-Amyloidogenic P3 Peptide: Implications for Membrane Insertion and Ion Channel Formation
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Structural convergence among diverse, toxic beta-sheet ion channels.
Recent studies show that an array of beta-sheet peptides, including N-terminally truncated Abeta peptides (Abeta(11-42/17-42)), K3 (a beta(2)-microglobulin fragment), and protegrin-1 (PG-1) peptides form ion channel-like structures and elicit single channel ion conductance when reconstituted in lipid bilayers and induce cell damage through cell calcium overload. Striking similarities are observed in the dimensions of these toxic channels irrespective of their amino acid sequences. However, the intriguing question of preferred channel sizes is still unresolved. Here, exploiting ssNMR-based, U-shaped, beta-strand-turn-beta-strand coordinates, we modeled truncated Abeta peptide (p3) channels with different sizes (12- to 36-mer). Molecular dynamics (MD) simulations show that optimal channel sizes of the ion channels presenting toxic ionic flux range between 16- and 24-mer. This observation is in good agreement with channel dimensions imaged by AFM for Abeta(9-42), K3 fragment, and PG-1 channels and highlights the bilayer-supported preferred toxic beta-channel sizes and organization, regardless of the peptide sequence