Bilayer Technique Research Articles

Exploring the Nature of Cationic Blocker Recognition by the Anthrax Toxin Channel

Obstructing conductive pathways of the channel-forming toxins with targeted blockers is a promising drug design approach. Nearly all tested positively charged ligands have been shown to reversibly block the cation-selective channel-forming protective antigen (PA63) component of the binary anthrax toxin. The cationic ligands with more hydrophobic surfaces, particularly those carrying aromatic moieties, inhibited PA63 more effectively. To understand the physical basis of PA63 selectivity for a particular ligand, detailed information is required on how the blocker structural elements (e.g., positively charged and aromatic groups) influence the molecular kinetics of the blocker/channel binding reactions. In this study, we address this problem using the high-resolution single-channel planar lipid bilayer technique. Several structurally distinct cationic blockers, namely per-6-S-(3-amino) propyl-β-cyclodextrin, per-6-S-(3-aminomethyl) benzyl-α-cyclodextrin, per-6-S-(3-aminomethyl) benzyl-β-cyclodextrin, per-6-S-(3-aminomethyl) benzyl-γ-cyclodextrin, methyltriphenylphosphonium ion, and G0 polyamidoamine dendrimer are tested for their ability to inhibit the heptameric and octameric PA63 variants and PA63F427A mutant. The F427 residues form a hydrophobic constriction region inside the channel, known as the “ϕ-clamp.” We show that the cationic blockers interact with PA63 through a combination of forces. Analysis of the binding reaction kinetics suggests the involvement of cation-π, Coulomb, and salt-concentration-independent π-π or hydrophobic interactions in the cationic cyclodextrin binding. It is possible that these blockers bind to the ϕ-clamp and are also stabilized by the Coulomb interactions of their terminal amino groups with the water-exposed negatively charged channel residues. In PA63F427A, only the suggested Coulomb component of the cyclodextrin interaction remains. Methyltriphenylphosphonium ion and G0 polyamidoamine dendrimer, despite being positively charged, interact primarily with the ϕ-clamp. We also show that seven- and eightfold symmetric cyclodextrins effectively block the heptameric and octameric forms of PA63 interchangeably, adding flexibility to the earlier formulated blocker/target symmetry match requirement.

Optical Sensing of Ion Flux through Biomimetic Carbon Nanotube Channels

Increasing demands for cheaper and more efficient water treatment methods have never been higher, as shortages of fresh water have become a growing pain in many regions worldwide. In theoretical research and simulations, carbon nanotubes have shown a potential in water treatment, desalination and energy conversion with high ion selectivity and enhanced water transport. In this research, we present an approach to investigate fluid transport across each single carbon nanotube channel by visualizing the ion and water flux under a Total Internal Reflection Fluorescence Microscopy (TIRF). Using a droplet interface bilayer (DIB) technique, we place an aqueous droplet (∼ 200 nanoliters) on an oil-agarose hydrogel interface, which brings together the lipid monolayers on the droplet and the interface to form a bilayer that mimics a cell membrane. We then incorporate the carbon nanotube channels in this bilayer. The ion and mass fluxes in each channel were characterized through the optical image of ion-sensitive fluorescence, which made it available to trace the flux through a large number of nanoscale channels individually instead of averaging them. The evanescent wave from TIRF also made it possible by eliminating the noise in the background and confining the illuminated region. We envision that our method will allow probing of fundamental transport properties through biomimetic carbon nanotube porins.

Ion Transport Through Large-Diameter DNA Origami Nanotube Channels Across Synthetic Membranes

Transport through nanoscale channels is a fundamental mechanism of exchange in living systems. The ability to direct such transport using membrane-based synthetic devices could make it possible to systematically study or control these transport processes. Techniques from structural DNA nanotechnology have recently made it possible to emulate the structural and functional aspects of naturally occurring membrane channels using easy-to-design DNA nanostructures. We have designed a system for controlled transport that begins with ∼10 nm-diameter DNA origami structures that forms pores in synthetic membranes. These DNA origami structures also serves as nucleation sites for DNA tile nanotubes that can grow to many microns in length, making it possible to study nanofluidic transport within extended self-assembled biomolecular channels. The DNA origami pores spontaneously insert into membranes formed using the droplet interface bilayer technique. Single-channel electrophysiological measurements indicate that these pores have ohmic conductances in the range of 1.0-1.2 nS. DNA nanotubes growing from the origami pore extend channel length without changing channel architecture produced channels with larger conductances. An artificial DNA origami shutter that when bound to the DNA origami pore closes off the internal diameter halves the channel's conductance. Our work thus demonstrates that DNA nanostructures several microns in diameter form channels in membranes and further how the transport ions across DNA origami nanostructures can be modulated by a DNA origami structure that serves as a shutter complex.

Photopolymerized microdomains in both lipid leaflets establish diffusive transport pathways across biomimetic membranes.

Controlled transport within a network of aqueous subcompartments provides a foundation for the construction of biologically-inspired materials. These materials are commonly assembled using the droplet interface bilayer (DIB) technique, adhering droplets together into a network of lipid membranes. DIB structures may be functionalized to generate conductive pathways by enhancing the permeability of pre-selected membranes, a strategy inspired by nature. Traditionally these pathways are generated by dissolving pore-forming toxins (PFTs) in the aqueous phase. A downside of this approach when working with larger DIB networks is that transport is enabled in all membranes bordering the droplets containing the PFT, instead of occurring exclusively between selected droplets. To rectify this limitation, photopolymerizable phospholipids (23:2 DiynePC) are incorporated within the aqueous phase of the DIB platform, forming conductive pathways in the lipid membranes post-exposure to UV-C light. Notably these pathways are only formed in the membrane if both adhered droplets contain the photo-responsive lipids. Patterned DIB networks can then be generated by controlling the lipid composition within select droplets which creates conductive routes one droplet thick. We propose that the incorporation of photo-polymerizable phospholipids within the aqueous phase of DIB networks will improve the resolution of the patterned conductive pathways and reduce diffusive loss within the synthetic biological network.

Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue.

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of glass-supported planar bilayers and in vitro-derived T-cell clones or lines1,2,3,4. How these findings apply to the primary human T cells isolated from blood or lymphoid tissues is not known, partly due to significant difficulties in obtaining a sufficient number of cells for analysis5. Here we address this through the development of a technique exploiting multichannel flow slides to build planar lipid bilayers containing activating and adhesion molecules. The low height of the flow slides promotes rapid cell sedimentation in order to synchronize cell:bilayer attachment, thereby allowing researchers to study the dynamic of the synaptic interface formation and the kinetics of the granules release. We apply this approach to analyze the synaptic interface of as few as 104 to 105 primary cryopreserved T cells isolated from lymph nodes (LN) and peripheral blood (PB). The results reveal that the novel planar lipid bilayer technique enables the study of the biophysical properties of primary human T cells derived from blood and tissues in the context of health and disease.

Lipid Composition and Endosomal pH Regulates Anthrax Lethal Toxin Uptake and PA63 Channel Behavior

The tight regulation of cellular pH is critical for the structure and function of cells and organelles. Besides, cells adjust bilayer biophysical parameters by controlling lipid composition and gradient across their plasma and intracellular organelle membranes. Both pH and membrane lipid composition change dramatically along the endocytic pathway, which is the internalization transport process that many bacterial toxins utilize to invade cells. In this study, we investigate the role of the lipid and pH environment in the uptake of the anthrax lethal toxin (composed of protective antigen (PA) and lethal factor (LF)). The anthrax toxin follows a multistep internalization process, which involves PA binding to its cellular receptors, proteolytic cleavage to PA63, oligomerization to form prepores, and LF binding followed by endocytosis. The acidic endosomal environment causes conformational changes in PA63 leading to its insertion into the early endosomal membrane and ion channel formation. The PA63-mediated delivery of the enzymatic LF to cytoplasm was suggested to take place later in the endocytic pathway via the back-fusion of intraluminal vesicles. Given the variations in lipid composition of the early endosome, the intraluminal vesicles, and the late endosomal limiting membrane, it is important to understand how these changes influence the anthrax toxin uptake. Using the planar lipid bilayer technique, we reconstituted single PA63 channels into neutral DOPC, DPhPC and DOPE and negatively-charged DOBMP, DPhPS membranes and varied the solution pH from 7.5 to 4.5 to mimic the changing physical environments of the endosomal pathway. The observed differences in the PA63 single-channel conductance, selectivity, and gating, and in LF binding and internalization were significant. These findings suggest that the anthrax toxin complex represents a robust universal model to study protein/lipid and protein/protein interactions in lipid membranes of different composition.

Membrane Transport Processes Analyzed by a Highly Parallel Nanopore Chip System at Single Protein Resolution.

Membrane protein transport on the single protein level still evades detailed analysis, if the substrate translocated is non-electrogenic. Considerable efforts have been made in this field, but techniques enabling automated high-throughput transport analysis in combination with solvent-free lipid bilayer techniques required for the analysis of membrane transporters are rare. This class of transporters however is crucial in cell homeostasis and therefore a key target in drug development and methodologies to gain new insights desperately needed. The here presented manuscript describes the establishment and handling of a novel biochip for the analysis of membrane protein mediated transport processes at single transporter resolution. The biochip is composed of microcavities enclosed by nanopores that is highly parallel in its design and can be produced in industrial grade and quantity. Protein-harboring liposomes can directly be applied to the chip surface forming self-assembled pore-spanning lipid bilayers using SSM-techniques (solid supported lipid membranes). Pore-spanning parts of the membrane are freestanding, providing the interface for substrate translocation into or out of the cavity space, which can be followed by multi-spectral fluorescent readout in real-time. The establishment of standard operating procedures (SOPs) allows the straightforward establishment of protein-harboring lipid bilayers on the chip surface of virtually every membrane protein that can be reconstituted functionally. The sole prerequisite is the establishment of a fluorescent read-out system for non-electrogenic transport substrates. High-content screening applications are accomplishable by the use of automated inverted fluorescent microscopes recording multiple chips in parallel. Large data sets can be analyzed using the freely available custom-designed analysis software. Three-color multi spectral fluorescent read-out furthermore allows for unbiased data discrimination into different event classes, eliminating false positive results. The chip technology is currently based on SiO2 surfaces, but further functionalization using gold-coated chip surfaces is also possible.

H2S detection using low-cost SnO2 nano-particle Bi-layer OFETs

In this article, a unique platform with an organic field-effect transistor (OFET) integrated with metal oxide nanoparticles for sensing of H2S gas is presented. Metal oxide nanoparticles such as SnO2 and ZnO synthesized using herbal techniques were used in the fabrication of OFETs using a bi-layer technique. The as-synthesized nanoparticles were characterized by Field Effect Scanning Electron Microscopy (FE-SEM), X-ray diffraction (XRD) and UV–vis Spectroscopy (UV–vis) to establish the material properties. We showed that the SnO2 based OFETs displayed better response for H2S at room temperature (25°C) compared to the OFETs fabricated with ZnO. The characterization of the sensors by using extracted electrical parameters like field-effect mobility (μ), On-Current (Ion), threshold voltage (VT) and saturation current (IDsat) establish the fact that the SnO2 based OFETs detect H2S gas at room temperature. Plausible mechanisms explaining the H2S gas detection by bi-layer film were discussed. On the other hand, the sensitivity of these OFETs against other reducing gases was less.

Çok Tabakalı Tablet Teknolojisi, Üretim Yöntemi, Değerlendirilmesi ve Kullanıldığı Çalışmalar

According to new developed technologies, drug industry is also triying to refresh and improve itself. With the conception of rational pharmacotherapy, new technologies becomes smarter than finding new active substances. Correspondingly as a new dosage system, multi-layer tablets that improved to serve this purpose, provides ease of use to patients, reduces the side effects and increases of effectiveness of active agent. Therefore multi-layer tablets are developed to improve both the quality of product and life. Conducted work studied for understanding the bi-layer tablet technique, evaluating the assesment parameters and to giving examples to used spaces.

Bilayer technique and nano-filled coating increase success of approximal ART restorations: a randomized clinical trial.

The high-viscosity consistency of glass ionomer cement (GIC) may lead to its incorrect adaptation into the cavity and therefore to restoration failure. To compare two different insertion techniques for GIC in approximal atraumatic restorative treatment (ART) restorations and two different surface protection materials. Approximal caries lesion in primary molars from 208 schoolchildren was randomly assigned into four groups: G1, conventional GIC insertion protected with petroleum jelly (PJ); G2, bilayer technique protected with PJ; G3 conventional GIC insertion protected with nano-filled particles coating for GIC (NPC); G4, bilayer technique protected with NPC. Restorations were evaluated after 1, 6, 12, 18, 24, and 36 months. Kaplan-Meier survival analysis and log-rank test were performed. Cox regression analysis (α = 5%) was used to verify the influence of clinical factors. Restoration survival was 52.8%. Log-rank test indicated a better survival of the bilayer technique restorations, compared to conventional restorations (P = 0.005), whereas the coated conventional restorations presented higher survival than the uncoated ones (P = 0.035). Cox regression analysis showed no influence of any clinical tested variables. The survival rate of the approximal ART restorations is positively influenced by the bilayer technique, and the application of nano-filled coating increases the longevity of the conventional approximal ART restorations.

Applications of nanoimprint lithography/hot embossing: a review

This review concentrates on the applications of nanoimprint lithography (NIL) and hot embossing for the fabrications of nanolectronic devices, nanophotonic metamaterials and other nanostructures. Technical challenges and solutions in NIL such as nanofabrication of templates, removal of residual resist, pattern displacement in thermal NIL arising from thermal expansion are first discussed. In the nanofabrication of templates, dry etch in plasma for the formation of multi-step structures and ultra-sharp tip arrays in silicon, nanophotonic chiral structures with high aspect ratio in SiC are demonstrated. A bilayer technique for nondestructive removal of residual resist in thermal NIL is described. This process is successfully applied for the fabrication of T-shape gates and functional high electron mobility transistors. However, pattern displacement intrinsically existing in thermal NIL/hot embossing owing to different thermal expansions in the template and substrate, respectively, limits its further development and scale-up. Low temperature even room temperature NIL (RTNIL) was then proposed on HSQ, trying to eliminate the pattern distortion by avoiding a thermal loop in the imprint. But, considerable pressure needed in RTNIL turned the major attentions to the development of UV-curing NIL in UV-curable monomers at low temperature. A big variety of applications by low-temperature UV-curing NIL in SU-8 are described, including high-aspect-ratio phase gratings, tagging technology by nanobarcode for DNA sequencing, nanofluidic channels, nanophotonic metamaterials and biosensors. Hot embossing, as a parallel technique to NIL, was also developed, and its applications on ferroelectric polymers as well as metals are reviewed. Therefore, it is necessary to emphasize that this review is mainly attempted to review the applications of NIL/embossing instead of NIL technique advances.

Pore forming activity of the potent RTX-toxin produced by pediatric pathogen Kingella kingae: Characterization and comparison to other RTX-family members

Pediatric septic arthritis in patients under age of four is frequently caused by the oral Gram-negative bacterium Kingella kingae. This organism may be responsible for a severe form of infective endocarditis in otherwise healthy children and adults. A major virulence factor of K. kingae is RtxA, a toxin that belongs to the RTX (Repeats-in-ToXin) group of secreted pore forming toxins. To understand the RtxA effects on host cell membranes, the toxin activity was studied using planar lipid bilayers. K. kingae strain PYKK081 and its isogenic RtxA-deficient strain, KKNB100, were tested for their ability to form pores in artificial membranes of asolectin/n-decane. RtxA, purified from PYKK081, was able to rapidly form pores with an apparent diameter of 1.9nm as measured by the partition of nonelectrolytes in the pores. The RtxA channels are cation-selective and showed strong voltage-dependent gating. In contrast to supernatants of PYKK081, those of KKNB100 did not show any pore forming activity. We concluded that RtxA toxin is the only secreted protein from K. kingae forming large channels in host cell membranes where it induces cation flux leading to programmed cell death. Furthermore, our findings suggested that the planar lipid bilayer technique can effectively be used to test possible inhibitors of RTX toxin activity and to investigate the mechanism of the toxin binding to the membrane.

Pseudo painting/air bubble technique for planar lipid bilayers.

A functional reconstitution of channel proteins in planar lipid bilayers is still very versatile to study structure/function correlates under well-defined conditions at the single protein level. In this study we present an improved planar lipid bilayer technique in which an air bubble is used for stabilizing unstable/leaky bilayers or for removing excess lipids. The bubble can also be used as a tool for reducing the number of channels in the bilayer with the goal of having only one active channel in the membrane. Stable planar lipid bilayers are formed within seconds to minutes. In the case of multiple channel insertion the air bubble can be used to reduce the number of channels within minutes. The simple improvement of the classical folding technique guarantees a very fast creation of stable bilayers even with difficult phospholipids in a conventional vertical bilayer set-up; it requires no modifications of the existing set-up. This technique is very easy to handle and guarantees successful single channel recordings for any kind of planar lipid bilayer experiment.

Wicking: A Rapid Method for Manually Inserting Ion Channels into Planar Lipid Bilayers

The planar lipid bilayer technique has a distinguished history in electrophysiology but is arguably the most technically difficult and time-consuming method in the field. Behind this is a lack of experimental consistency between laboratories, the challenges associated with painting unilamellar bilayers, and the reconstitution of ion channels into them. While there has be a trend towards automation of this technique, there remain many instances where manual bilayer formation and subsequent membrane protein insertion is both required and advantageous. We have developed a comprehensive method, which we have termed “wicking”, that greatly simplifies many experimental aspects of the lipid bilayer system. Wicking allows one to manually insert ion channels into planar lipid bilayers in a matter of seconds, without the use of a magnetic stir bar or the addition of other chemicals to monitor or promote the fusion of proteoliposomes. We used the wicking method in conjunction with a standard membrane capacitance test and a simple method of proteoliposome preparation that generates a heterogeneous mixture of vesicle sizes. To determine the robustness of this technique, we selected two ion channels that have been well characterized in the literature: CLIC1 and α-hemolysin. When reconstituted using the wicking technique, CLIC1 showed biophysical characteristics congruent with published reports from other groups; and α-hemolysin demonstrated Type A and B events when threading single stranded DNA through the pore. We conclude that the wicking method gives the investigator a high degree of control over many aspects of the lipid bilayer system, while greatly reducing the time required for channel reconstitution.

Application of Chitosan as a Barrier Coating on Coated Ivory Board

Chitosan solution was applied to coated ivory board as a barrier material, and the surface microstructure, oxygen resistance and water vapor permeability of chitosan-coated paper under different coating weight were studied. According to the images of scanning electron microscope(SEM) and Atomic force microscope(AFM), the coated ivory board surface has a smooth contour without pores and cracks after coating with chitosan. Increasing in coating hold-out of chitosan, the smoothness and the oxygen barrier properties of coated paper were improved considerably, but no improvement on water vapor resistance. An Oxygen transmission rate (OTR) of 119.0 cm3/m2•24h•0.1MPa was obtained when the coating weight of chitosan was 3.96 g/m2. Single-layer and double-layer techniques were used to coat coated ivory paper with chitosan; it was found that the OTR of paper, obtaining by double-layer coating technique, was lower than that of single-layer paper at similar coating weight. For the purpose of reducing water vapor transmission rate (WVTR) of chitosan-coated paper, Poly(vinyldene chloride)(PVDC) was applied on the chitosan-coated paper. Water vapor and oxygen barrier properties were enhanced as the coating weight of PVDC increased from 1.05 g/m2to 7.40 g/m2. While the chitosan and PVDC was coated on coated ivory paper through bi-layer technique for 1.96 g/m2and 7.40g/m2, respectively, the WVTR and OTR of paper decreased by 66.3% and 98.0% separately, compared to that of the chitosan-coated paper for 1.96g/m2.

Detection of lipid raft domains in neutral and anionic Langmuir monolayers and bilayers of complex lipid composition

In reference to the complexity and heterogeneity of cellular membranes, the structure and lateral ordering of lipid monolayers and bilayers composed of multi-component lipid mixtures have been investigated and compared in the present study. These complex model biomembrane systems represent valuable model systems, e.g. for studies of lipid–peptide interactions, where an integrated in situ multi-technique approach using both monolayer and bilayer techniques is required. A zwitterionic (3 components) and an anionic (5 components) heterogeneous model membrane system have been characterized that consist of saturated and unsaturated phospholipids as well as cholesterol. Lipid monolayers were analyzed by surface X-ray scattering techniques, and both the vertical structure (electron density profile) and the degree of in-plane ordering were determined as a function of surface pressure and temperature. The corresponding structure and lateral organisation of the bilayer membranes were characterized by atomic force microscopy. Both monolayers and bilayers reveal ordered domain formation, the monolayer ones being of much smaller size and different temperature stability, however. Furthermore, the charge density of the lipid monolayer has a drastic influence on the size of ordered domains as well as the intermolecular distances of the lipid molecules.

Property of large conductance Ca2+-activated K+ channels from Fasciola hepatica incorporated into planar lipid bilayer

Fasciola hepatica causes biliary epithelial hyperplasia and obstructive jaundice in humans and animals. Using a planar lipid bilayer technique, we further characterized the single channel property of large conductance K+-permeable channels that were previously identified from F. hepatica. The single channel conductance was 254.7±17.9pS under a symmetrical 200/200mM (cis/trans) KCl gradient. Open state probability (Po) varied from channel to channel at a given membrane potential and Ca2+ concentration, but increased with voltage (−60 to +40mV) and cis Ca2+ (1–200μM). Under a near bi-ionic condition of 200mM [K+]cis/200mM [Na+]trans, the permeability ratio of K+ to Na+ was 5.0. Charybdotoxin (1μM) inhibited Po, whereas tetraethylammonium reduced the conductance (KD=67.8mM). Taken together, the results show that the single channel properties of the large conductance K+-permeable channels in F. hepatica are similar to those of large conductance Ca2+-activated K+ (BK) channels in general, but distinct from typical BK channels in the extent of voltage- and Ca2+-dependence, as well as permeability to Na+. This study further reveals a variant BK channel in F. hepatica that could serve as a new drug target to treat fascioliasis.

Exploring protein import pores of cellular organelles at the single molecule level using the planar lipid bilayer technique

Proteins of living cells carry out their specialized functions within various subcellular membranes or aqueous spaces. Approximately half of all the proteins of a typical cell are transported into or across membranes. Targeting and transport to their correct subcellular destinations are essential steps in protein biosynthesis. In eukaryotic cells secretory proteins are transported into the endoplasmic reticulum before they are transported in vesicles to the plasma membrane. Virtually all proteins of the endosymbiotic organelles, chloroplasts and mitochondria, are synthesized on cytosolic ribosomes and posttranslationally imported. Genetic and biochemical techniques led to rather detailed knowledge on the subunit composition of the various protein transport complexes which carry out the membrane transport of the preproteins. Conclusive concepts on targeting and cytosolic transport of polypeptides emerged, while still few details on the molecular nature and mechanisms of the channel moieties of protein translocation complexes have been achieved. In this paper we will describe the history of how the individual subunits forming the channel pores of the chloroplast, mitochondrial and endoplasmic reticulum protein import machineries were identified and characterized by single channel electrophysiological techniques in planar bilayers. We will also highlight recent developments in the exploration of the molecular properties of protein translocating channels and the regulation of the diverse protein translocation systems using the planar bilayer technique.

A Cytotoxic Peptide from a Marine Sponge, Polytheonamide B; II. Properties for Ion Conduction and Voltage Dependent Gating

A 48-mer peptide from a marine sponge Theonella swinhoei, polytheonamide B (pTB), is highly cytotoxic against eukaryotic cells. Alternating D- and L-chirals throughout the molecule suggest formation of β-helix, which was supported by channel activity having high permeability to Cs+. The 48-mer β-helix is long enough to span the membrane and was shown to form the channel by single peptide. In this study we evaluated the ion conduction and gating properties of pTB channel by use of planar lipid bilayer technique. Single-channel I-V curve in CsCl solution exhibited weak outward rectification. Concentration-dependency of single-channel conductance of pTB channel was examined. For Cs+ permeation, clear saturation in unitary conductance was observed in the concentration range up to 3.0 M, which was contrast to gramicidin A (gA) channel. Gating manner of pTB channel was characteristic. Fast transition between open and closed state was observed, suggesting that the structural changes of single pTB molecule directly link to the gating. In addition to asymmetrical single-channel conductance, distinctive voltage-dependent gating was observed in single-channel and macroscopic current traces of pTB channels. On the basis of the structure of pTB channel, mechanism of ion conduction and voltage-dependent gating will be discussed.

Psalmotoxin-1 Docking to Human Acid-sensing Ion Channel-1

Acid-sensing ion channel-1 (ASIC-1) is a proton-gated ion channel implicated in nociception and neuronal death during ischemia. Recently the first crystal structure of a chicken ASIC was obtained. Expanding upon this work, homology models of the human ASICs were constructed and evaluated. Energy-minimized structures were tested for validity by in silico docking of the models to psalmotoxin-1, which potently inhibits ASIC-1 and not other members of the family. The data are consistent with prior radioligand binding and functional assays while also explaining the selectivity of PcTX-1 for homomeric hASIC-1a. Binding energy calculations suggest that the toxin and channel create a complex that is more stable than the channel alone. The binding is dominated by the coulombic contributions, which account for why the toxin-channel interaction is not observed at low pH. The computational data were experimentally verified with single channel and whole-cell electrophysiological studies. These validated models should allow for the rational design of specific and potent peptidomimetic compounds that may be useful for the treatment of pain or ischemic stroke.