ANALYSIS OF MULTICHANNEL SIGNALS USING A CHANNEL SIMULATOR

poster abstractsIn biological systems ion channels can be thought of as opening and clos-ing pores that allow the flow of ion through a membrane. Ion channels are responsible for intracellular communication and keeping an osmotic equilib-rium across cell membranes. Measurements of ion channel activities that generate multichannel events (the opening of more than one channel at a time) are important to understand, but difficult to analyze. In many cases the multichannel events are discarded instead of used in the analysis. The availability of a channel signal simulator offers an excellent opportunity to develop and test statistical models for analysis of multichannel signals. We have generated single channel traces for various open probabilities and then digitally superimposed these signals to obtain multichannel events. We then applied our simplified analysis to these multichannel traces to calculate sin-gle channel parameters such as the average ON and OFF times and their statistical distribution of the ON times. The average ON time as well as the ON time distribution matched the single channel input mean open time. The importance of our findings is that our proposed analysis will be able to use the statistical distribution of ON times, in addition to the traditional “dwell time” (or average ON time) parameter, to characterize ion channels

Functional Effects of Nanoparticle Exposure on Calu-3 Airway Epithelial Cells

High concentrations of manufactured carbon nanoparticles (CNP) are known to cause oxidative stress, inflammatory responses and granuloma formation in respiratory epithelia. To examine the effects of lower, more physiologically relevant concentrations, the human airway epithelial cell line, Calu-3, was used to evaluate potential alterations in transepithelial permeability and cellular function of airway epithelia after exposure to environmentally realistic concentrations of carbon nanoparticles. Three common carbon nanoparticles, fullerenes, single- and multi-wall carbon nanotubes (SWCNT, MWCNT) were used in these experiments. Electrophysiological measurements were performed to assay transepithelial electrical resistance (TEER) and epinephrine-stimulated chloride (Cl(-)) ion secretion of epithelial cell monolayers that had been exposed to nanoparticles for three different times (1 h, 24 h and 48 h) and over a 7 log unit range of concentrations. Fullerenes did not have any effect on the TEER or stimulated ion transport. However, the carbon nanotubes (CNT) significantly decreased TEER and inhibited epinephrine-stimulated Cl(-) secretion. The changes were time dependent and at more chronic exposures caused functional effects which were evident at concentrations substantially lower than have been previously examined. The functional changes manifested in response to physiologically relevant exposures would inhibit mucociliary clearance mechanisms and compromise the barrier function of airway epithelia

Fluorescence Measurements of Aromatic Amino Acids in the Presence of Lipid Membranes

Amphiphilic peptides are capable of finding their way to, and occasionally through, cellular membranes using a mechanism that includes specific amino acid sequences. Physical measurements of amino acid-lipid interactions are of interest for a quantitative description of peptide affinities to biological membranes. In this study, we investigate small peptide-lipid interactions using the fluorescence of the aromatic amino acids tyrosine (Tyr), tryptophan (Trp) and phenylalanine (Phe). Reference spectra in deuterated isopropanol solutions are obtained to mimic hydrophobic environments and are used to quantify the interaction of Lys-Tyr-Lys, Trp-Gly, and Gly-Phe with 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and palmitoyl-oleoyl phosphatidylserine (POPS) lipid membranes. These fluorescence data complement previously reported UV absorption data and have the advantage of eliminating background and scatter from solution. Together with NMR data, these results can be used to more fully characterize lipid-aromatic amino residue interactions

Reorganization of Ternary Lipid Mixtures of Non-Phosphorylated Phosphatidylinositol Interacting with Angiomotin

Phosphatidylinositol (PI) lipids are necessary for many cellular signaling pathways of membrane associated proteins, such as Angiomotin (Amot). The Amot family regulates cellular polarity, growth, and migration. Given the low concentration of PI lipids in these membranes, it is likely that such protein-membrane interactions are stabilized by lipid domains or small lipid clusters. By small-angle x-ray scattering, we show that non-phosphorylated PI lipids induce lipid de-mixing in ternary mixtures of phosphatidylcholine (PC) and phosphatidylethanolamine (PE), likely due to preferential interactions between the head groups of PE and PI. These results were obtained in the presence of buffer containing concentrations of Tris, HEPES, NaCl, EDTA, DTT, and Benzamidine at pH 8.0 that in previous work showed an ability to cause PC to phase separate but are necessary to stabilize Amot for in vitro experimentation. Collectively, this provided a framework for determining the effect of Amot on lipid organization. Using fluorescence spectroscopy, we were able to show that the association of Amot with this lipid platform causes significant reorganization of the lipid into a more homogenous organization. This reorganization mechanism could be the basis for Amot membrane association and fusigenic activity previously described in the literature and should be taken into consideration in future protein-membrane interaction studies

Conductance of Ideally Cation Selective Channel Depends on Anion Type

poster abstractGramicidin A (gA) is a transmembrane, cation selective ion channel that has been used in many biophysical studies of lipid bilayers, in particular for investigations of lipid-protein interactions and membrane electrostatics. In addition, it was found that ionic interactions with neutral lipid membranes also affect the kinetics of gA channels. Here we report measurements of gA ion-channels for a series of sodium and potassium salts that show an anion-dependence of gA conductance. We find that gA conductance varies significantly with the anion type with ClO4 and SCN producing distinctly larger conductance values than Cl, F, and H2PO4. These results can provide new insights into ion-lipid membrane interactions and ion channel functions in general

Phase Coexistence in Single-Lipid Membranes Induced by Buffering Agents

Recent literature has shown that buffers affect the interaction between lipid bilayers through a mechanism that involves van der Waals forces, electrostatics, hydration forces and membrane bending rigidity. This letter shows an additional peculiar effect of buffers on the mixed chain 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid bilayers, namely phase coexistence similar to what was reported by Rappolt et al. for alkali chlorides. The data presented suggest that one phase appears to dehydrate below the value in pure water, while the other phase swells as the concentration of buffer is increased. However, since the two phases must be in osmotic equilibrium with one another, this behavior challenges theoretical models of lipid interactions

Structural Changes of Alpha 1-Antitrypsin under Osmotic Pressure and in the Presence of Lipid Membranes

poster abstractAlpha 1-Antitrypsin (A1AT) is a glycoprotein that has been shown to have protective roles of lung cells against emphysema, a disease characterized by lung tissue destruction. Most known glycoproteins have been shown to play a role in cellular interactions but the exact role of the glycan chains is still under investigation. Previous electrophysiological measurements show that A1AT has a strong affinity to lipid bilayers perturbing the function of ion channels present in the membrane. We have performed contrastmatching small-angle neutron scattering (SANS) experiments to study the conformational changes of the glycosylated form of A1AT for different concentrations of the osmolyte poly(ethelene glycol) (PEG) and in the presence of two different lipid membranes: POPC and POPS. We also monitor the structural changes of the lipid vesicles in the presence of A1AT by SANS. Guinier fits were used as a first approximation to obtain the radius of gyration (Rg) of A1AT. Bragg peaks were used to study structural changes of lipid vesicles. We observed that the Rg of A1AT changes as a function of PEG concentration in solution and when in the presence of lipid vesicles. The deformations monitored through changes in A1AT’s Rg in the presence of lipid vesicles are compared to the deformations of the glycoprotein observed under osmotic pressure and to the structural changes observed in the lipid vesicles

Detecting Counterfeit Pharmaceuticals through UV Spectrophotometry

poster abstractAccording to the World Health Organization between 10%-30% of medicines, in Africa, Asia and South America, are counterfeit or sub-standard, affecting the health of millions of people. Currently, there is no effective way to check the quality of a medicine at the point of care, leaving many with treatable diseases at risk. The goal of this study is to identify UV-Vis (240nm - 500nm) absorbance patterns that would indicate if a drug is sub-standard or counterfeit. UV-Vis spectroscopy was selected as the method for testing due to the maturity and availability of the technology. Pure Acetaminophen and Tylenol were used as controls for proof of concept. Samples were prepared by dissolving different combinations of the pure active ingredient and adulterants such as cement, rice flour, vitamin C and lactose in three different types of solvents (H2O, 0.1 M HCl, 0.1 NaOH). Various concentrations (ranging from 0.01mg/ml to 0.04mg/ml) and mixing ratios were analyzed using a UV-Vis Spectrophotometer. It was found that adulterants significantly decrease the absorption of acetaminophen at 245nm by interacting with its benzene ring, while showing a slight increase in other parts of the spectrum. UV-Vis scans show that the amount of change in absorbance at specific wavelengths, coupled with characteristic wavelength shifts produced by different solvents, can be used for detection of counterfeit drugs. The methods presented here could be used for quality control of medicines at or near the point of care in parts of the world at higher risk of encountering defective pharmaceuticals