Nanoscale ion sequestration to determine the polarity selectivity of ion conductance in carriers and channels

© 2014 American Chemical Society. The nanoscale spacing between a tethered lipid bilayer membrane (tBLM) and its supporting gold electrode can be utilized to determine the polarity selectivity of the conduction of ion channels and ion carriers embedded in a membrane. The technique relies upon a bias voltage sequestering or eliminating ions, of a particular polarity, into or out of the aqueous electrolyte region between the gold electrode and the tethered membrane. A demonstration is given, using ac swept frequency impedance spectrometry, of the bias polarity dependence of the ionophore conductance of gramicidin A, a cationic selective channel, and valinomycin, a potassium ion selective carrier. We further use pulsed amperometry to show that the intrinsic voltage dependence of the ion conduction is actually selective of the polarity of the transported ion and not simply of the direction of the ionic current flow

The use of tethered bilayer lipid membranes to identify the mechanisms of antimicrobial peptide interactions with lipid bilayers

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This review identifies the ways in which tethered bilayer lipid membranes (tBLMs) can be used for the identification of the actions of antimicrobials against lipid bilayers. Much of the new research in this area has originated, or included researchers from, the southern hemisphere, Australia and New Zealand in particular. More and more, tBLMs are replacing liposome release assays, black lipid membranes and patch-clamp electrophysiological techniques because they use fewer reagents, are able to obtain results far more quickly and can provide a uniformity of responses with fewer artefacts. In this work, we describe how tBLM technology can and has been used to identify the actions of numerous antimicrobial agents

Structural Characterization of a Cation-Selective, Self-Assembled Peptide Pore in Planar Phospholipid Bilayers.

GALA is a 30-residue amphipathic peptide that self-assembles into multimeric transmembrane pores in a pH-dependent fashion. In this study, we characterize the size, multimeric structure, and cation selectivity of GALA pores in planar phospholipid bilayers using electrical impedance spectroscopy and molecular dynamics simulations. We demonstrate that in planar bilayers GALA pores are likely formed by six peptide monomers rather than eight to 12 monomers as previously reported for lipid vesicles. We further show that in planar bilayers, GALA pores exhibit previously unreported cation selectivity. We propose that the difference between the predicted pore structures in planar bilayers and lipid vesicles exemplifies the importance of phospholipid bilayer structural properties on the aggregation of transmembrane helical structures

New strategies to measure intracellular sodium concentrations

Fluorescent ion indicators are widely used to measure ion concentrations in living cells. However, despite considerable efforts in synthesizing new compounds, no ratiometric sodium indicator is available that can be excited at visible wavelengths. Ratiometric indicators have an advantage in that measured fluorescence intensities can be corrected for fluctuations of the indicator concentration and the illumination intensity, which is not possible when non-ratiometric indicators are used. One way to circumvent this problem is to measure fluorescence lifetimes, which are independent of these factors. Another way to overcome the disadvantages of a non-ratiometric indicator dye is to embed it, together with a reference dye, into nanoparticles. By relating the indicator fluorescence to the fluorescence of the reference dye, inhomogeneities in the nanosensor concentration or the illumination intensity can be cancelled out reliably. In this study we compare the benefits and drawbacks of these approaches. © 2010 Copyright SPIE - The International Society for Optical Engineering

Neurological effects in the offspring after switching from tobacco cigarettes to e-cigarettes during pregnancy in a mouse model.

BACKGROUND:Maternal smoking is currently a public health concern and has been associated with a number of complications in the offspring. E-cigarettes are gaining popularity as a 'safer' alternative to tobacco cigarettes during pregnancy, however, there are a limited number of studies to suggest that it is actually 'safe'. STUDY DESIGN:Balb/C female mice were exposed to ambient air (n = 8; Sham), or tobacco cigarette smoke (n = 8; SE) before gestation, during gestation and lactation. A third group was exposed to cigarette smoke before gestation followed by e-cigarette aerosols during gestation and lactation (n = 8; Switch). Male offspring (12-week old, n = 10-14/group) underwent behavioural assessments to investigate short-term memory, anxiety and activity using the novel object recognition (NOR) and elevated plus maze (EPM) tests. Brains were collected at postnatal day (P)1, P20 and Week13 for global DNA methylation, epigenetic gene expression, and neuronal cell counts. RESULTS:The offspring from mothers switching to e-cigarettes exhibited no change in exploration/activity, but showed a decrease in global DNA methylation, Aurora Kinase (Aurk) A and AurkB gene expression and a reduction in neuronal cell numbers in the cornu ammonis 1 region of the dorsal hippocampus compared to the SE group. CONCLUSIONS:Continuous tobacco cigarette smoke exposure during pregnancy resulted in marked neurological deficits in the offspring. Switching to e-cigarettes during pregnancy reduced these neurological deficits compared to cigarette smoke exposure. However, neurological changes were still observed, so we therefore conclude that e-cigarette use during pregnancy is not advised

Kalata B1 and Kalata B2 Have a Surfactant-Like Activity in Phosphatidylethanolomine-Containing Lipid Membranes

© 2017 American Chemical Society. Cyclotides are cyclic disulfide-rich peptides that are chemically and thermally stable and possess pharmaceutical and insecticidal properties. The activities reported for cyclotides correlate with their ability to target phosphatidylethanolamine (PE)-phospholipids and disrupt cell membranes. However, the mechanism by which this disruption occurs remains unclear. In the current study we examine the effect of the prototypic cyclotides, kalata B1 (kB1) and kalata B2 (kB2), on tethered lipid bilayer membranes (tBLMs) using swept frequency electrical impedance spectroscopy. We confirmed that kB1 and kB2 bind to bilayers only if they contain PE-phospholipids. We hypothesize that the increase in membrane conduction and capacitance observed upon addition of kB1 or kB2 is unlikely to result from ion channel like pores but is consistent with the formation of lipidic toroidal pores. This hypothesis is supported by the concentration dependence of effects of kB1 and kB2 being suggestive of a critical micelle concentration event rather than a progressive increase in conduction arising from increased channel insertion. Additionally, conduction behavior is readily reversible when the peptide is rinsed from the bilayer. Our results support a mechanism by which kB1 and kB2 bind to and disrupt PE-containing membranes by decreasing the overall membrane critical packing parameter, as would a surfactant, which then opens or increases the size of existing membrane defects. The cyclotides need not participate directly in the conductive pore but might exert their effect indirectly through altering membrane packing constraints and inducing purely lipidic conductive pores

Heterologously-expressed and Liposome-reconstituted Human Transient Receptor Potential Melastatin 4 Channel (TRPM4) is a Functional Tetramer

Mutation, irregular expression and sustained activation of the Transient Receptor Potential Channel, type Melastatin 4 (TRPM4), have been linked to various cardiovascular diseases. However, much remains unknown about the structure of this important ion channel. Here, we have purified a heterologously expressed TRPM4-eGFP fusion protein and investigated the oligomeric state of TRPM4-eGFP in detergent micelles using crosslinking, native gel electrophoresis, multi-angle laser light scattering and electron microscopy. Our data indicate that TRPM4 is tetrameric, like other TRP channels studied to date. Furthermore, the functionality of liposome reconstituted TRPM4-eGFP was examined using electrophysiology. Single-channel recordings from TRPM4-eGFP proteoliposomes showed inhibition of the channel using Flufenamic acid, a well-established inhibitor of TRPM4, suggesting that the channels are functional upon reconstitution. Our characterisation of the oligomeric structure of TRPM4 and the ability to reconstitute functional channels in liposomes should facilitate future studies into the structure, function and pharmacology of this therapeutically relevant channel

Cholic Acid-Based Antimicrobial Peptide Mimics as Antibacterial Agents

There is a significant and urgent need for the development of novel antibacterial agents to tackle the increasing incidence of antibiotic resistance. Cholic acid-based small molecular antimicrobial peptide mimics are reported as potential new leads to treat bacterial infection. Here, we describe the design, synthesis and biological evaluation of cholic acid-based small molecular antimicrobial peptide mimics. The synthesis of cholic acid analogues involves the attachment of a hydrophobic moiety at the carboxyl terminal of the cholic acid scaffold, followed by the installation of one to three amino acid residues on the hydroxyl groups present on the cholic acid scaffold. Structure–activity relationship studies suggest that the tryptophan moiety is important for high antibacterial activity. Moreover, a minimum of +2 charge is also important for antimicrobial activity. In particular, analogues containing lysine-like residues showed the highest antibacterial potency against Gram-positive S. aureus. All di-substituted analogues possess high antimicrobial activity against both Gram-positive S. aureus as well as Gram-negative E. coli and P. aeruginosa. Analogues 17c and 17d with a combination of these features were found to be the most potent in this study. These compounds were able to depolarise the bacterial membrane, suggesting that they are potential antimicrobial pore forming agents