Liposome-based Assays Research Articles

The translocation of a chloride channel from the Golgi to the plasma membrane helps plants adapt to salt stress

A key mechanism employed by plants to adapt to salinity stress involves maintaining ion homeostasis via the actions of ion transporters. While the function of cation transporters in maintaining ion homeostasis in plants has been extensively studied, little is known about the roles of their anion counterparts in this process. Here, we describe a mechanism of salt adaptation in plants. We characterized the chloride channel (CLC) gene AtCLCf, whose expression is regulated by WRKY transcription factor under salt stress in Arabidopsis thaliana. Loss-of-function atclcf seedlings show increased sensitivity to salt, whereas AtCLCf overexpression confers enhanced resistance to salt stress. Salt stress induces the translocation of GFP-AtCLCf fusion protein to the plasma membrane (PM). Blocking AtCLCf translocation using the exocytosis inhibitor brefeldin-A or mutating the small GTPase gene AtRABA1b/BEX5 (RAS GENES FROM RAT BRAINA1b homolog) increases salt sensitivity in plants. Electrophysiology and liposome-based assays confirm the Cl−/H+ antiport function of AtCLCf. Therefore, we have uncovered a mechanism of plant adaptation to salt stress involving the NaCl-induced translocation of AtCLCf to the PM, thus facilitating Cl− removal at the roots, and increasing the plant’s salinity tolerance.

Bacterial cytological profiling identifies transmembrane anion transport as the mechanism of action for a urea-based antibiotic

ABSTRACT There is a growing fear that bacteria are emerging that are resistant to most known antibiotics, which could lead to an increasing number of fatalities due to bacterial infections. There is therefore an urgent need for the development of novel antibacterial products, preferably with novel mode of actions for which there are no known resistance pathways. We have previously found serendipitously that a simple urea-based compound functions as a potent antibacterial agent. In this manuscript, we use bacterial cytological profiling (BCP) to determine its mechanism of action. BCP reveals that the urea is acting through a membrane-based pathway that is comparable to, but distinct from, the mechanism of cationophore calcimycin. Additional liposome-based assays suggest that the likely mechanism of action of this mono-urea is transmembrane anion transport. This finding highlights that synthetic anionophores could be used as potential antibiotics.

The fluoride permeation pathway and anion recognition in Fluc family fluoride channels.

Fluc family fluoride channels protect microbes against ambient environmental fluoride by undermining the cytoplasmic accumulation of this toxic halide. These proteins are structurally idiosyncratic, and thus the permeation pathway and mechanism have no analogy in other known ion channels. Although fluoride-binding sites were identified in previous structural studies, it was not evident how these ions access aqueous solution, and the molecular determinants of anion recognition and selectivity have not been elucidated. Using x-ray crystallography, planar bilayer electrophysiology, and liposome-based assays, we identified additional binding sites along the permeation pathway. We used this information to develop an oriented system for planar lipid bilayer electrophysiology and observed anion block at one of these sites, revealing insights into the mechanism of anion recognition. We propose a permeation mechanism involving alternating occupancy of anion-binding sites that are fully assembled only as the substrate approaches.

Correlating ion channel structure and function.

Recent developments in cryogenic electron microscopy (cryo-EM) led to an exponential increase in high-resolution structures of membrane proteins, and in particular ion channels. However, structures alone can only provide limited information about the workings of these proteins. In order to understand ion channel function and regulation in molecular detail, the obtained structural data need to be correlated to functional states of the same protein. Here, we describe several techniques that can be employed to study ion channel structure and function in vitro and under defined, similar conditions. Lipid nanodiscs provide a native-like environment for membrane proteins and have become a valuable tool in membrane protein structural biology and biophysics. Combined with liposome-based flux assays for the kinetic analysis of ion channel activity as well as electrophysiological recordings, researchers now have access to an array of experimental techniques allowing for detailed structure-function correlations using purified components. Two examples are presented where we put emphasis on the lipid environment and time-resolved techniques together with mutations and protein engineering to interpret structural data obtained from single particle cryo-EM on cyclic nucleotide-gated or Ca2+-gated K+ channels. Furthermore, we provide short protocols for all the assays used in our work so that others can adapt these techniques to their experimental needs. Comprehensive structure-function correlations are essential in order to pharmacologically target channelopathies.

The role of intracellular anionic phospholipids in the production of N-acyl-phosphatidylethanolamines by cytosolic phospholipase A2ɛ.

N-Acyl-phosphatidylethanolamines (NAPEs) represent a class of glycerophospholipids and serve as the precursors of bioactive N-acylethanolamines, including arachidonoylethanolamide (anandamide), palmitoylethanolamide and oleoylethanolamide. NAPEs are produced in mammals by N-acyltransferases, the enzymes which transfer an acyl chain of glycerophospholipids to the amino group of phosphatidylethanolamine. Recently, the ɛ isoform of cytosolic phospholipase A2 (cPLA2ɛ, also called PLA2G4E) was identified as Ca2+-dependent N-acyltransferase. We showed that the activity is remarkably stimulated by phosphatidylserine (PS) in vitro. In the present study, we investigated whether or not endogenous PS regulates the function of cPLA2ɛ in living cells. When PS synthesis was suppressed by the knockdown of PS synthases in cPLA2ɛ-expressing cells, the cPLA2ɛ level and its N-acyltransferase activity were significantly reduced. Mutagenesis studies revealed that all of C2, lipase and polybasic domains of cPLA2ɛ were required for its proper localization as well as the enzyme activity. Liposome-based assays showed that several anionic glycerophospholipids, including PS, phosphatidic acid and phosphatidylinositol 4,5-bisphosphate, enhance the Ca2+-dependent binding of purified cPLA2ɛ to liposome membrane and stimulate its N-acyltransferase activity. Altogether, these results suggested that endogenous PS and other anionic phospholipids affect the localization and enzyme activity of cPLA2ɛ.

Structural and functional studies on a prokaryotic homologue of the mammalian SLC7 cationic amino acid transporters

The Amino acid/Polyamine/Cationic transporter superfamily is among the largest of the sec-ondary active transporters. APC family members use either a proton or sodium gradient to drive substrate translocation across the membrane. Members of the APC superfamily are responsible for the uptake of amino acids and are found in all kingdoms of life. Amino acids, and their derivatives, are crucial for both prokaryotic and eukaryotic cell biology, being the building blocks of a variety of macromolecules and play important roles in metabolism and cell signalling. Therefore, the systems responsible for their transport are important targets of further study, in particular to gain greater insight into how these proteins recognise and discriminate between different types of amino acids. The transport mechanism of several APC members has been elucidated using a variety of structural, biophysical and functional approaches. However, these studies have focussed on sodium coupled transporters due to their function as neurotransmitter transporters. Here, the crystal structure of the first pro-ton-coupled amino acid transporter of the APC superfamily, from Geobacillus kaustophilus, GkApcT, is presented. GkApcT closely related to the mammalian cationic amino acid trans-porters (CATs) of the SLC7 family, responsible for the transport arginine and lysine into the cell. The mechanism of transport was further investigated using liposome-based functional assays to characterise the substrate specificity and kinetics. The crystal structure was used to guide a detailed site-directed mutagenesis study, which identified several conserved res-idues involved in substrate binding and proton-coupling. The current structure serves as a template to further understand proton coupled amino acid transport in bacteria and to un-derstand mammalian amino acid transport via the SLC7 and SLC36 families.

Protein⁻Phospholipid Interaction Motifs: A Focus on Phosphatidic Acid.

Cellular membranes are composed of thousands of different lipids usually maintained within a narrow range of concentrations. In addition to their well-known structural and metabolic roles, signaling functions for many lipids have also emerged over the last two decades. The latter largely depend on the ability of particular classes of lipids to interact specifically with a great variety of proteins and to regulate their localization and activity. Among these lipids, phosphatidic acid (PA) plays a unique role in a large repertoire of cellular activities, most likely in relation to its unique biophysical properties. However, until recently, only incomplete information was available to model the interaction between PA and its protein partners. The development of new liposome-based assays as well as molecular dynamic simulation are now providing novel information. We will review the different factors that have shown to modulate the capacity of PA to interact with specific domains in target proteins.

Liposome Preparation for the Analysis of Lipid-Receptor Interaction and Efferocytosis.

Efficient phagocytosis of apoptotic cells (efferocytosis) is essential for immune homeostasis. Phospholipids exposed on the surface of apoptotic cells, such as phosphatidylserine, supply important "eat-me" signals. Liposomes are lipid bilayer vesicles that can be generated from one or several types of phospholipids of interest. Thus, these vesicles offer versatility, flexibility, and, importantly, a three-dimensional structure for studying the interaction between lipids and their receptors as well as the lipid-receptor interaction-mediated signaling events controlling efferocytosis by cells like professional phagocytes. Here, we describe methods to prepare liposomes, perform liposome-based lipid-receptor binding assays, use liposomes to block efferocytosis, and utilize liposome-coated beads as apoptotic cell surrogates for phagocytosis. © 2018 by John Wiley & Sons, Inc.

Ca2+ releases E-Syt1 autoinhibition to couple ER-plasma membrane tethering with lipid transport.

The extended synaptotagmins (E-Syts) are endoplasmic reticulum (ER) proteins that bind the plasma membrane (PM) via C2 domains and transport lipids between them via SMP domains. E-Syt1 tethers and transports lipids in a Ca2+-dependent manner, but the role of Ca2+ in this regulation is unclear. Of the five C2 domains of E-Syt1, only C2A and C2C contain Ca2+-binding sites. Using liposome-based assays, we show that Ca2+ binding to C2C promotes E-Syt1-mediated membrane tethering by releasing an inhibition that prevents C2E from interacting with PI(4,5)P2-rich membranes, as previously suggested by studies in semi-permeabilized cells. Importantly, Ca2+ binding to C2A enables lipid transport by releasing a charge-based autoinhibitory interaction between this domain and the SMP domain. Supporting these results, E-Syt1 constructs defective in Ca2+ binding in either C2A or C2C failed to rescue two defects in PM lipid homeostasis observed in E-Syts KO cells, delayed diacylglycerol clearance from the PM and impaired Ca2+-triggered phosphatidylserine scrambling. Thus, a main effect of Ca2+ on E-Syt1 is to reverse an autoinhibited state and to couple membrane tethering with lipid transport.

The Pore-Forming Protein Gasdermin D Regulates Interleukin-1 Secretion from Living Macrophages

The interleukin-1 (IL-1) family cytokines are cytosolic proteins that exhibit inflammatory activity upon release into the extracellular space. These factors are released following various cell death processes, with pyroptosis being a common mechanism. Recently, it was recognized that phagocytes can achieve a state of hyperactivation, which is defined by their ability to secrete IL-1 while retaining viability, yet it is unclear how IL-1 can be secreted from living cells. Herein, we report that the pyroptosis regulator gasdermin D (GSDMD) was necessary for IL-1β secretion from living macrophages that have been exposed to inflammasome activators, such as bacteria and their products or host-derived oxidized lipids. Cell- and liposome-based assays demonstrated that GSDMD pores were required for IL-1β transport across an intact lipid bilayer. These findings identify a non-pyroptotic function for GSDMD, and raise the possibility that GSDMD pores represent conduits for the secretion of cytosolic cytokines under conditions of cell hyperactivation.

A comprehensive lipid binding and activity validation of a cancer-specific peptide-peptoid hybrid PPS1

We recently identified a peptide-peptoid hybrid, PPS1, which recognizes lipids that have an overall negative charge, such as phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidic acid (PA), and phosphatidylinositol (PI), but that does not bind to neutral lipids, such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), and sphingomyelin (SM). The simple dimeric version of PPS1, PPS1D1, displayed strong cytotoxicity to cancer cells over normal cells in vitro and tumor burden in vivo. In this study, we comprehensively characterized the direct binding and activity of PPS1 on PS, PG, and PA using liposome-based assays and lung cancer cell lines that express these negatively charged lipids. First, the fluorescence polarization (FP) binding studies of fluoresceinated-PPS1 (PPS1-FITC) to PS-, PG-, and PA-containing PC-liposomes showed that the binding of PPS1 to PC-liposomes increased as concentrations of these lipids increased. In terms of activity, PPS1D1 induced the release of calcein from large, unilamellar PC-liposomes containing 15–30% PS, PG, and PA. PPS1D1 had no activity when the liposomes were composed of 100% PC. This effect was higher at 30% lipids than 15%, and the EC50 for PG and PA were higher than that of PS, indicating that PPS1D1 is more specific towards PS. PPS1D1 binds to and induces significant cytotoxicity in lung cancer cell lines H1693, HCC95, and H1395, which express negatively charged lipids, but had no effect on normal HBEC30KT cells, which has mostly PC in the outer layer. In addition, a series of previously developed PPS1D1 derivatives, which retain or lose activity, were tested with these liposome-based assays, and the data were equivalent to previous observations. This study provides comprehensive binding and activity validations of a unique peptide-peptoid hybrid, PPS1, on negatively charged lipids PS, PA, and PG that are elevated on cancer cell surfaces relative to normal human cell surfaces.

PS059 Prevalence of Essential Hypertension and Assessment of Cardiac Risk in Pakistani Adults – The Reveal Study

We recently identified a peptide-peptoid hybrid, PPS1, which recognizes lipids that have an overall negative charge, such as phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidic acid (PA), and phosphatidylinositol (PI), but that does not bind to neutral lipids, such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), and sphingomyelin (SM). The simple dimeric version of PPS1, PPS1D1, displayed strong cytotoxicity to cancer cells over normal cells in vitro and tumor burden in vivo. In this study, we comprehensively characterized the direct binding and activity of PPS1 on PS, PG, and PA using liposome-based assays and lung cancer cell lines that express these negatively charged lipids. First, the fluorescence polarization (FP) binding studies of fluoresceinated-PPS1 (PPS1-FITC) to PS-, PG-, and PA-containing PC-liposomes showed that the binding of PPS1 to PC-liposomes increased as concentrations of these lipids increased. In terms of activity, PPS1D1 induced the release of calcein from large, unilamellar PC-liposomes containing 15–30% PS, PG, and PA. PPS1D1 had no activity when the liposomes were composed of 100% PC. This effect was higher at 30% lipids than 15%, and the EC50 for PG and PA were higher than that of PS, indicating that PPS1D1 is more specific towards PS. PPS1D1 binds to and induces significant cytotoxicity in lung cancer cell lines H1693, HCC95, and H1395, which express negatively charged lipids, but had no effect on normal HBEC30KT cells, which has mostly PC in the outer layer. In addition, a series of previously developed PPS1D1 derivatives, which retain or lose activity, were tested with these liposome-based assays, and the data were equivalent to previous observations. This study provides comprehensive binding and activity validations of a unique peptide-peptoid hybrid, PPS1, on negatively charged lipids PS, PA, and PG that are elevated on cancer cell surfaces relative to normal human cell surfaces.

A Comparative Study on Techniques for the Determination of Permeabilization Events on Charged and Uncharged Lipid Bilayers

Host defense peptides (HDPs) are small effector molecules of the innate immune system building up the first defense barrier when it comes to infections. Infectious diseases are commonly treated with antibiotics leading to an increase in antibiotic resistances among clinically relevant pathogens. Therefore, understanding the mode of function of alternative agents with antibiotic function is undeniable for the successful development of new antibiotics opening new treatment options. To understand recognition-, recruiting- and permeabilization-processes on membrane surfaces a set of structurally diverse peptides is investigated regarding their membrane active function on charged and uncharged model membranes. Lipid bilayers are reconstituted from pure DOPC to mimic uncharged membranes and from POPE/POPG in a ratio of 4:1 to mimic the inner leaflet of the bactericidal cell envelope with an overall negative surface charge. Permeabilization events induced by the antimicrobial peptides LL32, Arenicin-1 and hBD3-l as well as the ionophore Nonactin and α-Hemolysin of Staphylococcus aureus are investigated with respect to the membrane systems stated above, buffer composition (high-salt vs. low-salt) and temperature conditions. A combination of a complex array of biophysical techniques is taken into account for studying permeabilization events with nearly no limitations - MONTAL-MUELLER-Setup for planar lipid bilayers, tethered membrane technology for supported bilayers, liposome-based assays (Calcein release assay, potassium iodide quenching, FRET-intercalation assay) and a chip-based pore spanning dye release assay. These methods allow to investigate the whole spectrum ranging from single pore characterization to the statistical analysis of either large membrane areas or a huge number of single membranes.

A light-regulated synthetic ion channel constructed by an azobenzene modified hydraphile

Biological ion channels are key molecules for cellular regulation and communication. To mimic the structure and functions of nature ion channels, a new class of light-regulated transmembrane ion channels was reported based on tri(macrocycle) hydraphile and azobenzene photoswitch (hydraphile 1). The liposome-based proton transport assays showed that hydraphile 1 exhibited excellent transmembrane activity (Y), and Ymax arrived 0.7 at 40 μmol/L. The successful isomerization of azobenzene moiety was confirmed and qualified by UV and NMR spectra. Upon alternative irradiation of 365 nm UV light and 450 nm visible light, the transmembrane activity of hydraphile 1 was regulated between 0.35 and 0.5, reversibly. All the obtained results have demonstrated the promise of developing excellent synthetic ion channels with ion gating properties based on simple molecular design.

Parts-per-Million of Polyethylene Glycol as a Non-Interfering Blocking Agent for Homogeneous Biosensor Development

Many homogeneous assays are complicated by the adsorption of probe molecules by the surface of reaction vessels, which are often made of polypropylene or polystyrene-based plastics. To solve this problem, many protein and surfactant-based blocking agents are used. However, these blockers may interfere with intended assays by sequestering transition-metal ions, inducing protein denaturing, generating air bubbles or making pores in membranes. Coating surfaces with polyethylene glycol (PEG) through covalent linkages has been proven to be an effective method to minimize protein adsorption. However, this method is more difficult to apply on plastic surfaces and is quite expensive. While unmodified PEG is often considered as a nonadsorbing polymer, in this Technical Note, we report that PEG at very low concentration (ppm level) can still effectively block plastic surfaces. This method works for DNA, protein, and liposome-based assays as long as the molecular weight of PEG is greater than 2000. PEG works because of multivalent hydrophobic interaction from its repeating methylene units. This Technical Note will not only facilitate biosensor development, but also enhance our understanding of the interaction between various molecules and plastic surfaces.

Dissecting quasi-equivalence in nonenveloped viruses: membrane disruption is promoted by lytic peptides released from subunit pentamers, not hexamers.

Nonenveloped viruses often invade membranes by exposing hydrophobic or amphipathic peptides generated by a proteolytic maturation step that leaves a lytic peptide noncovalently associated with the viral capsid. Since multiple copies of the same protein form many nonenveloped virus capsids, it is unclear if lytic peptides derived from subunits occupying different positions in a quasi-equivalent icosahedral capsid play different roles in host infection. We addressed this question with Nudaurelia capensis omega virus (NωV), an insect RNA virus with an icosahedral capsid formed by protein α, which undergoes autocleavage during maturation, producing the lytic γ peptide. NωV is a unique model because autocatalysis can be precisely initiated in vitro and is sufficiently slow to correlate lytic activity with γ peptide production. Using liposome-based assays, we observed that autocatalysis is essential for the potent membrane disruption caused by NωV. We observed that lytic activity is acquired rapidly during the maturation program, reaching 100% activity with less than 50% of the subunits cleaved. Previous time-resolved structural studies of partially mature NωV particles showed that, during this time frame, γ peptides derived from the pentamer subunits are produced and are organized in a vertical helical bundle that is projected toward the particle surface, while identical polypeptides in quasi-equivalent subunits are produced later or are in positions inappropriate for release. Our functional data provide experimental support for the hypothesis that pentamers containing a central helical bundle, observed in different nonenveloped virus families, are a specialized lytic motif.

Structural Determinants for Plant Annexin−Membrane Interactions

The interactions of two plant annexins, annexin 24(Ca32) from Capsicum annuum and annexin Gh1 from Gossypium hirsutum, with phospholipid membranes have been characterized using liposome-based assays and adsorption to monolayers. These two plant annexins show a preference for phosphatidylserine-containing membranes and display a membrane binding behavior with a half-maximum calcium concentration in the sub-millimolar range. Surprisingly, the two plant annexins also display calcium-independent membrane binding at levels of 10-20% at neutral pH. This binding is regulated by three conserved surface-exposed residues on the convex side of the proteins that play a pivotal role in membrane binding. Due to quantitative differences in the membrane binding behavior of N-terminally His-tagged and wild-type annexin 24(Ca32), we conclude that the N-terminal domain of plant annexins plays an important role, reminiscent of the findings in their mammalian counterparts. Experiments elucidating plant annexin-mediated membrane aggregation and fusion, as well as the effect of these proteins on membrane surface hydrophobicity, agree with findings from the membrane binding experiments. Results from electron microscopy reveal elongated rodlike assemblies of plant annexins in the membrane-bound state. It is possible that these structures consist of protein molecules directly interacting with the membrane surface and molecules that are membrane-associated but not in direct contact with the phospholipids. The rodlike structures would also agree with the complex data from intrinsic protein fluorescence. The tubular lipid extensions suggest a role in the membrane cytoskeleton scaffolding or exocytotic processes. Overall, this study demonstrates the importance of subtle changes in an otherwise conserved annexin fold where these two plant annexins possess distinct modalities compared to mammalian and other nonplant annexins.

Sorting nexin 5 is localized to a subdomain of the early endosomes and is recruited to the plasma membrane following EGF stimulation

Sorting nexins are a large family of proteins that contain the phosphoinositide-binding Phox homology (PX) domain. A number of sorting nexins are known to bind to PtdIns3P, which mediates their localization to membranes of the endocytic pathway. We show here that sorting nexin 5 (SNX5) can be recruited to two distinct membrane compartments. In non-stimulated cells, the PX domain was independently targeted to endosomal structures and colocalized with full-length SNX5. The membrane binding of the PX domain was inhibited by the PI 3-kinase inhibitor, wortmannin. Although SNX5 colocalized with a fluid-phase marker and was found predominantly within a PtdIns3P-rich endosomal domain, very little colocalization was observed between SNX5 and the PtdIns3P-binding protein, EEA1. Using liposome-based binding assays, we have shown that the PX domain of SNX5 interacts not only with PtdIns3P but also with PtdIns3,4P2. In response to EGF stimulation, either the SNX5-PX domain or full-length SNX5 was rapidly recruited to the plasma membrane. The localization of SNX1, which does not bind PtdIns3,4P2, was unaffected by EGF signalling. Therefore, SNX5 is localized to a subdomain of the early endosome distinct from EEA1 and, following EGF stimulation and elevation of PtdIns3,4P2, is also transiently recruited to the plasma membrane. These results indicate that SNX5 may have functions not only associated with endosomal sorting but also with the phosphoinositide-signalling pathway.

Elevated Function of Blood Clotting Factor VIIa Mutants That Have Enhanced Affinity for Membranes: BEHAVIOR IN A DIFFUSION-LIMITED REACTION

Blood clotting factor VIIa is involved in the first step of the blood coagulation cascade, as a membrane-associated enzyme in complex with tissue factor (TF). Factor VIIa is also an important therapeutic agent for hemophilia where its function may include TF-independent as well as TF-dependent mechanisms. This study compared the activity of wild type factor VIIa (WT-VIIa) with that of a mutant with elevated affinity for membrane (P10Q/Q32E, QE-VIIa). Phospholipid and cell-based assays showed the mutant to have up to 40-fold higher function than WT-VIIa in both TF-dependent and TF-independent reactions. Tissue factor-dependent reactions displayed the maximum enhancement when binding had reached equilibrium in competition with another TF-binding protein. In liposome-based assays, the association rate of WT-VIIa with TF occurred at a physical maximum and could not be improved by site-directed mutagenesis. A practical consequence was identical function of WT-VIIa and QE-VIIa in assays that depended entirely on assembly kinetics. Thus, factor VIIa mutants provided unique reagents for probing the mechanism of factor VIIa action. They may also offer superior agents for therapy.

Self-Assembling Cyclic β3-Peptide Nanotubes as Artificial Transmembrane Ion Channels

A new class of self-assembling transmembrane ion channels based on cyclic β3-peptides is described. Cyclic peptide subunits were designed to adopt flat, ring shaped conformations and stack through extensive backbone-backbone hydrogen bonding to form tubular channel structures. Candidate channel-forming peptides cyclo[(-β3-HTrp)4-] 1, cyclo[(-β3-HTrp-β-HLeu)2-] 2, and cyclo[(-β3-HLeu)4-] 3 were examined in liposome-based proton transport assays and single channel conductance experiments. Compounds 1 and 2 exhibited remarkable ion transport activities with single-channel K+ conductance of 56 pS for peptide 1, while compound 3 was inactive, possibly due to its poor solubility. Additionally, the putative structure of transmembrane channels formed by peptides 1 and 2 was supported by FT-IR spectroscopy of membrane-peptide preparations. The present system not only complements that of channel-forming cyclic d,l-α-peptides previously reported from this laboratory but also is expected to exhibit novel properties a...