Exogenous lysophospholipids with large head groups perturb clathrin‐mediated endocytosis

In this study, we have investigated how clathrin‐dependent endocytosis is affected by exogenously added lysophospholipids (LPLs). Addition of LPLs with large head groups strongly inhibits transferrin (Tf) endocytosis in various cell lines, while LPLs with small head groups do not. Electron and total internal reflection fluorescence microscopy (EM and TIRF) reveal that treatment with lysophosphatidylinositol (LPI) with the fatty acyl group C18:0 leads to reduced numbers of invaginated clathrin‐coated pits (CCPs) at the plasma membrane, fewer endocytic events per membrane area and increased lifetime of CCPs. Also, endocytosis of Tf becomes dependent on actin upon LPI treatment. Thus, our results demonstrate that one can regulate the kinetics and properties of clathrin‐dependent endocytosis by addition of LPLs in a head group size‐ and fatty acyl‐dependent manner. Furthermore, studies performed with optical tweezers show that less force is required to pull membrane tubules outwards from the plasma membrane when LPI is added to the cells. The results are in agreement with the notion that insertion of LPLs with large head groups creates a positive membrane curvature which might have a negative impact on events that require plasma membrane invagination, while it may facilitate membrane bending toward the cell exterior.      

Novel bioactive glycerol-based lysophospholipids: New data – New insight into their function

Based on the results of research conducted over last two decades, lysophospholipids (LPLs) were observed to be not only structural components of cellular membranes but also biologically active molecules influencing a broad variety of processes such as carcinogenesis, neurogenesis, immunity, vascular development or regulation of metabolic diseases. With a growing interest in the involvement of extracellular lysophospholipids in both normal physiology and pathology, it has become evident that those small molecules may have therapeutic potential. While lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) have been studied in detail, other LPLs such as lysophosphatidylglycerol (LPG), lysophosphatidylserine (LPS), lysophosphatidylinositol (LPI), lysophosphatidylethanolamine (LPE) or even lysophosphatidylcholine (LPC) have not been elucidated to such a high degree. Although information concerning the latter LPLs is sparse as compared to LPA and S1P, within the last couple of years much progress has been made. Recently published data suggest that these compounds may regulate fundamental cellular activities by modulating multiple molecular targets, e.g. by binding to specific receptors and/or altering the structure and fluidity of lipid rafts. Therefore, the present review is devoted to novel bioactive glycerol-based lysophospholipids and recent findings concerning their functions and possible signaling pathways regulating physiological and pathological processes.     

Regulation of transmembrane ion transport by reaction products of phospholipase A2. I. Effects of lysophospholipids on mitochondrial Ca2+ transport

Lysophospholipids inhibited mitochondrial Ca2+ uptake, induced a net Ca2+ efflux, and thereby increased the extramitochondrial Ca2+ concentration. The inhibitory potency decreased in the order lysophosphatidylcholine (LPC) = lysophosphatidylglycerol (LPG) greater than lysophosphatidylinositol (LPI) greater than lysophosphatidylserine (LPS) much greater than lysophosphatidylethanolamine (LPE). This relative order is in inverse relation to the ability of the various phospholipid head-groups to build up intermolecular hydrogen bonds with neighbouring membrane lipids. This indicates that changes in Ca2+ transport induced by lysophospholipids are mediated by the interaction of the lysophospholipids with the mitochondrial membrane bilayer structure. The mitochondrial membrane potential, which is the main driving force for mitochondrial Ca2+ uptake, was affected in the same order by the various lysophospholipids. This reduction of the mitochondrial membrane potential may be the underlying cause for the inhibition of the mitochondrial Ca2+ uniport and the resulting release of Ca2+ from the mitochondria.     

Capacitation of bovine spermatozoa by lysophospholipids and trypsin

Bovine spermatozoa were incubated in vitro with lysophosphatidylserine (LPS), lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), lysophosphatidylinositol (LPI), or trypsin. Capacitation of sperm was evaluated by penetration of the zonae pellucidae of dead bovine oocytes. Capacitation times could be shortened to 3 h or less by treatment of spermatozoa with each of these lysophospholipids (LPLs) (P < .05). The maximum oocyte penetration percentages for individual LPLs were 40% for 10 μM LPS, 24% for 160 μM LPC, 31% for 320 μM LPE, and 19% for 320 μM LPI. Capacitation also was facilitated (P < .01) by trypsin treatment of spermatozoa. Spermatozoa treated with 250 or 2,500 units/ml of trypsin penetrated more oocytes (17 and 18%) than spermatozoa treated with 0 or 25 units/ml of trypsin (0 and 3%). Spermatozoa treated with increasing concentrations of LPL showed a decrease in both the percentage of intact acrosomes and of progressively motile spermatozoa. Increasing levels of trypsin in the incubation medium also led to a decrease (P < .05) in the percentages of intact acrosomes and a decrease (P < .01) in the percentages of progressively motile spermatozoa. Percentages of live, ovulated oocytes fertilized by spermatozoa incubated for 1 h in LPS (86%, 6/7) were not different from those incubated for 24 h in control medium (71%, 5/7). Percentages of oocytes fertilized with both of these capacitation treatments were higher (P < .05) than for oocytes exposed or killed or uncapacitated sperm. Rapid induction of capacitation and the acrosome reaction can be accomplished by exogenous treatment of bovine sperm with lysophospholipids or trypsin.     

Relaxation spectra of gramicidin dimerization in a lipid bilayer membrane

The kinetics of formation and dissociation of gramicidin dimers in a lipid bilayer membrane have been studied by pressure-jump and electric field-jump methods. The traditional AC-coupled pressure-jump apparatus has been modified so that a known DC-voltage drop is maintained across a Teflon cell divided by a septum with a hole for membrane formation. From the response of the amplified output voltage after the pressure release, information about the kinetics of channel (dimer) formation is obtained. In addition, using the same apparatus, electric field-jump measurements were performed on the gramicidin/membrane system. In asolectin/7-dehydrocholesterol (5:1) membranes at 25 +/- 0.1 degrees C, the best fit to the pressure-jump data gives a dimer dissociation rate constant of 0.5 +/- 0.3 s-1. The standard volume change for dimerization determined from the amplitude of the pressure-jump experiments is -66 +/- 35 cm3/mol. Rate data determined by the electric field-jump method are consistent with the pressure-jump values; results obtained with either technique are compatible with other determinations of the kinetics of dimerization on gramicidin/membrane systems.     

Bioactive lysophospholipids and mesangial cell intracellular signaling pathways: role in the pathobiology of kidney disease

Lysophosphatidic acid (LPA), lyso-phosphatidylcholine (LPC), and sphingosine-1-phosphate (S1P) are major biologically active lysophospholipids (LPLs) that are produced by activated platelets, monocyte/macrophages, and many types of mammalian cells. LPLs have been shown to induce a wide array of physiological and pathophysiological properties including cellular differentiation, proliferation, migration, extracellular matrix deposition, change in morphology, and chemotactic responses. The recent cloning and identification of G protein-coupled receptors as specific receptors for LPLs created a great deal of interest in LPLs signaling and diverse biological responses. The pathobiological role of LPLs has been implicated in a number of pathological states and human diseases including atherosclerosis, glomerulosclerosis, post-ischemic renal failure, polycystic kidney disease, and ovarian cancer. Although the research in this area is growing at an enormous rate, this review is specifically focused on the recent understanding of the pathophysiological properties of LPA and LPC with special reference to kidney diseases, and their specific G-protein-coupled receptors and intracellular signaling pathways.     

The equivalence of fluctuation analysis and chemical relaxation measurements: a kinetic study of ion pore formation in thin lipid membranes

(1) Autocorrelation measurements were made of the current fluctuations due to ion-conducting channels produced by gramicidin A in black lipid membranes. (2) Relaxation experiments using a voltage jump were made on the same system. (3) Reciprocal time constants were determined by both methods over a 10⁸ fold range in membrane conductivity. starting from the single channel level. (4) The mean squared amplitudes of the fluctuations were determined from the autocorrelation functions. (5) The data were tentatively rationalized on the basis of a hypothetical dimerization reaction, assuming that gramicidin A dimers form conducting channels. The same forward rate constants are obtained, via (3), by both methods (1) and (2). The backward rate constant agrees excellently with direct measurements of the mean life time of a conducting channel. (6) The unit channel conductance and - assuming again a dimerization - the equilibrium constant can be obtained from the fluctuation amplitude distribution.     

Emerging lysophospholipid mediators,lysophosphatidylserine, lysophosphatidylthreonine,lysophosphatidylethanolamine and lysophosphatidylglycerol

It is now widely accepted that lysophospholipids (LPLs), a product of the phospholipase A reaction, function as mediators through G-protein-coupled receptors. Notably, recent studies of lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) have revealed their essential roles in vivo. On the other hand, other LPLs such as lysophosphatidylserine (LPS), lysophosphatidylthreonine (LPT), lysophosphatidylethanolamine (LPE), lysophosphatidylinositol (LPI) and lysophosphatidylglycerol (LPG) have been reported to show lipid mediator-like responses both in vivo (LPS and LPT) and in vitro (LPS, LPT, LPE and LPG), while very little is known about their receptor, synthetic enzyme and patho-physiological roles. In this review, we summarize the actions of these LPLs as lipid mediators including LPS, LPT, LPE and LPG.     

Calcium-dependent association of annexins with lipid bilayers modifies gramicidin A channel parameters

In order to examine whether calcium-dependent binding of annexin to acidic phospholipids could change the lipid bilayer environment sufficiently to perturb channel-mediated transmembrane ion-transport, gramicidin A channel activity in planar lipid bilayers was investigated in the presence of calcium and annexins II, III or V. The experiments were performed with membranes consisting of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine in 300 mM KCl solution buffered to pH 7.4 and with either 0.1 or 1 mM calcium added to the solution. Annexin (1 microM) was subsequently applied to the cis side of the membrane. All three annexins (II, III and V) when tested at 1 mM calcium decreased the gramicidin single-channel conductance. Annexins II and III increased the mean lifetime of the channels whereas annexin V seemed to have no influence on the mean lifetime. Since the lifetime of gramicidin A channels is a function of the rate constant for dissociation of the gramicidin dimer, which is dependent on the physical properties of the lipid phase, binding of annexins II and III seems to stabilize the gramicidin channel owing to a change of the bilayer structure.     

Gramicidin channels that have no tryptophan residues.

In order to understand how aromatic residues modulate the function of membrane-spanning proteins, we examined the role of the four tryptophans in gramicidin A (gA) in determining the average duration and permeability characteristics of membrane-spanning gramicidin channels; the tryptophan residues were replaced by tyrosine (gramicidin T, gT), tyrosine O-benzyl ether [gramicidin T(Bzl), gT(Bzl)], naphthylalanine (gramicidin N, gN), and phenylalanine (gramicidin M enantiomer, gM-). These analogues form channels with durations and conductances that differ some 10- and 16-fold, respectively. The single-channel conductance was invariably decreased by the Trp----Yyy replacement, and the relative conductance alterations were similar in phosphatidylcholine (DPhPC) and monoglyceride (GMO) bilayers. The duration variations exhibited a more complex pattern, which was quite different in the two membrane environments: in DPhPC bilayers, gN channels have an average duration that is approximately 2-fold longer than that of gA channels; in GMO bilayers, the average duration of gN channels is about one-tenth that of gA channels. The sequence-dependent alterations in channel function do not result from alterations in the channels' peptide backbone structure, because heterodimers can form between the different analogues and gramicidine A, and there is no energetic cost associated with heterodimer formation [cf. Durkin, J. T., Koeppe, R. E., II, & Andersen, O. S. (1990) J. Mol. Biol. 211, 221]. The alterations in permeability properties are consistent with the notion that Trp residues alter the energy profile for ion permeation through long-range electrostatic interactions.     

The effects of bilayer thickness and tension on gramicidin single-channel lifetime

Measurements have been made of gramicidin single-channel lifetimes in monoacylglycerol bilayers chosen so that their thickness ranged from above to below the length of the gramicidin channel. Contact angles, electrical capacities and bulk-phase interfacial tensions have also been determined for these systems. The mean channel lifetime decreased with the hydrocarbon thickness of the membrane until the latter reached 2.2 nm, after which the lifetime was relatively constant. A theoretical model has been proposed which relates the mean channel lifetime (or dissociation constant) to both the thickness and the tension of the bilayers. The analysis of the present results and of those of previous studies has led to the idea that aggregates of water molecules may play an important r?le in the dissociation of the gramicidin channel.     

Effect of the dipole potential of a bilayer lipid membrane on gramicidin channel dissociation kinetics.

A technique of measuring of the light-induced transients of the gramicidin-mediated electric current across a membrane in the presence of a photosensitizer has been applied for the study of the effect of agents modifying the dipole potential of a bilayer lipid membrane (phloretin, 6-ketocholestanol, and RH421) on the processes of the gramicidin channel dissociation and formation. It is shown that phloretin, known to lower the dipole potential, decelerates the flash-induced decrease in the current, whereas 6-ketocholestanol and RH421, known to raise the dipole potential, accelerate the current decrease. It is revealed that the addition of phloretin leads to a decrease in the dissociation rate constant, whereas addition of either 6-ketocholestanol or RH421 causes an increase in this constant. Single-channel data show that phloretin brings about an increase in the lifetime of the gramicidin channels, whereas RH421 produces a more complicated effect. It is conclude that the dipole potential affects the process of channel dissociation, presumably via the influence on the movement of the dipoles of gramicidin molecules through the layer of the dipole potential drop near the membrane-water interface.     

The structure of the gramicidin A transmembrane channel

Gramicidin A is a linear polypeptide antibiotic that facilitates the diffusion of monovalent cations across lipid bilayer membranes by forming channels. It has been proposed that the conducting channel is a dimer which is in equilibrium with nonconducting monomers in the membrane. To directly test this model in several independent ways, we have prepared and purified a series of gramicidin C derivatives. All of these derivatives are fully active analogs of gramicidin A, and each derivative has a useful chromophore esterified to the phenolic hydroxyl of tyrosine #11. Simultaneous conductance and fluorescence measurements on planar lipid bi-layer membranes containing dansyl gramicidin C yielded four conclusions: (1) A plot of the logarithm of the membrane conductance versus the logarithm of the membrane fluorescence had a slope of 2.0 ± 0.3, over a concentration range for which nearly all the gramicidin was monomeric. Hence, the active channel is a dimer of the nonconducting species. (2) In a membrane in which nearly all of the gramicidin was dimeric, the number of channels was approximately equal to the number of dimers. Thus, most dimers are active channels and so it should be feasible to carry out spectroscopic studies of the conformation of the transmembrane channel. (3) The association constant for dimerization is more than 1,000-fold larger in a glycerolester membrane with 26 Å-hydrocarbon thickness than in a 47 Å-glycerolester membrane. The dimerization constant in a 48 Å-phosphatidyl choline membrane was 200 times larger than in a 47 Å-glycerolester membrane, showing that it depends on the type of lipid as well as on the thickness of the hydrocarbon core. (4) We were readily able to detect 10^?14 mole cm^?2 of dansyl gramicidin C in a bilayer membrane, which corresponds to 60 fluorescent molecules per square μm. The fluorescent techniques described here should be sufficiently sensitive for fluorescence studies of reconstituted gates and receptors in planar bilayer membranes. An alternative method of determining the number of molecules of gramicidin in the channel is to measure the fraction of hybrid channels present in a mixture of 2 chemically different gramicidins. The single-channel conductance of p-phenylazo-benzene-sulfonyl ester gramicidin C (PABS gramicidin C) was found to be 0.68 that of gramicidin A. In membranes containing a mixture of these 2 gramicidins, a hybrid channel was evident in addition to 2 pure channels. The hybrid channel conductance was 0.82 that of gramicidin A. Fluorescence energy transfer from dansyl gramicidin C to diethylamino-phenylazobenzene-sulfonyl ester gramicidin C (DPBS gramicidin C), provided an independent way to measure the fraction of hybrid channels on liposomes. For both techniques the fraction of hybrid channels was found to be 2ad where a² and d² were the fractions of the 2 kinds of pure channels. This result strongly supports a dimer channel and the hybrid data excludes the possibility of a tetramer channel. The study of hybrid species by conductance and fluorescence techniques should be generally useful in elucidating the subunit structure of oligomeric assemblies in membranes. The various models which have been proposed for the conformation of the gramicidin transmembrane channel are briefly discussed.     

Genistein can modulate channel function by a phosphorylation-independent mechanism: importance of hydrophobic mismatch and bilayer mechanics

Genistein, a generic tyrosine kinase inhibitor, has been used extensively as a tool to investigate the possible regulation of membrane function by tyrosine phosphorylation. Genistein, in micromolar concentrations, alters the function of numerous ion channels and other membrane proteins, but only in few cases has it been demonstrated that the changes in membrane protein (ion channel) function are due to changes in a protein's phosphorylation status. The major common denominator characterizing proteins that are modulated by genistein seems to be that they are imbedded into, and span, the bilayer component of the plasma membrane. We therefore explored whether genistein could alter ion channel function by a bilayer-mediated mechanism and examined genistein's effect on gramicidin A (gA) channels in planar phospholipid bilayers. gA channels form by transmembrane dimerization of two nonconducting subunits, and genistein potentiates gA channel activity by increasing the appearance rate and prolonging the lifetime of bilayer-spanning gA dimers. That is, genistein shifts the equilibrium between nonconducting monomers and conducting dimers in favor of the bilayer-spanning dimers; the changes in channel activity therefore cannot be due to changes in bilayer fluidity. To obtain further insights into the mechanism underlying this modulation of gA channel function, we examined the effects of genistein on channels formed by gA analogues that differ in amino acid sequence. For a given channel length, the effects of genistein on gA dimerization do not depend on the specific sequence, or the chirality, of the channel-forming gA analogues. In contrast, when we change the channel length (by decreasing or increasing the number of amino acid residues in the sequence), or the bilayer thickness (by changing methylene groups in the acyl chains), the magnitude of genistein's effect increases with increasing hydrophobic mismatch between the channel length and the bilayer thickness. These results strongly suggest that genistein alters bilayer mechanical properties, which in turn modulates channel function. This bilayer-mediated mechanism is likely to apply to other pharmacological reagents and membrane proteins.     

Temperature-jump and voltage-jump experiments at planar lipid membranes support an aggregational (micellar) model of the gramicidin A ion channel

Summary The kinetics of formation and dissociation of channels formed by gramicidin A and two analogues in planar lipid membranes was studied using a laser temperature-jump technique developed earlier [Brock, W., Stark, G., Jordan, P.C. (1981),Biophys. Chem. 13:329–348]. The time course of the electric current was found to agree with a single exponential term plus a linear drift. In case of gramicidin A the relaxation time was identical to that reported for V-jump experiments [Bamberg, E., Läuger, P. (1973),J. Membrane Biol. 11:177–194], which were interpreted on the basis of a dimerization reaction. The same results were obtained for gramicidin A and for chemically dimerized malonyl-bis-desformylgramicidin. It is therefore suggested that the dimerization represents a parallel association of two dimers to a tetramer. There is evidence that the tetramer, contrary to the presently favored dimer hypothesis, is the smallest conductance unit of an active gramicidin channel. An additional V-jump-induced relaxation process of considerably larger time constant is interpreted as a further aggregation of gramicidin dimers.Abbreviations GA gramicidin A - OPG O-pyromellitylgramicidin A - MBDG malonyl-bis-desformylgramicidin     

Effects of green tea catechins on gramicidin channel function and inferred changes in bilayer properties

Green tea's health benefits have been attributed to its major polyphenols, the catechins: (-)-epigallocatechin gallate (EGCG), (-)-epicatechin gallate (ECG), (-)-epigallocatechin (EGC), and epicatechin (EC). Catechins (especially EGCG) modulate a wide range of biologically important molecules, including many membrane proteins. Yet, little is known about their mechanism(s) of action. We tested the catechins' bilayer-modifying potency using gramicidin A (gA) channels as molecular force probes. All the catechins alter gA channel function and modify bilayer properties, with a 500-fold range in potency (EGCG>ECG?EGC>EC). Additionally, the gallate group causes current block, as evident by brief downward current transitions (flickers).     

Single Molecule Methods for Monitoring Changes in Bilayer Elastic Properties

Membrane protein function is regulated by the cell membrane lipid composition. This regulation is due to a combination of specific lipid-protein interactions and more general lipid bilayer-protein interactions. These interactions are particularly important in pharmacological research, as many current pharmaceuticals on the market can alter the lipid bilayer material properties, which can lead to altered membrane protein function. The formation of gramicidin channels are dependent on conformational changes in gramicidin subunits which are in turn dependent on the properties of the lipid. Hence the gramicidin channel current is a reporter of altered properties of the bilayer due to certain compounds. Open in a separate windowClick here to view.^{(63M, flv)}     

Lysophospholipids and ATP mutually suppress maturation and release of IL-1 beta in mouse microglial cells using a Rho-dependent pathway

The P2X7 receptor (P2X7R), an ATP-gated ion channel, plays essential roles in the release and maturation of IL-1beta in microglial cells in the brain. Previously, we found that lysophosphatidylcholine (LPC) potentiated P2X7R-mediated intracellular signals in microglial cells. In this study, we determined whether the lysophospholipids, i.e., LPC and sphingosylphosphorylcholine (SPC), modulate the ATP-induced release and processing of IL-1beta mediated by P2X7R in mouse MG6 microglial cells. LPC or SPC alone induced the release of precursor (pro-IL-1beta) and mature IL-1beta (mIL-1beta) from LPS-primed MG6 cells, possibly due to lytic functions. However, these lysophospholipids inhibited ATP-induced caspase-1 activation that is usually followed by the release of mIL-1beta. Conversely, ATP inhibited the release of pro-IL-1beta and mIL-1beta induced by LPC/SPC. This suggests that lysophospholipids and ATP mutually suppressed each function to release IL-1beta. P2X7R activation resulted in microtubule reorganization in the MG6 cells that was blocked in the presence of LPC and SPC. LPC/SPC reduced the amount of activated RhoA after stimulation with ATP, implying that these lysophospholipids block ATP-induced microtubule reorganization by interfering with RhoA activation. In addition, the microtubule inhibitor colchicine inhibited ATP-induced release of mIL-1beta similar to that of LPC and SPC. This suggests that the impairment of the microtubule reassembly may be associated with the inhibitory effects of LPC/SPC on ATP-induced mIL-1beta release. Mutual suppression by ATP and LPC/SPC on the maturation of IL-1beta was observed in LPS-primed primary microglia. Collectively, these data suggest opposing functions by lysophospholipids, either proinflammatory or anti-inflammatory, in regard to the maturation and release of IL-1beta from microglial cells.     

Dimerization constant and single-channel conductance of Gramicidin in thylakoid membranes

Summary The effect of the pore-forming antibiotic gramicidin on pure lipid membranes is well characterized. We studied its action in protein-rich thylakoid membranes that contain less than 25% (wt/wt) acyl lipids. A transmembrane voltage was induced by flashing light, and its decay was measured and interpreted to yield the distribution of gramicidin over thylakoids, its dimerization constant and its single-channel conductance in this membrane. The distribution of gramicidin over the ensemble of thylakoids was immediately homogeneous when the antibiotic was added under stirring, while it became homogeneous only after 20 min in a stirred suspension that was initially heterogeneous. The dimerization constant, 5×10¹⁴ cm²/mol, was about 10 times larger than in pure lipid membranes. This was attributed to the upconcentration of gramicidin in the small fractional area of protein free lipid bilayer and further by a preference of gramicidin for stacked portions of the membrane. The latter bears important consequences with regard to bioenergetic studies with this ionophore. As gramicidin was largely dimerized from a concentration of 1 nm (in the suspension) on, the membrane's conductance then increased linearly as a function of added gramicidin. When the negative surface potential at the thylakoid membrane was screened, the conductance of a single gramicidin dimer agreed well with figures reported for bilayers from neutral lipid (about 0.5 pS at 10 mm NaCl). The modulation of the conductance by the surface potential in spinach versus pea thylakoids and between different preparations is discussed in detail.We would like to thank Ms. H. Kenneweg for photographs. financial support by the DFG (SFB 171/B3) is gratefully acknowledged.This paper is dedicated to the Late Prof. Peter Läger.     

Small-Molecule Photostabilizing Agents are Modifiers of Lipid Bilayer Properties

Small-molecule photostabilizing or protective agents (PAs) provide essential support for the stability demands on fluorescent dyes in single-molecule spectroscopy and fluorescence microscopy. These agents are employed also in studies of cell membranes and model systems mimicking lipid bilayer environments, but there is little information about their possible effects on membrane structure and physical properties. Given the impact of amphipathic small molecules on bilayer properties such as elasticity and intrinsic curvature, we investigated the effects of six commonly used PAs—cyclooctatetraene (COT), para-nitrobenzyl alcohol (NBA), Trolox (TX), 1,4-diazabicyclo[2.2.2]octane (DABCO), para-nitrobenzoic acid (pNBA), and n-propyl gallate (nPG)—on bilayer properties using a gramicidin A (gA)-based fluorescence quench assay to probe for PA-induced changes in the gramicidin monomer↔dimer equilibrium. The experiments were done using fluorophore-loaded large unilamellar vesicles that had been doped with gA, and changes in the gA monomer↔dimer equilibrium were assayed using a gA channel-permeable fluorescence quencher (Tl⁺). Changes in bilayer properties caused by, e.g., PA adsorption at the bilayer/solution interface that alter the equilibrium constant for gA channel formation, and thus the number of conducting gA channels in the large unilamellar vesicle membrane, will be detectable as changes in the rate of Tl⁺ influx—the fluorescence quench rate. Over the experimentally relevant millimolar concentration range, TX, NBA, and pNBA, caused comparable increases in gA channel activity. COT, also in the millimolar range, caused a slight decrease in gA channel activity. nPG increased channel activity at submillimolar concentrations. DABCO did not alter gA activity. Five of the six tested PAs thus alter lipid bilayer properties at experimentally relevant concentrations, which becomes important for the design and analysis of fluorescence studies in cells and model membrane systems. We therefore tested combinations of COT, NBA, and TX; the combinations altered the fluorescence quench rate less than would be predicted assuming their effects on bilayer properties were additive. The combination of equimolar concentrations of COT and NBA caused minimal changes in the fluorescence quench rate.