Alter Lipid Bilayer Properties Research Articles

Tyrocidine potentiates the pore-forming ability of the linear gramicidins.

Antimicrobial peptides (AMPs) play an essential role in the innate immune system. They alter lipid bilayer properties, but they have intracellular effects as well. While microbes may become resistant to AMPs, the mechanisms are likely to differ from those developed against more conventional antibiotics. Given the pressing nature of increasing antibiotic resistance, we explored physicochemical mechanisms by which AMPs alter bilayer properties, with the aim to optimize future drug design, including minimizing the acquisition of resistance. To this end, we focused on the antibiotics produced by Brevibacillus brevis, a Gram-positive soil bacterium, a mixture of cyclic decapeptides (the tyrocidines), and linear pentadecapeptides (the gramicidins). The tyrocidines tend to be cytotoxic, but the linear gramicidins were the first antibiotics to be used and continue to be used in topical medications. The bactericidal activity of gramicidin results from the formation of cation-selective pores, which are sensitive to the physicochemical properties of the bilayer in which they are embedded. We therefore explored whether the tyrocidines might potentiate gramicidin channel formation through perturbation of membrane properties. Indeed, the tyrocidines enhance the channel-forming potency of gramicidin in both zwitterionic membranes formed by 1,2-Dierucoyl-sn-Glycero-3-Phosphocholine (DEPC) and negatively charged membranes formed by a 3:1 mixture of DEPC and 1,2-Dierucoyl-sn-Glycero-3-Phosphoglycerol (DEPG). Using dynamic light scattering (DLS) and zeta potential measurements to assess the LUV size distributions and net charge, we conclude that tyrocidine binds with high affinity to negatively charged DEPC:DEPG vesicles. Yet, the potentiation of gramicidin activity was greater in DEPC than in DEPC:DEPG membranes, likely due to an organizing effect of the cationic tyrocidines. Compared to the cyclic decapeptide gramicidin S, the tyrocidines behave similarly but are more potent.

Bilayer-modifying potency of antipsychotics as a function of pH.

Antipsychotic drugs (APDs) have complex pharmacological profiles with a range of putative membrane protein targets and off-target effects. We have previously shown that APDs, including chlorpromazine (CPZ) and trifluoperazine (TFP), alter lipid bilayer properties as detected by bilayer-spanning gramicidin channels, which raises the possibility that at least part of their non-specific effects may be mediated by the bilayer. Following Malheiros et al. (1998) and Ahyayaych et al. (2003), who showed that the bilayer affinity of a titratable drug depends on its charge, being higher for neutral molecules, we explored the role of charge on the bilayer-modifying potencies of CPZ and TFP at pHs between 4 and 10. (Short-term exposure to pH 10 does not cause measurable changes in bilayer properties.) Using dynamic and electrophoretic light scattering to monitor partitioning of the charged species, we found that increasing pH produced a decrease in the magnitude of the zeta potential for both CPZ and TFP with little effect on the particle size distribution. Using gramicidin channels as probes, we found that the bilayer-modifying potencies of the drugs varied with changes in pH: the potency of CPZ increased modestly with increasing pH, while the potency of TFP varied little at low concentrations (3 and 10 µM) but decreased as pH increased at 30 µM. From experiments changing the total lipid concentration in the system, we found that the apparent partition coefficient of CPZ increased from pH 4 to pH 10, whereas that for TFP decreased. We conclude that the bilayer-modifying potency of a titratable drug is different for the neutral and charged species, with the charged species being more bilayer-modifying per molecule in the membrane.

Antimicrobial peptides alter lipid bilayer properties before disrupting membrane barrier property.

Antimicrobial peptides (AMPs) have been the subject of inquiry since the initial search for antibiotics in the ­first half of 20th century; the first antibiotic used in humans was the linear pentadecapeptide gramicidin. Much of this interest comes from the modest frequency of bacterial resistance against AMPs. Though AMPs differ in sequence and structure, the majority share three core physicochemical properties; they are short, cationic and amphipathic. Moreover, enantiomers of AMPs tend to have similar potencies. These common features have led to a focus on the bacterial membrane as a target for AMPs. High concentrations of AMPs cause gross bilayer defects, through pore formation or micellar breakdown. At lower concentrations, AMPs have effects on bacterial physiology that appear unrelated to membrane permeabilization, suggesting that they may act through bilayer-mediated regulation (BMR) of membrane protein function. If correct, this offers a mechanism of action that integrates the known membrane-modifying effects of AMPs with their ability to broadly alter bacterial physiology at sub-pore forming concentrations. We tested this hypothesis using melittin, gramicidin S, and indolicidin, three AMPs with distinct structures, that indeed alter bilayer properties at concentrations below those required for pore formation. Contrary to expectation, AMP induced BMR is attenuated in anionic bilayers despite increased AMP binding, suggesting an organizing effect of membrane-bound counterions. We further found that amphiphilic bilayer modifiers promote membrane permeabilization, suggesting that BMR may be responsible for the cooperativity of AMP-induced permeabilization. Two membrane-impermeant detergents, CHAPS and zwittergent, promote pore formation despite inhibiting AMP binding, suggesting a role for leaflet-specific lateral pressure differences. These results suggest a new mechanism by which AMPs exert their bactericidal effect, which may guide antibiotic design and provide a path for circumventing bacterial resistance.

Bilayer-modifying potency of antipsychotics as a function of pH

Chlorpromazine (CPZ) and trifluoperazine (TFP) are amphipathic antipsychotic drugs with a range of putative membrane protein targets, as well as off-target effects that underlie their complex pharmacological profiles. We have shown that antipsychotics alter lipid bilayer properties, as reported by the bilayer-spanning gramicidin channels, which raises the possibility that CPZ and TFP may exert, at least part of their action by altering lipid bilayer properties. Here we explore the role of charge on CPZ’s and TFP’s bilayer-modifying potency following Malheiros et al.

Membranes Matter: Predicting Drug Toxicity

It remains a challenge to predict whether a new drug candidate will have undesirable side effects. Here we explore a general mechanism that may cause side effects, namely that many biologically active molecules, including drugs and drug-leads, are amphiphiles that partition into lipid bilayers. This may alter bilayer physical properties, which may cause undesirable changes in cell function that, if large enough, may cause toxicity. Thus, it may be possible to predict whether a compound will have important off-target effects based on the compound's bilayer-modifying potential. Using a gramicidin-based fluorescence assay (GBFA), which reports how a compound alters the gramicidin monomer <-> dimer equilibrium, we have shown that many drugs and drug-leads alter lipid bilayer properties at the concentrations where these compounds become indiscriminate modifiers of membrane protein function and are likely to have off target effects. We pursued this question in a blinded study on a library of 488 compounds (289 non-toxic, 199 toxic) that had been tested for cytotoxicity with “high-content” screening. We found that the GBFA can be used to predict cellular toxicity, with a false positive rate of 5%. We are currently investigating why these false positives arise. We hypothesize that their toxicity is due to direct protein interactions and are examining the impact of albumin on their bilayer perturbing effects. We also explore a computational approach to gain insight into which physicochemical parameters drive a compound's bilayer-perturbing propensity. We can predict which compounds will produce bilayer-modifying effects at either extreme (i.e. little membrane effect or significant membrane perturbation) with 90% accuracy. Our results support a mechanism by which amphiphiles exert their toxicity, namely by altering lipid bilayer physical properties and that this in vitro measurement could be used as a warning sign for off-target biological effects in drug discovery efforts.

Fluorinated Alcohols’ Effects on Lipid Bilayer Properties

Fluorinated alcohols (fluoroalcohols) have physicochemical properties that make them excellent solvents of peptides, proteins, and other compounds. Like other alcohols, fluoroalcohols also alter membrane protein function and lipid bilayer properties and stability. Thus, the questions arise: how potent are fluoroalcohols as lipid-bilayer-perturbing compounds, could small residual amounts that remain after adding compounds dissolved in fluoroalcohols alter lipid bilayer properties sufficiently to affect membranes and membrane protein function, and do they behave like other alcohols? To address these questions, we used a gramicidin-based fluorescence assay to determine the bilayer-modifying potency of selected fluoroalcohols: trifluoroethanol (TFE), HFIP, and perfluoro-tert-butanol (PFTB). These fluoroalcohols alter bilayer properties in the low (PFTB) to high (TFE) mM range. Using the same assay, we determined the bilayer partitioning of the alcohols. When referenced to the aqueous concentrations, the fluoroalcohols are more bilayer perturbing than their nonfluorinated counterparts, with the largest fluoroalcohol, PFTB, being the most potent and the smallest, TFE, the least. When referenced to the mole fractions in the membrane, however, the fluoroalcohols have equal or lesser bilayer-perturbing potency than their nonfluorinated counterparts, with TFE being more bilayer perturbing than PFTB. We compared the fluoroalcohols’ molecular level bilayer interactions using atomistic molecular dynamics simulations and showed how, at higher concentrations, they can cause bilayer breakdown using absorbance measurements and 31P nuclear magnetic resonance.

Membranes Matter: Predicting Drug Toxicity

It remains a challenge to predict whether a new drug candidate will have undesirable side effects. Many biologically active molecules, including drugs and drug-leads, are amphiphiles that partition into lipid bilayers, which may alter bilayer physical properties, thereby modulating membrane protein function. Such bilayer-modifying molecules may be promiscuous modifiers of membrane protein function, raising the possibility that they will have off-target effects. Thus, it may be possible to predict whether a compound will have important off-target effects based on quantitative studies of the compound's bilayer-modifying potential. We developed an assay to quantify the bilayer-modifying potential of large numbers of compounds. Using a gramicidin-based fluorescence assay (GBFA), which reports how a compound alters the conformational equilibrium of a membrane embedded reporter protein and thus indicates global changes in bilayer properties. We have shown that many drugs and drug-leads alter lipid bilayer properties at the concentrations where these compounds become indiscriminate modifiers of membrane protein function. We pursued this question in a blinded study on a library of 504 compounds (204 non-toxic, 300 toxic) that had been tested for cytotoxicity in “high-content” screening assays using the GBFA and found that the changes in fluorescence quench rate can be used to predict cellular toxicity, with a false positive rate of only 5%. These results support a mechanism by which amphiphiles exert their toxicity, namely by altering lipid bilayer physical properties and that such an in vitro measurement could be used as a warning sign for off-target biological effects in drug discovery efforts. We are currently employing machine-learning techniques to better understand which physicochemical parameters influence a small molecule's bilayer perturbing potency.

Divergent effects of anesthetics on lipid bilayer properties and sodium channel function.

General anesthetics revolutionized medicine by allowing surgeons to perform more complex and much longer procedures. This widely used class of drugs is essential to patient care, yet their exact molecular mechanism(s) are incompletely understood. One early hypothesis over a century ago proposed that nonspecific interactions of anesthetics with the lipid bilayer lead to changes in neuronal function via effects on membrane properties. This model was supported by the Meyer-Overton correlation between anesthetic potency and lipid solubility and despite more recent evidence for specific protein targets, in particular ion-channels, lipid bilayer-mediated effects of anesthetics is still under debate. We therefore tested a wide range of chemically diverse general anesthetics on lipid bilayer properties using a sensitive and functional gramicidin-based assay. None of the tested anesthetics altered lipid bilayer properties at clinically relevant concentrations. Some anesthetics did affect the bilayer, though only at high supratherapeutic concentrations, which are unlikely relevant for clinical anesthesia. These results suggest that anesthetics directly interact with membrane proteins without altering lipid bilayer properties at clinically relevant concentrations. Voltage-gated Na+ channels are potential anesthetic targets and various isoforms are inhibited by a wide range of volatile anesthetics. They inhibit channel function by reducing peak Na+ current and shifting steady-state inactivation toward more hyperpolarized potentials. Recent advances in crystallography of prokaryotic Na+ channels, which are sensitive to volatile anesthetics, together with molecular dynamics simulations and electrophysiological studies will help identify potential anesthetic interaction sites within the channel protein itself.

Clinical concentrations of chemically diverse general anesthetics minimally affect lipid bilayer properties

General anesthetics have revolutionized medicine by facilitating invasive procedures, and have thus become essential drugs. However, detailed understanding of their molecular mechanisms remains elusive. A mechanism proposed over a century ago involving unspecified interactions with the lipid bilayer known as the unitary lipid-based hypothesis of anesthetic action, has been challenged by evidence for direct anesthetic interactions with a range of proteins, including transmembrane ion channels. Anesthetic concentrations in the membrane are high (10-100 mM), however, and there is no experimental evidence ruling out a role for the lipid bilayer in their ion channel effects. A recent hypothesis proposes that anesthetic-induced changes in ion channel function result from changes in bilayer lateral pressure that arise from partitioning of anesthetics into the bilayer. We examined the effects of a broad range of chemically diverse general anesthetics and related nonanesthetics on lipid bilayer properties using an established fluorescence assay that senses drug-induced changes in lipid bilayer properties. None of the compounds tested altered bilayer properties sufficiently to produce meaningful changes in ion channel function at clinically relevant concentrations. Even supra-anesthetic concentrations caused minimal bilayer effects, although much higher (toxic) concentrations of certain anesthetic agents did alter lipid bilayer properties. We conclude that general anesthetics have minimal effects on bilayer properties at clinically relevant concentrations, indicating that anesthetic effects on ion channel function are not bilayer-mediated but rather involve direct protein interactions.

General Anesthetics Minimally affect Lipid Bilayer Properties at Clinical Concentrations

General anesthetics are widely used but their exact molecular mechanisms are incompletely understood. One early model, known as the unitary lipid-based hypothesis of anesthetic action, proposed that nonspecific anesthetic interactions with the lipid bilayer altered neuronal function through effects on membrane properties. This model, which is based largely on the Meyer-Overton correlation between anesthetic potency and lipid solubility, has been challenged by more recent evidence supporting direct interactions of anesthetics with a range of proteins, in particular transmembrane ion channels. The available results, however, do not exclude anesthetic modulation of ion channel function through interactions with the lipid bilayer. We therefore tested the effects of a range of chemically diverse general anesthetics (diethyl ether, desflurane, sevoflurane, isoflurane, halothane, chloroform, bromoform, cyclopropane, F3, ketamine, etomidate and propofol) and related nonanesthetics (F6 and flurothyl, predicted to be anesthetic based on their lipid solubilities) on lipid bilayer properties using a sensitive gramicidin-based assay. We show that none of the anesthetics or nonanesthetics tested significantly altered lipid bilayer properties at clinically relevant concentrations. Even at supra-anesthetic concentrations, most anesthetics produced only minimal bilayer-mediated effects, though some were able to alter lipid bilayer properties at supra-therapeutic (toxic) concentrations. We conclude that the effects of general anesthetics at clinically relevant concentrations involve minimal changes in lipid bilayer properties. Therefore clinical anesthesia is likely to result from direct interactions of anesthetics with membrane proteins.Supported by GM058055 (HCH) and GM021347 (OSA).

Regulation of KcsA by Bilayer-Modifying Molecules

The coupling between membrane proteins and their surrounding lipid bilayer make understanding the membrane lipid regulation of membrane proteins key to understanding their function. The membrane lipid regulation of membrane protein function ranges from specific lipid binding to more general lipid bilayer regulation. The latter mechanism arises from energetic coupling between the lipid bilayer and membrane protein where protein conformational change will alter the organization of the adjacent bilayer. To gain insight into, and tease apart, the specific and general mechanism(s) of membrane protein regulation by the membrane lipids, we examine how the prototypical bacterial potassium channel, KcsA, is regulated by bilayer modifying compounds and drugs using a fluorescence-based technique. The concentration ranges at which the compounds alter the lipid bilayer is determined using gramicidin channels as probes. Comparing the concentration ranges at which molecules alter KcsA function and lipid bilayer properties, enable us to determine whether the underlying mechanism primarily involves specific interactions with KcsA or is bilayer-mediated. Most compounds tested slowed activation and accelerated inactivation at concentrations below those where they alter bilayer properties, suggesting that KcsA likely has a non-specific site that allows hydrophobic molecules to bind and alter channel properties. This site may be the fenestrations proposed to be located on the cytosolic side of aqueous pore of potassium channels (and evident in the x-ray structure). The same compounds alter KcsA function at the concentrations where they alter lipid bilayer properties. These, bilayer-modifying, molecules slow recovery from inactivation at concentrations at which they alter bilayer properties. These results suggest that conformational changes responsible for recovery from inactivation are regulated by a bilayer-mediated mechanism. Our results point to a complicated relationship between direct and bilayer-mediated mechanisms, which both are rather non-specific and may affect a diverse range of ion channels.

Membranes Matter: Predicting Drug Toxicity

It remains a challenge to predict whether a new drug candidate will have undesirable side effects. Many biologically active molecules, including drugs and drug-leads, are amphiphiles that partition into lipid bilayers, which will tend to alter bilayer physical properties, thereby modulating membrane protein function. Such bilayer-modifying molecules may be promiscuous modifiers of membrane protein function, raising the possibility that they have off-target effects. Consequently, it may be possible to predict whether a compound will have off-target effects based on quantitative studies on the compound's bilayer-modifying potential. To address this question, we developed an assay to quantify the bilayer-modifying potential of large numbers of compounds using a gramicidin-based fluorescence assay (GBFA), which reports how a compound alters the gramicidin monomer ↔ dimer equilibrium. Using this assay, we have shown that many drug and drug-leads alter lipid bilayer properties at the concentrations where these compounds become indiscriminate modifiers of membrane protein function. Such indiscriminate modifiers of membrane protein function are likely to have off target effects; we pursued this question in a study on libraries of compounds that had been tested for toxicity in “high-content” screening assays that quantified cellular ATP levels, nuclear morphology, nuclear membrane integrity, and apoptosis in immortalized liver, lung, and neuronal cell lines. We tested 524 total compounds comprising four libraries (one non-toxic library and three mixed libraries of non-toxic and toxic compounds; the libraries were “blinded” until the results of the GBFA were known) and found that the GBFA predicts cellular toxicity, with minimal assignment of false positives. These results suggest that in vitro measurement could be used as a warning sign for off-target biological effects in drug discovery efforts and, further, provides a mechanism by which amphiphiles exert their toxicity, namely by altering lipid bilayer physical properties.

Bilayer Modifying Effects of Antipsychotics of Different Generations

Antipsychotics are used to manage the symptoms of schizophrenia (delusions, hallucinations, etc.), to treat bipolar mania, and are commonly used as adjective agents in the treatment of depression. They are known to alter the function of diverse membrane proteins, and for certain antipsychotics (e.g. quintepine), this polypharmacology is thought to be desirable. Because antipsychotics are amphiphiles that modulate the function of different, structurally unrelated membrane proteins, we investigated whether antipsychotics alter lipid bilayer properties at concentrations where they alter membrane protein function. To this end, we used a gramicidin-based fluorescence assay (GBFA) as well as single-channel electrophysiology and we find that antipsychotics like chlorpromazine increase the lifetime and appearance rate of gramicidin (gA) channels, thus shifting the gA monomer dimer equilibrium toward the conducting dimers. We are currently examining other commonly prescribed antipsychotics with the goal of exploring whether there are systematic differences between antipsychotics from the first and second generations. The expectation is that the newer antipsychotics will be less bilayer active and therefore would be expected to have fewer undesired effects. Our other studies have found that an increased alteration of bilayer properties, above a given threshold, may indicate toxicity. Thus, we were keen to learn whether antipsychotics substantially altered bulk bilayer properties and more specifically, we hope to observe whether or not there is a correlation between how well an antipsychotic is tolerated (and thus more commonly prescribed) and its bilayer modifying propensity.

Predicting Drug Toxicity: Early Detection of Likely Failures in Drug Development

It remains a challenge to predict whether or not a new drug candidate will have undesirable side-effects in the clinic. Many biologically active molecules, including drugs and drug-leads, are amphiphiles that partition into lipid bilayers, which may alter bilayer physical properties, thereby modulating membrane protein function. Such bilayer-modifying molecules may be promiscuous modifiers of membrane protein function, raising the possibility that they have off-target effects. Thus, it may be possible to predict whether a compound will have important off-target effects based on quantitative studies on the compound's bilayer-modifying potential. We developed an assay to quantify the bilayer-modifying potential of large numbers of compounds. Using a gramicidin-based fluorescence assay (GBFA), which reports how a compound alters the gramicidin monomer dimer equilibrium, we have shown that many drug and drug-leads alter lipid bilayer properties at the concentrations where these compounds become indiscriminate modifiers of membrane protein function. Such indiscriminate modifiers of membrane protein function are likely to have off target effects; we pursued this question in a study on a library of compounds that had been tested for cytotoxicity in “high-content” screening assays that quantified cellular ATP levels, nuclear morphology, nuclear membrane integrity, and apoptosis in immortalized liver, lung, and neuronal cell lines. We tested 134 compounds (40 non-toxic, 40 moderately toxic and 54 highly toxic) using the GBFA (the library was “blinded” until the results of the GBFA were known) and found that the GBFA predicts cellular toxicity, with the Area Under The Curve of the Receiver-Operating Characteristic being 0.84. These results further support a mechanism by which amphiphiles exert their toxicity, namely by altering lipid bilayer physical properties and that such an in vitro measurement could be used as a warning sign for off-target biological effects in drug discovery efforts.

A General Mechanism for Off-Target Effects: Studies with Amiodarone and other Antiarrhythmics

Atrial fibrillation (AF) is the most prevalent type of arrhythmia and is associated with significant mortality. Amiodarone, owing to its efficacy and minimal proarrhythmic effects, is the most commonly prescribed antiarrhythmic agent. Though amiodarone is considered a class III, repolarization-prolonging, antiarrhythmic that inhibits potassium channels involved in restoring the membrane excitable state, it also alters the function of many other structurally and functionally diverse membrane proteins. This concomitant regulation of multiple membrane proteins by amiodarone results in complex therapeutic and toxicity profiles. Other antiarrhythmics, such as dronedarone, have similar multiple membrane protein targets. Though such a multipronged mechanism for treating AF appears to be desired, it is not clear how amiodarone, and other antiarrhythmics exert their therapeutic actions and regulate a diverse range of membrane proteins at similar concentrations. Chatelane et al. (1989) found that amiodarone and propranolol alter lipid bilayer properties, and that amiodarone does so at therapeutic concentrations. We therefore took advantage of the gramicidin (gA) channels’ sensitivity to changes in the lipid bilayer properties to determine whether the commonly used antiarrhythmics amiodarone, dronedarone, propranolol and pindolol perturb the lipid bilayer properties and at which concentrations. Using a gA-based fluorescence assay and single-channel electrophysiology to explore the antiarrhythmics’ effects on the lipid bilayer, we found that amiodarone and dronedarone are potent bilayer modifiers, propranolol has intermediate activity, and pindolol is the least potent. Moreover, amiodarone and propranolol increase bilayer elasticity. Because amiodarone and dronedarone alter the lipid bilayer at their therapeutic concentrations, where they act on multiple membrane proteins, our results suggest that their multi-target effects may involve a lipid bilayer-mediated mechanism. This underscores the need to further explore the role of bilayer-mediated mechanism in therapeutic as well as toxic effects of antiarrhythmics agents.

Volatile anesthetics inhibit sodium channels without altering bulk lipid bilayer properties.

Although general anesthetics are clinically important and widely used, their molecular mechanisms of action remain poorly understood. Volatile anesthetics such as isoflurane (ISO) are thought to alter neuronal function by depressing excitatory and facilitating inhibitory neurotransmission through direct interactions with specific protein targets, including voltage-gated sodium channels (Na(v)). Many anesthetics alter lipid bilayer properties, suggesting that ion channel function might also be altered indirectly through effects on the lipid bilayer. We compared the effects of ISO and of a series of fluorobenzene (FB) model volatile anesthetics on Na(v) function and lipid bilayer properties. We examined the effects of these agents on Na(v) in neuronal cells using whole-cell electrophysiology, and on lipid bilayer properties using a gramicidin-based fluorescence assay, which is a functional assay for detecting changes in lipid bilayer properties sensed by a bilayer-spanning ion channel. At clinically relevant concentrations (defined by the minimum alveolar concentration), both the FBs and ISO produced prepulse-dependent inhibition of Na(v) and shifted the voltage dependence of inactivation toward more hyperpolarized potentials without affecting lipid bilayer properties, as sensed by gramicidin channels. Only at supra-anesthetic (toxic) concentrations did ISO alter lipid bilayer properties. These results suggest that clinically relevant concentrations of volatile anesthetics alter Na(v) function through direct interactions with the channel protein with little, if any, contribution from changes in bulk lipid bilayer properties. Our findings further suggest that changes in lipid bilayer properties are not involved in clinical anesthesia.

General Anesthetics do not Alter Lipid Bilayer Properties at Clinically Relevant Concentrations

General anesthetics are a widely used class of drugs but, despite their clinical use for >160 years, their exact molecular mechanism(s) remain to be elucidated. A mechanism proposed early on was direct interaction with the lipid bilayer, in some unspecified manner to alter cellular function, which lead to the unitary lipid-based hypothesis of anesthetic action. More recent studies show that general anesthetics interact specifically with various proteins, in particular membrane-embedded ion channels. For example, the inhibition of voltage-gated sodium channels by volatile anesthetics leads to reduced neurotransmitter release in excitable cells. But, though a number of anesthetic targets have been identified, it remains unclear whether the bilayer per se may be involved as well. We therefore examined whether various general anesthetics (isoflurane, sevoflurane, halothane, desflurane, chloroform, diethyl ether, F3, cyclopropane, ketamine and etomidate) and related nonanesthetics (F6 and flurothyl) alter lipid bilayer properties at clinically relevant concentrations. The effects on lipid bilayer properties were tested using the gramicidin-based fluorescence assay (GBFA). The results show that none of the anesthetics or nonanesthetics tested altered lipid bilayer properties at the clinical concentration of 1 MAC (minimal alveolar concentration) with a membrane mole-fraction ranging from 1x10∧-3 (for F6) to 0.1 (for diethyl ether and sevoflurane). Even at two- to four-fold higher concentrations only minimal effects on the bilayer were observed; at much higher (supratherapeutic) concentrations, however, certain anesthetic agents did alter lipid bilayer properties. These results suggest that general anesthetics do not alter ion channel function by altering lipid bilayer properties in a manner that is sensed by a bilayer-spanning channel at clinically relevant concentrations.

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.

Phosphoinositides alter lipid bilayer properties

Phosphatidylinositol-4,5-bisphosphate (PIP2), which constitutes ∼1% of the plasma membrane phospholipid, plays a key role in membrane-delimited signaling. PIP2 regulates structurally and functionally diverse membrane proteins, including voltage- and ligand-gated ion channels, inwardly rectifying ion channels, transporters, and receptors. In some cases, the regulation is known to involve specific lipid–protein interactions, but the mechanisms by which PIP2 regulates many of its various targets remain to be fully elucidated. Because many PIP2 targets are membrane-spanning proteins, we explored whether the phosphoinositides might alter bilayer physical properties such as curvature and elasticity, which would alter the equilibrium between membrane protein conformational states—and thereby protein function. Taking advantage of the gramicidin A (gA) channels’ sensitivity to changes in lipid bilayer properties, we used gA-based fluorescence quenching and single-channel assays to examine the effects of long-chain PIP2s (brain PIP2, which is predominantly 1-stearyl-2-arachidonyl-PIP2, and dioleoyl-PIP2) on bilayer properties. When premixed with dioleoyl-phosphocholine at 2 mol %, both long-chain PIP2s produced similar changes in gA channel function (bilayer properties); when applied through the aqueous solution, however, brain PIP2 was a more potent modifier than dioleoyl-PIP2. Given the widespread use of short-chain dioctanoyl-phosphoinositides, we also examined the effects of diC8-phosphoinositol (PI), PI(4,5)P2, PI(3,5)P2, PI(3,4)P2, and PI(3,4,5)P3. The diC8 phosphoinositides, except for PI(3,5)P2, altered bilayer properties with potencies that decreased with increasing head group charge. Nonphosphoinositide diC8 phospholipids generally were more potent bilayer modifiers than the polyphosphoinositides. These results show that physiological increases or decreases in plasma membrane PIP2 levels, as a result of activation of PI kinases or phosphatases, are likely to alter lipid bilayer properties, in addition to any other effects they may have. The results further show that exogenous PIP2, as well as structural analogues that differ in acyl chain length or phosphorylation state, alters lipid bilayer properties at the concentrations used in many cell physiological experiments.

Statins Alter Lipid Bilayer Properties

Statins are widely prescribed to manage hypercholesterolemia. Statins exert their primary mechanism of action by inhibiting the HMG-CoA reductase, thereby preventing cholesterol synthesis. In addition to this canonical action they also alter the function of diverse membrane proteins. Because statins are amphiphiles that modulate the function of different, structurally unrelated membrane proteins, we investigated whether statins could alter lipid bilayer properties at concentrations where they alter membrane protein function. To this end, we used the gramicidin-based fluorescence assay (GBFA) as well as single-channel electrophysiology. We found that atorvastatin, fluvastatin, lovastatin, mevastatin, pravastatin, pitavastatin, and simvastatin all increased the rate of fluorescence quenching, meaning that they shifted the gramicidin (gA) monomer dimer equilibrium toward the formation of conducting dimers. Statins thus alter lipid bilayer properties, with fluvastatin being the most active and rosuvastatin the least active. When examined using single-channel electrophysiology, simvastatin, pravastatin, and fluvastatin increased the lifetime and appearance rate of gA channels with fluvastatin being the most active and pravastain being the least active. We observe larger effects on the shorter channels; the hydrophobic mismatch dependant effects indicate a change in bilayer elasticity. We conclude that statins alter lipid bilayer properties by a common mechanism, an increase in bilayer elasticity (independent of any changes in membrane cholesterol concentration) and that specific channel-statin interactions need not be the sole mechanism underlying the statins’ actions on membrane protein function.