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.     

Alcohols reduce lateral membrane pressures: predictions from molecular theory

We explore the effects of alcohols on fluid lipid bilayers using a molecular theory with a coarse-grained model. We show that the trends predicted from the theory in the changes in area per lipid, alcohol concentration in the bilayer, and area compressibility modulus, as a function of alcohol chain length and of the alcohol concentration in the solvent far from the bilayer, follow those found experimentally. We then use the theory to study the effect of added alcohol on the lateral pressure profile across the membrane, and find that added alcohol reduces the surface tensions at both the headgroup/solvent and headgroup/tailgroup interfaces, as well as the lateral pressures in the headgroup and tailgroup regions. These changes in lateral pressures could affect the conformations of membrane proteins, providing a nonspecific mechanism for the biological effects of alcohols on cells.     

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).     

The 2004 Biophysical Society-Avanti Award in Lipids address: roles of bilayer structure and elastic properties in peptide localization in membranes

This review details how bilayer structural/elastic properties impact three distinct areas of biological significance. First, the partitioning of melittin into bilayers and melittin-induced bilayer leakage depended strongly on bilayer composition. The incorporation of cholesterol into phosphatidylcholine bilayers decreased melittin-induced leakage from 73 to 3%, and bilayers composed of lipopolysaccharide (LPS), the main lipid on the surface of Gram-negative bacteria, also had low (3%) melittin-induced leakage. Second, transbilayer peptides of different hydrophobic lengths were largely excluded from bilayer microdomains (“rafts”) enriched in sphingomyelin (SM) and cholesterol, even when the length of the transbilayer peptide domain matched the hydrocarbon thickness of the raft bilayer. This is likely due to the large area compressibility modulus of SM:cholesterol bilayers. Third, the major water barrier of skin, the extracellular lamellae of the stratum corneum, was found to contain tightly packed asymmetric lipid bilayers with cholesterol located preferentially on one side of the bilayer and a unique skin ceramide containing an unsaturated acyl chain on the opposite side. We argue that, in each of these three areas, key factors are differences in lipid hydrocarbon chain packing for different lipids, particularly the tight hydrocarbon chain packing caused by cholesterol’s strong interaction with saturated chains.     

Adaptation of membrane lipids to alcohols.

The effects of alcohols of different chain lengths on the fatty acid composition of Escherichia coli K-12 have been examined. My results indicate that these cells radically change their fatty acid composition when grown in the presence of alcohols. These changes represent an adaptive membrane alteration compensating for the direct physicochemical interaction of alcohols with the membrane. Similar adaptive responses of membrane lipids are proposed as a possible biochemical basis for tolerance to alcohol and related drugs.     

Antioxidant activity of hydroxytyrosyl esters studied in liposome models

The properties and the antioxidant activity of a series of hydroxytyrosyl esters having different carbon chain lengths (C4, C8, C12 and C18) have been measured in phosphatidylcholine model membrane (liposomes) using specific probes for the bilayer and liposome lumen microenvironment, i.e., 1,6-diphenyl-1,3,5-hexatriene (DPH) and 2′,7′-dichlorodihydrofluorescein (H₂DCF), respectively.Antioxidants self-assembly and their interaction with liposomes has been evaluated by light scattering, fluorescence, turbidimetry, gel filtration chromatography and microfiltration measurements, allowing the determination of critical aggregation concentration, bound fraction, capacity of crossing the lipid bilayer.The distribution of hydroxytyrosyl long chain esters has been proved to depend quite specifically on their lipophilic chain length, and this turns to have deep effects on their antioxidant behaviour. Shedding new light on the cut off effect and antioxidant behaviour of phenolipids, this study also put forward the relevance of cell-free liposome-based cellular models, like giant liposomes, for further characterization of analogous systems.     

The membrane environment modulates self-association of the human GpA TM domain—Implications for membrane protein folding and transmembrane signaling

The influence of lipid bilayer properties on a defined and sequence-specific transmembrane helix-helix interaction is not well characterized yet. To study the potential impact of changing bilayer properties on a sequence-specific transmembrane helix-helix interaction, we have traced the association of fluorescent-labeled glycophorin A transmembrane peptides by fluorescence spectroscopy in model membranes with varying lipid compositions. The observed changes of the glycophorin A dimerization propensities in different lipid bilayers suggest that the lipid bilayer thickness severely influences the monomer-dimer equilibrium of this transmembrane domain, and dimerization was most efficient under hydrophobic matching conditions. Moreover, cholesterol considerably promotes self-association of transmembrane helices in model membranes by affecting the lipid acyl chain ordering. In general, the order of the lipid acyl chains appears to be an important factor involved in determining the strength and stability of transmembrane helix-helix interactions. As discussed, the described influences of membrane properties on transmembrane helix-helix interactions are highly important for understanding the mechanism of transmembrane protein folding and functioning as well as for gaining a deeper insight into the regulation of signal transduction via membrane integral proteins by bilayer properties.     

Regulation of channel function due to physical energetic coupling with a lipid bilayer

Regulation of membrane protein functions due to hydrophobic coupling with a lipid bilayer has been investigated. An energy formula describing interactions between lipid bilayer and integral ion channels with different structures, which is based on the screened Coulomb interaction approximation, has been developed. Here the interaction energy is represented as being due to charge-based interactions between channel and lipid bilayer. The hydrophobic bilayer thickness channel length mismatch is found to induce channel destabilization exponentially while negative lipid curvature linearly. Experimental parameters related to channel dynamics are consistent with theoretical predictions. To measure comparable energy parameters directly in the system and to elucidate the mechanism at an atomistic level we performed molecular dynamics (MD) simulations of the ion channel forming peptide–lipid complexes. MD simulations indicate that peptides and lipids experience electrostatic and van der Waals interactions for short period of time when found within each other’s proximity. The energies from these two interactions are found to be similar to the energies derived theoretically using the screened Coulomb and the van der Waals interactions between peptides (in ion channel) and lipids (in lipid bilayer) due to mainly their charge properties. The results of in silico MD studies taken together with experimental observable parameters and theoretical energetic predictions suggest that the peptides induce ion channels inside lipid membranes due to peptide–lipid physical interactions. This study provides a new insight helping better understand of the underlying mechanisms of membrane protein functions in cell membrane leading to important biological implications.     

Direct determination of a membrane-peptide interface using the nuclear magnetic resonance cross-saturation method

Membrane-peptide interactions are involved in many crucial biological and pharmacological activities. To clarify the interaction mode of membrane-peptide complexes, it is important to analyze both the dynamic properties and the contact residues of the membrane-bound peptide. In this study, we investigated the dynamic properties of a peptide bound to a lipid bilayer, using relaxation and amide-water exchange analyses, and directly determined the membrane-peptide interface, using the cross-saturation method. For the models of a lipid bilayer and a peptide, isotropic bicelles and mastoparan were used, respectively. The results indicate that mastoparan had a heterogeneous distribution of motion over various timescales and interacted with the lipid bilayer by using its hydrophobic side; the molecule was located within the lipid bilayer rather than on the surface, as thought previously. This study shows that the cross-saturation method is useful for determining the interface of not only protein-protein but also membrane-peptide complexes.     

Gramicidin-based Fluorescence Assay; for Determining Small Molecules Potential for Modifying Lipid Bilayer Properties

Many drugs and other small molecules used to modulate biological function are amphiphiles that adsorb at the bilayer/solution interface and thereby alter lipid bilayer properties. This is important because membrane proteins are energetically coupled to their host bilayer by hydrophobic interactions. Changes in bilayer properties thus alter membrane protein function, which provides an indirect way for amphiphiles to modulate protein function and a possible mechanism for "off-target" drug effects. We have previously developed an electrophysiological assay for detecting changes in lipid bilayer properties using linear gramicidin channels as probes ^3,12. Gramicidin channels are mini-proteins formed by the transbilayer dimerization of two non-conducting subunits. They are sensitive to changes in their membrane environment, which makes them powerful probes for monitoring changes in lipid bilayer properties as sensed by bilayer spanning proteins. We now demonstrate a fluorescence assay for detecting changes in bilayer properties using the same channels as probes. The assay is based on measuring the time-course of fluorescence quenching from fluorophore-loaded large unilamellar vesicles due to the entry of a quencher through the gramicidin channels. We use the fluorescence indicator/quencher pair 8-aminonaphthalene-1,3,6-trisulfonate (ANTS)/Tl⁺ that has been successfully used in other fluorescence quenching assays ^5,13. Tl⁺ permeates the lipid bilayer slowly ⁸ but passes readily through conducting gramicidin channels ^1,14. The method is scalable and suitable for both mechanistic studies and high-throughput screening of small molecules for bilayer-perturbing, and potential "off-target", effects. We find that results using this method are in good agreement with previous electrophysiological results ¹².Download video file.^{(69M, mov)}     

The effect of benzyl alcohol and cholesterol on the acyl chain order and alkane solubility of bimolecular phosphatidylcholine membranes

An investigation was made of the effects of cholesterol and benzyl alcohol on the partitioning of n-alkanes between lipid bilayer membranes and bulk lipid/alkane solutions (in the torus). Bilayers were formed from solutions containing alkanes of different chain lengths, together with phosphatidylcholine and cholesterol in varying proportions. The partitioning of the alkanes was determined from measurements of the very low frequency (1 Hz) capacitance of the membranes. Perturbation of the internal membrane structure by the inclusion of cholesterol and benzyl alcohol produced very significant changes in the n-alkane partition coefficient, cholesterol causing a decrease and benzyl alcohol an increase in the alkane partitioning into the bilayer. A correlation exists between the effects of these compounds on the alkane partitioning and their effect on the segmental chain order of the acyl chains in the bilayer and this correlation is consistent with a statistical-mechanical model of the lipid/alkane bilayers in the liquid crystalline state. The perturbation by cholesterol and benzyl alcohol of the internal structure of membranes bears on the conflicting reports of the effects of these substances on artificial lipid bilayers and could also be relevant to their known physiological effects.     

Investigations into the membrane activity of arenicin antimicrobial peptide AA139

Arenicin-3 is an amphipathic β-hairpin antimicrobial peptide that is produced by the lugworm Arenicola marina. In this study, we have investigated the mechanism of action of arenicin-3 and an optimized synthetic analogue, AA139, by studying their effects on lipid bilayer model membranes and Escherichia coli bacterial cells. The results show that simple amino acid changes can lead to subtle variations in their interaction with membranes and therefore alter their pre-clinical potency, selectivity and toxicity. While the mechanism of action of arenicin-3 is primarily dependent on universal membrane permeabilization, our data suggest that the analogue AA139 relies on more specific binding and insertion properties to elicit its improved antibacterial activity and lower toxicity, as exemplified by greater selectivity between lipid composition when inserting into model membranes i.e. the N-terminus of AA139 seems to insert deeper into lipid bilayers than arenicin-3 does, with a clear distinction between zwitterionic and negatively charged lipid bilayer vesicles, and AA139 demonstrates a cytoplasmic permeabilization dose response profile that is consistent with its greater antibacterial potency against E. coli cells compared to arenicin-3.     

Phosphatidyl alcohols: Effect of head group size on domain forming properties and interactions with sterols

In this study, we have examined the membrane properties and sterol interactions of phosphatidyl alcohols varying in the size of the alcohol head group coupled to the sn-3-linked phosphate. Phosphatidyl alcohols of interest were dipalmitoyl derivatives with methanol (DPPMe), ethanol (DPPEt), propanol (DPPPr), or butanol (DPPBu) head groups. The Phosphatidyl alcohols are biologically relevant, because they can be formed in membranes by the phospholipase D reaction in the presence of alcohol. The melting behavior of pure phosphatidyl alcohols and mixtures with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or cholesterol was assessed using high sensitivity differential scanning calorimetry (DSC). DPPMe had the highest melting temperature (∼ 49 °C), whereas the other phosphatidyl alcohols had similar melting temperatures as DPPC (∼ 40-41 °C). All phosphatidyl alcohols, except DPPMe, also showed good miscibility with DPPC. The effects of cholesterol on the melting behavior and membrane order in multilamellar bilayer vesicles were assessed using steady-state anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) and DSC. The ordering effect of cholesterol in the fluid phase was lower for all phosphatidyl alcohols as compared to DPPC and decreased with increasing head group size. The formation of ordered domains containing the phosphatidyl alcohols in complex bilayer membranes was determined using fluorescence quenching of DPH or the sterol analogue cholesta-5,7,(11)-trien-3-beta-ol (CTL). The phosphatidyl alcohols did not appear to form sterol-enriched ordered domains, whereas DPPMe, DPPEt appeared to form ordered domains in the temperature window examined (10-50 °C). The partitioning of CTL into bilayer membranes containing phosphatidyl alcohols was to a small extent increased for DPPMe and DPPEt, but in general, sterol interactions were weak or unfavorable for the phosphatidyl alcohols. Our results show that the biophysical and sterol interacting properties of phosphatidyl alcohols, having identical acyl chain structures, are markedly dependent on the size of the head group.     

Lag phase during the action of phospholipase A2 on phosphatidylcholine modified by alkanols

Theaction of pig pancreatic phospholipase A2 (EC 3.1.1.4) on phosphatidylcholine bilayer is studied under a variety of substrate modification conditions including the incorporation of long chain alcohols (hexanol and several isomeric octanols) into the bilayer. The rate of hydrolysis shows a biphasic dependence upon the concentration of the activating alcohol. The hexanol to lipid molar ratio in the bilayer is approximately 1.4:1 at the optimal alkanol concentration. The lag phase at the beginning of hydrolysis has been shown to depend upon the nature of the bilayer as modified by different alkanols and by intrinsic differences in the unilamellar vesicles (approximate diameter approximately 250 A) compared to the multilamellar vesicles. The rate constant for the activation process responsible for the lag period is first order and does not depend upon the concentration of the enzyme, substrate, alkanol, and calcium. These and other experiments are interpreted in terms of a hypothesis that the pancreatic phospholipase interacts with the bilayer by a catalytic and a recognition site. The data suggest that the packing of the interface regulates the interaction of both the catalytic and the recognition site. It is postulated that the biphasic activation profile as a function of hexanol concentration may be a consequence of two-site interactions between the enzyme and the substrate interface.     

Phenethyl alcohol disorders phospholipid acyl chains and promotes translocation of the mitochondrial precursor protein apocytochrome c across a lipid bilayer.

The interaction of phenethyl alcohol with model membranes and its effect on translocation of the chemically prepared mitochondrial precursor protein apocytochrome c across a lipid bilayer was studied. Phenethyl alcohol efficiently penetrates into monolayers and causes acyl chain disordering judged from deuterium nuclear magnetic resonance measurements with specific acyl chain-deuterated phospholipids. Translocation of apocytochrome c across a phospholipid bilayer was stimulated on addition of phenethyl alcohol indicating that the efficiency of translocation of this precursor protein is enhanced due to a disorder of the acyl chain region of the bilayer.     

Structure-activity relationship of alkanols as mosquito larvicides with novel findings regarding their mode of action

Primary alcohols, from methanol to eicosanol, were applied to water for control of larval stage mosquitoes. By applying the alkanols as soluble solutions rather than as insoluble monolayers, and by trapping larvae under glass in assays that isolated them from the surface phenomena believed to be responsible for death by suffocation, we have shown that the action of alkanols against mosquito larvae is biochemical in nature, not just physical. Primary alcohols are known to act as general anesthetics, with increasing potency correlated to increasing chain length until a point of cutoff is reached, usually at dodecanol (C12), after which activity disappears entirely. In mosquitoes, we found that activity levels off after undecanol (C11) but does not disappear until after pentadecanol (C15), that it is reversible, and that chain length plays a role not only in potency, but also in the time needed to manifest toxic effects. We used sonication, a surfactant, temperature, and the introduction of double bonds to manipulate activity around the cutoff, suggesting that it is at least partially a function of solubility. Mosquitoes appear to be the first animal for which cutoff has been demonstrated to occur at a chain length beyond C12, offering new insights into the molecular basis of anesthetic cutoff and suggesting the possibility that alkanols might be used for selective pest control. Alkanols are stable, colorless, inexpensive, biodegradable and essentially non-toxic to humans, making them promising candidates for pest management programs.     

Calculation of intermolecular interaction strengths in the P beta' phase in lipid bilayers. Implications for theoretical models.

The existence of the P beta' phase in certain lipid bilayers is evidence that molecular interactions between lipids are capable of producing unusual large-scale structures at or near biological conditions. The problem of identifying the specific intermolecular interactions responsible for the structures requires construction of theoretical models capable of clear predictions of the observable consequences of postulated intermolecular interactions. To this end we have carried out a twofold modeling effort aimed at understanding the ripple phase. First, we have performed detailed numerical calculations of potential energies of interaction between pairs and triplets of lipid molecules having different chain tilt angles and relative vertical alignments. The calculations support the notion that chain tilting in the gel phase is a result of successive 3-5-A displacements of neighboring molecules perpendicular to the bilayer plane rather than long-range cooperative chain tilting. Secondly, we have used these results as a guide to formulate a new lattice model for lipid bilayer condensed phases. The new model is less complex than our earlier model and it includes interactions which are, based on the energy calculations, more likely to be responsible for the ripple phase. In a certain limit the model maps onto the chiral clock model, a model of much interest in condensed matter theory. In this limit the model exhibits a chain-tilted ordered phase followed by (as temperature increases) a modulated phase followed by a disordered phase. Within this limit we discuss the properties of the model and compare structures of the modulated phase exhibited by the model with experimental data for the P beta' phase in lipid bilayers.     

Transmembrane Lipid Migration in Planar Asymmetric Bilayer Membranes

Planar asymmetric bilayer membranes, formed by apposing a monolayer of the neutral lipid glyceroldioleate (GDO) with one of the negatively charged lipid oleyl acid phosphate (OAP), were used to measure the rate of transmembrane OAP migration. The assay for this lipid flip-flop was the interaction of Ca²⁺ ions with negatively charged lipids which causes membranes to break: when Ca²⁺ is added to the compartment limited initially by the neutral lipid, flip-flop of the charged lipid eventually results in membrane breakdown. At 22 ± 2°C, in the absence of an externally applied electric field, an upper limit to the half time of OAP flip-flop was measured as 18.7 h, with a tentative lower limit of 14.4 h.     

A Molecular Target for an Alcohol Chain-Length Cutoff

Despite the widespread consumption of ethanol, mechanisms underlying its anesthetic effects remain uncertain. n-Alcohols induce anesthesia up to a specific chain length and then lose potency—an observation known as the “chain-length cutoff effect.” This cutoff effect is thought to be mediated by alcohol binding sites on proteins such as ion channels, but where these sites are for long-chain alcohols and how they mediate a cutoff remain poorly defined. In animals, the enzyme phospholipase D (PLD) has been shown to generate alcohol metabolites (e.g., phosphatidylethanol) with a cutoff, but no phenotype has been shown connecting PLD to an anesthetic effect. Here we show loss of PLD blocks ethanol-mediated hyperactivity in Drosophila melanogaster (fruit fly), demonstrating that PLD mediates behavioral responses to alcohol in vivo. Furthermore, the metabolite phosphatidylethanol directly competes for the endogenous PLD product phosphatidic acid at lipid-binding sites within potassium channels [e.g., TWIK-related K⁺ channel type 1 (K2P2.1, TREK-1)]. This gives rise to a PLD-dependent cutoff in TREK-1. We propose an alcohol pathway where PLD produces lipid-alcohol metabolites that bind to and regulate downstream effector molecules including lipid-regulated potassium channels.     

Alcohol action on membrane ion channels gated by extracellular ATP (P2X receptors).

Extracellular adenosine 5'-triphosphate (ATP) has been reported to produce excitatory actions in the nervous system, such as excitatory postsynaptic potentials or currents in both central and peripheral neurons, via activation of a class of ATP-gated membrane ion channels designated P2X receptors. This article reviews studies of alcohol effects on these receptor-channels. Ethanol has been found to inhibit ATP-gated ion channel function by shifting the agonist concentration-response curve to the right in a parallel manner, increasing the EC50 without affecting Emax of this curve. To distinguish whether this inhibition involves competitive antagonism of agonist action or a decrease in the affinity of the agonist binding site, the kinetics of activation and deactivation of agonist-activated current were studied. Ethanol was found to decrease the time-constant of deactivation of ATP-gated ion channels without affecting the time-constant of activation, indicating that ethanol inhibits the function of these receptors by an allosteric decrease in the affinity of the agonist binding site. The inhibition of ATP-gated ion channel function by a number of alcohols was found to exhibit a distinct cutoff effect that appeared to be related to the molecular volume of the alcohols. For alcohols with a molecular volume of < or = 42.2 ml/mol, potency for inhibiting ATP-activated current was correlated with lipid solubility (order of potency: 1-propanol = trifluoroethanol > monochloroethanol > ethanol > methanol). However, despite increased lipid solubility, alcohols with a molecular volume of > or = 46.1 ml/mol (1-butanol, 1-pentanol, trichloroethanol, and dichloroethanol) were without effect on the ATP-activated current. This cutoff effect has been interpreted as evidence that alcohols inhibit the function of ATP-gated ion channels by interacting with a hydrophobic pocket of circumscribed dimensions on the receptor protein. To evaluate the localization of this presumed alcohol binding site, the effect of the intracellular application of ethanol was studied on the inhibition of ATP-activated current by extracellularly applied ethanol. The intracellular application of 100 mM ethanol did not affect the inhibition of current by 100 mM extracellular ethanol, suggesting that the alcohol inhibition of ATP-gated ion channel function involves the extracellular domain of the receptor. Finally, recent studies suggest that the alcohol sensitivity of ATP-gated channels may be regulated by physiological mechanisms.