Tuning ion channel mechanosensitivity by asymmetry of the transbilayer pressure profile

Mechanical stimuli acting on the cellular membrane are linked to intracellular signaling events and downstream effectors via different mechanoreceptors. Mechanosensitive (MS) ion channels are the fastest known primary mechano-electrical transducers, which convert mechanical stimuli into meaningful intracellular signals on a submillisecond time scale. Much of our understanding of the biophysical principles that underlie and regulate conversion of mechanical force into conformational changes in MS channels comes from studies based on MS channel reconstitution into lipid bilayers. The bilayer reconstitution methods have enabled researchers to investigate the structure-function relationship in MS channels and probe their specific interactions with their membrane lipid environment. This brief review focuses on close interactions between MS channels and the lipid bilayer and emphasizes the central role that the transbilayer pressure profile plays in mechanosensitivity and gating of these fascinating membrane proteins.     

The SPFH domain-containing proteins: more than lipid raft markers

Membrane microdomains with distinct lipid compositions, called lipid rafts, represent a potential mechanism for compartmentalizing cellular functions within the plane of biological membranes. SPFH domain-containing proteins are found in lipid raft microdomains in diverse cellular membranes. The functions of these proteins are just beginning to be elucidated. Recent advances in the understanding of structural features and their roles within lipid rafts include a potential function for SPFH proteins in the formation of membrane microdomains and lipid raft-associated processes, such as endocytosis and mechanosensation.     

Transmembrane extension and oligomerization of the CLIC1 chloride intracellular channel protein upon membrane interaction

Chloride intracellular channel proteins (CLICs) differ from most ion channels as they can exist in both soluble and integral membrane forms. The CLICs are expressed as soluble proteins but can reversibly autoinsert into the membrane to form active ion channels. For CLIC1, the interaction with the lipid bilayer is enhanced under oxidative conditions. At present, little evidence is available characterizing the structure of the putative oligomeric CLIC integral membrane form. Previously, fluorescence resonance energy transfer (FRET) was used to monitor and model the conformational transition within CLIC1 as it interacts with the membrane bilayer. These results revealed a large-scale unfolding between the C- and N-domains of CLIC1 as it interacts with the membrane. In the present study, FRET was used to probe lipid-induced structural changes arising in the vicinity of the putative transmembrane region of CLIC1 (residues 24-46) under oxidative conditions. Intramolecular FRET distances are consistent with the model in which the N-terminal domain inserts into the bilayer as an extended α-helix. Further, intermolecular FRET was performed between fluorescently labeled CLIC1 monomers within membranes. The intermolecular FRET shows that CLIC1 forms oligomers upon oxidation in the presence of the membranes. Fitting the data to symmetric oligomer models of the CLIC1 transmembrane form indicates that the structure is large and most consistent with a model comprising approximately six to eight subunits.     

Membrane lipids: where they are and how they behave

Throughout the biological world, a 30 A hydrophobic film typically delimits the environments that serve as the margin between life and death for individual cells. Biochemical and biophysical findings have provided a detailed model of the composition and structure of membranes, which includes levels of dynamic organization both across the lipid bilayer (lipid asymmetry) and in the lateral dimension (lipid domains) of membranes. How do cells apply anabolic and catabolic enzymes, translocases and transporters, plus the intrinsic physical phase behaviour of lipids and their interactions with membrane proteins, to create the unique compositions and multiple functionalities of their individual membranes?     

Visualizing membrane microdomains by Laurdan 2-photon microscopy

Lateral segregation of cell membrane components gives rise to microdomains with a different structure within the membrane. Most prominently, lipid rafts are defined as domains in liquid ordered phase whereas surrounding membranes are more fluid. Here we review a 2-photon fluorescence microscopy approach, which allows the visualization of membrane fluidity. The fluorescent probe Laurdan exhibits a blue shift in emission with increasing membrane condensation caused by an alteration in the dipole moment of the probe as a consequence of exclusion of water molecules from the lipid bilayer. The quantification of membrane order is achieved by the Generalized Polarization (GP) values, which are defined as normalized intensity ratios of two emission channels. GP images are therefore not biased by probe concentrations and membrane ruffles. Furthermore, Laurdan reports membrane structure independently from the lipid and protein cargo of the membrane domains. We give examples where Laurdan microscopy was instrumental in quantifying the formation of condensed membrane domains and their cellular requirements. Moreover we discuss how microdomains identified by Laurdan microscopy are consistent with domains identified by other methodologies and put GP images in the context of current raft hypotheses.     

Contrasting roles of oxidized lipids in modulating membrane microdomains

Lipid rafts display a lateral heterogeneity forming membrane microdomains that hold a fundamental role on biological membranes and are indispensable to physiological functions of cells. Oxidative stress in cellular environments may cause lipid oxidation, changing membrane composition and organization, thus implying in effects in cell signaling and even loss of homeostasis. The individual contribution of oxidized lipid species to the formation or disruption of lipid rafts in membranes still remains unknown. Here, we investigate the role of different structures of oxidized phospholipids on rafts microdomains by carefully controlling the membrane composition. Our experimental approach based on fluorescence microscopy of giant unilamellar vesicles (GUV) enables the direct visualization of the impact of hydroperoxidized POPC lipid (referred to as POPCOOH) and shortened chain lipid PazePC (1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine) on phase separation. We found that the molecular structure of oxidized lipid is of paramount importance on lipid mixing and/or demixing. The hydrophobic mismatch promoted by POPCOOH coupled to its cylindrical molecular shape favor microdomains formation. In contrast, the conical shape of PazePC causes disarrangement of lipid 2D organized platforms. Our findings contribute to better unraveling how oxidized phospholipids can trigger formation or disruption of lipid rafts. As a consequence, phospholipid oxidation may indirectly affect association or dissociation of key biomolecules in the rafts thus altering cell signaling and homeostasis.     

Regulation of sodium channel function by bilayer elasticity: the importance of hydrophobic coupling. Effects of Micelle-forming amphiphiles and cholesterol

Membrane proteins are regulated by the lipid bilayer composition. Specific lipid-protein interactions rarely are involved, which suggests that the regulation is due to changes in some general bilayer property (or properties). The hydrophobic coupling between a membrane-spanning protein and the surrounding bilayer means that protein conformational changes may be associated with a reversible, local bilayer deformation. Lipid bilayers are elastic bodies, and the energetic cost of the bilayer deformation contributes to the total energetic cost of the protein conformational change. The energetics and kinetics of the protein conformational changes therefore will be regulated by the bilayer elasticity, which is determined by the lipid composition. This hydrophobic coupling mechanism has been studied extensively in gramicidin channels, where the channel-bilayer hydrophobic interactions link a "conformational" change (the monomer<-->dimer transition) to an elastic bilayer deformation. Gramicidin channels thus are regulated by the lipid bilayer elastic properties (thickness, monolayer equilibrium curvature, and compression and bending moduli). To investigate whether this hydrophobic coupling mechanism could be a general mechanism regulating membrane protein function, we examined whether voltage-dependent skeletal-muscle sodium channels, expressed in HEK293 cells, are regulated by bilayer elasticity, as monitored using gramicidin A (gA) channels. Nonphysiological amphiphiles (beta-octyl-glucoside, Genapol X-100, Triton X-100, and reduced Triton X-100) that make lipid bilayers less "stiff", as measured using gA channels, shift the voltage dependence of sodium channel inactivation toward more hyperpolarized potentials. At low amphiphile concentration, the magnitude of the shift is linearly correlated to the change in gA channel lifetime. Cholesterol-depletion, which also reduces bilayer stiffness, causes a similar shift in sodium channel inactivation. These results provide strong support for the notion that bilayer-protein hydrophobic coupling allows the bilayer elastic properties to regulate membrane protein function.     

Reconstitution of Homomeric GluA2flop Receptors in Supported Lipid Membranes: FUNCTIONAL AND STRUCTURAL PROPERTIES*

AMPA receptors (AMPARs) are glutamate-gated ion channels ubiquitous in the vertebrate central nervous system, where they mediate fast excitatory neurotransmission and act as molecular determinants of memory formation and learning. Together with detailed analyses of individual AMPAR domains, structural studies of full-length AMPARs by electron microscopy and x-ray crystallography have provided important insights into channel assembly and function. However, the correlation between the structure and functional states of the channel remains ambiguous particularly because these functional states can be assessed only with the receptor bound within an intact lipid bilayer. To provide a basis for investigating AMPAR structure in a membrane environment, we developed an optimized reconstitution protocol using a receptor whose structure has previously been characterized by electron microscopy. Single-channel recordings of reconstituted homomeric GluA2_flop receptors recapitulate key electrophysiological parameters of the channels expressed in native cellular membranes. Atomic force microscopy studies of the reconstituted samples provide high-resolution images of membrane-embedded full-length AMPARs at densities comparable to those in postsynaptic membranes. The data demonstrate the effect of protein density on conformational flexibility and dimensions of the receptors and provide the first structural characterization of functional membrane-embedded AMPARs, thus laying the foundation for correlated structure-function analyses of the predominant mediators of excitatory synaptic signals in the brain.     

Thermosensing via transmembrane protein–lipid interactions

Cell membranes are composed of a lipid bilayer containing proteins that cross and/or interact with lipids on either side of the two leaflets. The basic structure of cell membranes is this bilayer, composed of two opposing lipid monolayers with fascinating properties designed to perform all the functions the cell requires. To coordinate these functions, lipid composition of cellular membranes is tailored to suit their specialized tasks. In this review, we describe the general mechanisms of membrane–protein interactions and relate them to some of the molecular strategies organisms use to adjust the membrane lipid composition in response to a decrease in environmental temperature. While the activities of all biomolecules are altered as a function of temperature, the thermosensors we focus on here are molecules whose temperature sensitivity appears to be linked to changes in the biophysical properties of membrane lipids. This article is part of a Special Issue entitled: Lipid–protein interactions.     

Determinants of voltage-gated potassium channel surface expression and localization in Mammalian neurons

Neurons strictly regulate expression of a wide variety of voltage-dependent ion channels in their surface membranes to achieve precise yet dynamic control of intrinsic membrane excitability. Neurons also exhibit extreme morphological complexity that underlies diverse aspects of their function. Most ion channels are preferentially targeted to either the axonal or somatodendritic compartments, where they become further localized to discrete membrane subdomains. This restricted accumulation of ion channels enables local control of membrane signaling events in specific microdomains of a given compartment. Voltage-dependent K+ (Kv) channels act as potent modulators of diverse excitatory events such as action potentials, excitatory synaptic potentials, and Ca2+ influx. Kv channels exhibit diverse patterns of cellular expression, and distinct subtype-specific localization, in mammalian central neurons. Here we review the mechanisms regulating the abundance and distribution of Kv channels in mammalian neurons and discuss how dynamic regulation of these events impacts neuronal signaling.     

Evidence for a mechanism by which omega-3 polyunsaturated lipids may affect membrane protein function

We have calculated the lateral pressure profile from well-converged, experimentally validated, molecular dynamics simulations of hydrated lipid bilayer membranes containing highly polyunsaturated fatty acids. The three simulations, each 30 ns in length, contain omega-3 fatty acids, omega-6 fatty acids, and a mixture of omega-3 fatty acids and cholesterol and were continued from previously published simulations that demonstrated excellent agreement with a wide variety of experimental measurements. We find that the distribution of lateral stress within the hydrophobic core of the membrane is sensitively dependent on the degree of chain unsaturation and on the presence of cholesterol. Replacing omega-3 fatty acids with omega-6 chains, or incorporating cholesterol into the membrane, shifts the repulsive lateral chain pressure away from the lipid/water interface toward the bilayer interior. This may support a previously proposed mechanism by which lipid composition may affect conformational equilibrium for integral membrane proteins.     

Probing ground and excited states of phospholamban in model and native lipid membranes by magic angle spinning NMR spectroscopy

In this paper, we analyzed the ground and excited states of phospholamban (PLN), a membrane protein that regulates sarcoplasmic reticulum calcium ATPase (SERCA), in different membrane mimetic environments. Previously, we proposed that the conformational equilibria of PLN are central to SERCA regulation. Here, we show that these equilibria detected in micelles and bicelles are also present in native sarcoplasmic reticulum lipid membranes as probed by MAS solid-state NMR. Importantly, we found that the kinetics of conformational exchange and the extent of ground and excited states in detergent micelles and lipid bilayers are different, revealing a possible role of the membrane composition on the allosteric regulation of SERCA. Since the extent of excited states is directly correlated to SERCA inhibition, these findings open up the exciting possibility that calcium transport in the heart can be controlled by the lipid bilayer composition. This article is part of a Special Issue entitled: Membrane protein structure and function.     

A marriage of convenience: beta-subunits and voltage-dependent K+ channels

The movement of ions across cell membranes is essential for a wide variety of fundamental physiological processes, including secretion, muscle contraction, and neuronal excitation. This movement is possible because of the presence in the cell membrane of a class of integral membrane proteins dubbed ion channels. Ion channels, thanks to the presence of aqueous pores in their structure, catalyze the passage of ions across the otherwise ion-impermeable lipid bilayer. Ion conduction across ion channels is highly regulated, and in the case of voltage-dependent K(+) channels, the molecular foundations of the voltage-dependent conformational changes leading to the their open (conducting) configuration have provided most of the driving force for research in ion channel biophysics since the pioneering work of Hodgkin and Huxley (Hodgkin, A. L., and Huxley, A. F. (1952) J. Physiol. 117, 500-544). The voltage-dependent K(+) channels are the prototypical voltage-gated channels and govern the resting membrane potential. They are responsible for returning the membrane potential to its resting state at the termination of each action potential in excitable membranes. The pore-forming subunits (alpha) of many voltage-dependent K(+) channels and modulatory beta-subunits exist in the membrane as one component of macromolecular complexes, able to integrate a myriad of cellular signals that regulate ion channel behavior. In this review, we have focused on the modulatory effects of beta-subunits on the voltage-dependent K(+) (Kv) channel and on the large conductance Ca(2+)- and voltage-dependent (BK(Ca)) channel.     

生物膜结构研究的一些进展

膜蛋白三维结构的解析存在很多困难.最近几年由于一些通道(如K⁺通道,Cl^-通道,水通道Aquaporin 1等)和泵（如Ca^2＋泵）的结晶获得成功,这些膜蛋白具有原子分辨率三维结构的解析才得以完成,从而基本阐明一些极性分子和离子选择性通过生物膜的分子机理.在膜脂结构方面,动物细胞质膜膜脂的分布是不均匀的.近年来已多方面证明,质膜具有一些被命名为“脂筏（lipid rafts）”和“质膜微囊（Caveolae）”的微区.它们富含鞘脂和胆固醇。简单介绍了这些脂质微区的大小、组分以及动态变化.根据研究结果,这类脂质微区含有大量信号分子,很可能具有信号传递中心的作用.此外,对脂筏在膜运送过程中的作用也进行一些评述.     

Determinants of Voltage-Gated Potassium Channel Surface Expression and Localization in Mammalian Neurons

Neurons strictly regulate expression of a wide variety of voltage-dependent ion channels in their surface membranes to achieve precise yet dynamic control of intrinsic membrane excitability. Neurons also exhibit extreme morphological complexity that underlies diverse aspects of their function. Most ion channels are preferentially targeted to either the axonal or somatodendritic compartments, where they become further localized to discrete membrane subdomains. This restricted accumulation of ion channels enables local control of membrane signaling events in specific microdomains of a given compartment. Voltage-dependent K⁺ (Kv) channels act as potent modulators of diverse excitatory events such as action potentials, excitatory synaptic potentials, and Ca²⁺ influx. Kv channels exhibit diverse patterns of cellular expression, and distinct subtype-specific localization, in mammalian central neurons. Here we review the mechanisms regulating the abundance and distribution of Kv channels in mammalian neurons and discuss how dynamic regulation of these events impacts neuronal signaling.     

Conformational and orientation studies of artificial ion channels incorporated into lipid bilayers

The conformational and orientation studies in lipid bilayers of 21 amino acid peptides bearing six crown ethers are reported. The compounds were designed to form artificial ion channels by stacking the crown rings, and were shown to be functional in bilayer membranes. We used Fourier transform infrared spectroscopy and CD spectropolarimetry to study the conformation of the peptides in solution and in lipid bilayers. These studies revealed that hexacrown peptides retain their alpha-helical conformation when incorporated in a lipid bilayer environment. Attenuated total reflectance spectroscopy was used to investigate the orientation of the peptides in a lipid bilayer. Results demonstrated that the peptides are not oriented at a fixed angle in membrane, but rather are in incorporation equilibrium between an active state parallel to the lipid chain and an inactive state adsorbed at the surface of the bilayer. From these results, we propose a model for the channel activity and the gating mechanism of these hexacrown peptides in bilayer membranes.     

Lipid rafts and little caves. Compartmentalized signalling in membrane microdomains.

Lipid rafts are liquid-ordered membrane microdomains with a unique protein and lipid composition found on the plasma membrane of most, if not all, mammalian cells. A large number of signalling molecules are concentrated within rafts, which have been proposed to function as signalling centres capable of facilitating efficient and specific signal transduction. This review summarizes current knowledge regarding the composition, structure, and dynamic nature of lipid rafts, as well as a number of different signalling pathways that are compartmentalized within these microdomains. Potential mechanisms through which lipid rafts carry out their specialized role in signalling are discussed in light of recent experimental evidence.     

Lipid composition and the lateral pressure profile in bilayers

The mechanisms by which variations in the lipid composition of cell membranes influence the function of membrane proteins are not yet well understood. In recent work, a nonlocal thermodynamic mechanism was suggested in which changes in lipid composition cause a redistribution of lateral pressures that in turn modulates protein conformational (or aggregation) equilibria. In the present study, results of statistical thermodynamic calculations of the equilibrium pressure profile and bilayer thickness are reported for a range of lipids and lipid mixtures. Large redistributions of lateral pressure are predicted to accompany variation in chain length, degree and position of chain unsaturation, head group repulsion, and incorporation of cholesterol and interfacially active solutes. Combinations of compositional changes are found that compensate with respect to bilayer thickness, thus eliminating effects of hydrophobic mismatch, while still effecting significant shifts of the pressure profile. It is also predicted that the effect on the pressure profile of addition of short alkanols can be reproduced with certain unnatural lipids. These results suggest possible roles of cholesterol, highly unsaturated fatty acids and small solutes in modulating membrane protein function and suggest unambiguous experimental tests of the pressure profile hypothesis. As a test of the methodology, calculated molecular areas and area elastic moduli are compared with experimental and simulation results.     

The influence of membrane lateral pressures on simple geometric models of protein conformational equilibria

The function of many intrinsic membrane proteins requires a conformational transition that is often strongly influenced by the molecular composition of the bilayer in which the protein is embedded. Recently, a mechanism for this shift in conformational equilibrium was suggested, in which it is argued that a shift in distribution of lateral pressures of the bilayer resulting from a change in lipid composition alters the amount of mechanical work of the protein conformational transition, if the change in the cross-sectional area profile of the protein varies with depth within the bilayer. As there is little information on the change in shape of the transmembrane region of any protein, various simple geometric models are considered. For both a generic model, and more specific models that approximate likely cooperative rearrangements of alpha-helices in bundles, it is found that the conformational equilibrium depends on the first and second integral moments of the lateral pressure distribution. In addition to revealing the possible physical underpinnings of the well-known correlation between protein activity and the 'nonlamellar' tendency of bilayer lipids, this dependence on moments of the pressure profile allows for prediction of the relative effects of different lipid compositional changes even in the absence of information on specific protein shape changes. Effects of variation in acyl chain length, degree and position of cis-unsaturation, and addition of cholesterol and small interfacially-active solutes (n-alkanols) are compared.     

bSUM: A bead-supported unilamellar membrane system facilitating unidirectional insertion of membrane proteins into giant vesicles

Fused or giant vesicles, planar lipid bilayers, a droplet membrane system, and planar-supported membranes have been developed to incorporate membrane proteins for the electrical and biophysical analysis of such proteins or the bilayer properties. However, it remains difficult to incorporate membrane proteins, including ion channels, into reconstituted membrane systems that allow easy control of operational dimensions, incorporation orientation of the membrane proteins, and lipid composition of membranes. Here, using a newly developed chemical engineering procedure, we report on a bead-supported unilamellar membrane (bSUM) system that allows good control over membrane dimension, protein orientation, and lipid composition. Our new system uses specific ligands to facilitate the unidirectional incorporation of membrane proteins into lipid bilayers. Cryo–electron microscopic imaging demonstrates the unilamellar nature of the bSUMs. Electrical recordings from voltage-gated ion channels in bSUMs of varying diameters demonstrate the versatility of the new system. Using KvAP as a model system, we show that compared with other in vitro membrane systems, the bSUMs have the following advantages: (a) a major fraction of channels are orientated in a controlled way; (b) the channels mediate the formation of the lipid bilayer; (c) there is one and only one bilayer membrane on each bead; (d) the lipid composition can be controlled and the bSUM size is also under experimental control over a range of 0.2–20 µm; (e) the channel activity can be recorded by patch clamp using a planar electrode; and (f) the voltage-clamp speed (0.2–0.5 ms) of the bSUM on a planar electrode is fast, making it suitable to study ion channels with fast gating kinetics. Our observations suggest that the chemically engineered bSUMs afford a novel platform for studying lipid–protein interactions in membranes of varying lipid composition and may be useful for other applications, such as targeted delivery and single-molecule imaging.