Jens A. Lundbæk

Lipid bilayer regulation of membrane protein function: gramicidin channels as molecular force probes

Membrane protein function is regulated by the host lipid bilayer composition. This regulation may depend on specific chemical interactions between proteins and individual molecules in the bilayer, as well as on non-specific interactions between proteins and the bilayer behaving as a physical entity with collective physical properties (e.g. thickness, intrinsic monolayer curvature or elastic moduli). Studies in physico-chemical model systems have demonstrated that changes in bilayer physical properties can regulate membrane protein function by altering the energetic cost of the bilayer deformation associated with a protein conformational change. This type of regulation is well characterized, and its mechanistic elucidation is an interdisciplinary field bordering on physics, chemistry and biology. Changes in lipid composition that alter bilayer physical properties (including cholesterol, polyunsaturated fatty acids, other lipid metabolites and amphiphiles) regulate a wide range of membrane proteins in a seemingly non-specific manner. The commonality of the changes in protein function suggests an underlying physical mechanism, and recent studies show that at least some of the changes are caused by altered bilayer physical properties. This advance is because of the introduction of new tools for studying lipid bilayer regulation of protein function. The present review provides an introduction to the regulation of membrane protein function by the bilayer physical properties. We further describe the use of gramicidin channels as molecular force probes for studying this mechanism, with a unique ability to discriminate between consequences of changes in monolayer curvature and bilayer elastic moduli.

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 (β-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.

Spring Constants for Channel-Induced Lipid Bilayer Deformations Estimates Using Gramicidin Channels

Hydrophobic interactions between a bilayer and its embedded membrane proteins couple protein conformational changes to changes in the packing of the surrounding lipids. The energetic cost of a protein conformational change therefore includes a contribution from the associated bilayer deformation energy (DeltaGdef0), which provides a mechanism for how membrane protein function depends on the bilayer material properties. Theoretical studies based on an elastic liquid-crystal model of the bilayer deformation show that DeltaGdef0 should be quantifiable by a phenomenological linear spring model, in which the bilayer mechanical characteristics are lumped into a single spring constant. The spring constant scales with the protein radius, meaning that one can use suitable reporter proteins for in situ measurements of the spring constant and thereby evaluate quantitatively the DeltaGdef0 associated with protein conformational changes. Gramicidin channels can be used as such reporter proteins because the channels form by the transmembrane assembly of two nonconducting monomers. The monomerleft arrow over right arrow dimer reaction thus constitutes a well characterized conformational transition, and it should be possible to determine the phenomenological spring constant describing the channel-induced bilayer deformation by examining how DeltaGdef0 varies as a function of a mismatch between the hydrophobic channel length and the unperturbed bilayer thickness. We show this is possible by analyzing experimental studies on the relation between bilayer thickness and gramicidin channel duration. The spring constant in nominally hydrocarbon-free bilayers agrees well with estimates based on a continuum analysis of inclusion-induced bilayer deformations using independently measured material constants.

Ion channels as tools to monitor lipid bilayer-membrane protein interactions: gramicidin channels as molecular force transducers.

Publisher Summary Numerous studies show that membrane protein function depends on the bilayer lipid composition. Specifically, the function of integral membrane proteins varies with lipid bilayer thickness and monolayer equilibrium curvature. In most cases, there is only modest chemical specificity in these membrane lipid-protein interactions. This lack of chemical specificity, together with the large number of lipid types that are found in the membranes of any given cell, has caused difficulties for attempts to understand the way the function of integral membrane proteins is affected by the bilayer lipid composition. These difficulties arose in part because the results were interpreted within the framework of the Singer-Nicolson fluid mosaic membrane model. A weakness of the fluid mosaic membrane model was that the lipid bilayer component was assumed to be a passive entity only—that is, a permeability barrier that separated the extracellular and intracellular aqueous phases. The view of the lipid bilayer as a sheet of liquid hydrocarbon led to the notion of bilayer fluidity as an important determinant of protein function. An important, but neglected consequence of the liquid-crystalline organization of lipid bilayers is that one needs to incorporate the bilayer material properties (thickness and compression modulus, curvature and bending modulus) into a description of membrane protein organization and function. Similarly, one needs to consider specifically the importance of geometric packing criteria for lipid-protein interactions and protein function.

Lipid Bilayer–mediated Regulation of Ion Channel Function by Amphiphilic Drugs

Drugs that at pico- to nanomolar concentration regulate ion channel function by high-affinity binding to their cognate receptor often have a “secondary pharmacology,” in which the same molecule at low micromolar concentrations regulates a diversity of membrane proteins in an apparently

Amphiphile regulation of ion channel function by changes in the bilayer spring constant

Many drugs are amphiphiles that, in addition to binding to a particular target protein, adsorb to cell membrane lipid bilayers and alter intrinsic bilayer physical properties (e.g., bilayer thickness, monolayer curvature, and elastic moduli). Such changes can modulate membrane protein function by altering the energetic cost (DeltaG(bilayer)) of bilayer deformations associated with protein conformational changes that involve the protein-bilayer interface. But amphiphiles have complex effects on the physical properties of lipid bilayers, meaning that the net change in DeltaG(bilayer) cannot be predicted from measurements of isolated changes in such properties. Thus, the bilayer contribution to the promiscuous regulation of membrane proteins by drugs and other amphiphiles remains unknown. To overcome this problem, we use gramicidin A (gA) channels as molecular force probes to measure the net effect of amphiphiles, at concentrations often used in biological research, on the bilayer elastic response to a change in the hydrophobic length of an embedded protein. The effects of structurally diverse amphiphiles can be described by changes in a phenomenological bilayer spring constant (H(B)) that summarizes the bilayer elastic properties, as sensed by a bilayer-spanning protein. Amphiphile-induced changes in H(B), measured using gA channels of a particular length, quantitatively predict changes in lifetime for channels of a different length--as well as changes in the inactivation of voltage-dependent sodium channels in living cells. The use of gA channels as molecular force probes provides a tool for quantitative, predictive studies of bilayer-mediated regulation of membrane protein function by amphiphiles.

GABAA Receptor Function is Regulated by Lipid Bilayer Elasticity

Docosahexaenoic acid (DHA) and other polyunsaturated fatty acids (PUFAs) promote GABA(A) receptor [(3)H]-muscimol binding, and DHA increases the rate of GABA(A) receptor desensitization. Triton X-100, a structurally unrelated amphiphile, similarly promotes [(3)H]-muscimol binding. The mechanism(s) underlying these effects are poorly understood. DHA and Triton X-100, at concentrations that affect GABA(A) receptor function, increase the elasticity of lipid bilayers measured as decreased bilayer stiffness using gramicidin channels as molecular force transducers. We have previously shown that membrane protein function can be regulated by amphiphile-induced changes in bilayer elasticity and hypothesized that GABA(A) receptors could be similarly regulated. We therefore studied the effects of four structurally unrelated amphiphiles that decrease bilayer stiffness (Triton X-100, octyl-beta-glucoside, capsaicin, and DHA) on GABA(A) receptor function in mammalian cells. All the compounds promoted GABA(A) receptor [(3)H]-muscimol binding by increasing the binding capacity of high-affinity binding without affecting the associated equilibrium binding constant. A semiquantitative analysis found a similar quantitative relation between the effects on bilayer stiffness and [(3)H]-muscimol binding. Membrane cholesterol depletion, which also decreases bilayer stiffness, similarly promoted [(3)H]-muscimol binding. In whole-cell voltage-clamp experiments, Triton X-100, octyl-beta-glucoside, capsaicin, and DHA all reduced the peak amplitude of the GABA-induced currents and increased the rate of receptor desensitization. The effects of the amphiphiles did not correlate with the expected changes in monolayer spontaneous curvature. We conclude that GABA(A) receptor function is regulated by lipid bilayer elasticity. PUFAs may generally regulate membrane protein function by affecting the elasticity of the host lipid bilayer.

Linear rate-equilibrium relations arising from ion channel-bilayer energetic coupling

Linear rate-equilibrium (RE) relations, also known as linear free energy relations, are widely observed in chemical reactions, including protein folding, enzymatic catalysis, and channel gating. Despite the widespread occurrence of linear RE relations, the principles underlying the linear relation between changes in activation and equilibrium energy in macromolecular reactions remain enigmatic. When examining amphiphile regulation of gramicidin channel gating in lipid bilayers, we noted that the gating process could be described by a linear RE relation with a simple geometric interpretation. This description is possible because the gating process provides a well-understood reaction, in which structural changes in a bilayer-embedded model protein can be studied at the single-molecule level. It is thus possible to obtain quantitative information about the energetics of the reaction transition state and its position on a spatial coordinate. It turns out that the linear RE relation for the gramicidin monomer-dimer reaction can be understood, and the quantitative relation between changes in activation energy and equilibrium energy can be interpreted, by considering the effects of amphiphiles on the changes in bilayer elastic energy associated with channel gating. We are not aware that a similar simple mechanistic explanation of a linear RE relation has been provided for a chemical reaction in a macromolecule. RE relations generally should be useful for examining how amphiphile-induced changes in bilayer properties modulate membrane protein folding and function, and for distinguishing between direct (e.g., due to binding) and indirect (bilayer-mediated) effects.

Channel Function and Channel-Lipid Bilayer Interactions

Ion channels are integral membrane proteins that form aqueous pores, which span the lipid bilayer moiety of biological membranes and provide for highly selective transfer of ions across the membrane. Ion transfer through the pore can occur at very high rates, and it is possible to use electrophysiological measuring techniques to record the function of single channels in real time. Ion channels are therefore useful for examining many aspects of macromolecular dynamics. The control of channel function is due to transitions between different channel states (conformations). The distribution between these states is determined by the channel’s intrinsic characteristics and by its interactions with the (membrane) environment, neither of which are well understood. We show, using the well-characterized gramicidin A channel as an example, that membrane control of channel function can be rationalized by considering the energetics of channel-bilayer interactions.

Ion distributions in brain during ischemia.

The function of the central nervous system-and other organs-depends upon preservation of ionic gradients across cell membranes. In nervous tissue, the ion gradients are especially important since generation of action potentials and synaptic processes relies on transfer of ions across the plasma membrane. This report describes the fact that anoxia profoundly changes the brain interstitial ion milieu. Impaired ATP regeneration starts the chain of events that cause a breakdown of ion homeostasis. The pronounced ionic changes are not caused by impaired ion pumping but rather by opening of ion channels-probably most importantly via release of transmitter substances. Despite the severity of the ionic changes, the brain interstitial ion environment is readily normalized after the anoxic episode. The ionic disturbances are not the cause of the functional deficits encountered in anoxia but are probably of significance in the irreversible neuronal damage evolving after anoxia.