2

Membrane Lipid PolymorphismRelationship to Bilayer Properties and Protein Function

Richard M. Epand

SummaryBilayers are the most familiar arrangement of phospholipids. However, even as bilayers, phospholipids can

arrange themselves in a variety of morphologies from essentially flat structures found in large liposomes or whenadhered to a flat solid support, to the curved structures found in small liposomes or as bicontinuous cubic phases.Phospholipids can also arrange themselves as curved monolayers, such as in the hexagonal phase, and they can evenform spherical or ellipsoid-shaped micelles. A number of factors will determine the final morphology of a lipidaggregate including the structure of the lipid, the nature of the lipid headgroup and its degree of hydration, and thetemperature. In addition to being interesting in its own right, the property of lipid polymorphism can be applied tounderstand how fundamental intrinsic curvature properties of a membrane alter the physical properties of a mem-brane bilayer. This, in turn, will affect the functional characteristics of membrane proteins, with several possiblemechanisms explaining the coupling of membrane properties with protein function.

Key Words: Bilayer; cubic phase; curvature strain; hexagonal phase; interfacial enzyme catalysis; lateral pres-sure profile; lipid polymorphism.

1. IntroductionIn the past couple of years, there has been an increased interest in the role of lipids in biol-

ogy. There are several reasons for this. In part, it is a component of the “systems biology”approach in which classes of biochemical molecules are considered together, so that onetakes into account the interactions among components. This has led to the establishment ofseveral fields such as genomics, proteomics and, more recently, lipidomics (1), among others.Several groups are now attempting to coordinate approaches to the study of lipidomics, per-haps the first of which was the Lipidmaps Consortium, which led to a suggested new classi-fication system for lipids (2). There has also been technical advances in the applications ofmass spectroscopy to lipid analysis (3). Combined with these approaches, there has been agreater appreciation for the roles of certain lipids as secondary messengers as well as theimportance of lipids in modulating the functional properties of membranes. There has alsobeen a long-standing interest in the role of dietary lipids on human health. This includes thepopular concern about the impact of cholesterol and triglycerides in the diet and their possi-ble relationship to atherosclerosis.

More recently, there has been increased attention to the detrimental health consequencesof trans-fatty acids (4–8), as well as the beneficial effects of ω-3 fatty acids (9–12). Many ofthe biological effects of dietary lipids are a consequence of altering the biophysical properties

From: Methods in Molecular Biology, vol. 400: Methods in Membrane LipidsEdited by: A. M. Dopico © Humana Press Inc., Totowa, NJ

15

of the host’s cell membranes (nonspecific effect), rather than to a more specific effect. Therelationship between lipid polymorphic properties and membrane function that will be ana-lyzed in this article is an example of a nonspecific effect.

2. Lipid Phases2.1. Normal vs Inverted

Pure phospholipids are capable of undergoing transformations from one shape or morphol-ogy to another. This is termed lipid polymorphism, i.e., the ability of lipids to take on struc-tures of different shapes. Some other recent reviews have pointed to the relationship betweenlipid polymorphism and membrane function (13,14). Lipid phases are divided into two gen-eral types: normal and inverted phases. Normal phases are those in which the polar moiety ofthe lipid faces outward from the lipid structure, whereas the nonpolar portion of the moleculemakes up the structure core. The lipid arrangement is opposite in inverted phases: the polargroups face inward and the nonpolar portion occupies the exterior of the structure. Normaland inverted phases are also referred to as type I and type II phases, respectively. Specificexamples will be illustrated below.

2.2. Bilayer/Lamellar Phase

Lipids in biological membranes are arranged primarily, if not exclusively, as bilayers. Thisis sometimes referred to as a lamellar phase, to distinguish it from bicontinuous cubic phases(see Subheading 2.3.) in which the lipid is also arranged as a bilayer. The difference is thatthe cubic phase is a three-dimensional structure, whereas the common kind of bilayer isessentially a flat, two-dimensional structure. Phospholipids that form flat bilayers sponta-neously stack when hydrated, and form repeating lamellae of multilamellar vesicles (MLVs).Hence, these types of flat bilayers are often referred to as lamellar phases to distinguish themfrom bilayers that form cubic phases.

A flat bilayer that is not infinitely large would have hydrophobic edges, which would beexposed to water. This would be a markedly destabilizing feature, although ruptured cellmembranes are suggested to contain fragments of bilayer flat pieces with exposed edges.Because these fragments are large, the edges include only a small fraction of the total surfacearea. A nascent high-density lipoprotein is also made up of a flat disk of phospholipid bilayerprotected at the edges by plasma apolipoproteins. However, these are exceptions, and a muchmore common situation is that lipids in the lamellar phase form spherical vesicles that do nothave exposed edges. When these vesicles are stacked, one inside the other, like an onion, thestructure is called an MLV. These structures are so large (on the order of microns) that on amolecular scale they are locally almost perfectly flat.

MLVs can be converted into unilamellar vesicles. Unilamellar vesicles have advantages forstudying membrane functions such as transport across the bilayer or membrane fusion,because these processes would not be complicated by several events occurring simultane-ously in stacks of bilayers. One way to make unilamellar vesicles is by extrusion throughpolycarbonate filters having pores of uniform size. This process of extrusion produces singlewall, large unilamellar vesicles (LUVs). The size of LUVs can be varied by choosing poly-carbonate filters having different pore diameters. However, LUVs with diameters more thanabout 200 nm are generally contaminated with vesicles that have more than one lamella. It isalso difficult to make vesicles smaller than 50 nm in diameter by this method. The smallestvesicles that can form are usually made by sonication, producing Small Unilamellar Vesicles

16 Epand

(SUV). These have diameters of the order of 20 nm. The bilayer in SUVs has greater curvaturethan that found in larger vesicles, simply because of the morphology of the particle. Thenature of this curvature in SUV is opposite for the two monolayers of the bilayer. The outermonolayer is curved with an expanded headgroup cross-sectional area, whereas the innermonolayer has a contracted headgroup cross-sectional area. The opposite is the case for thehydrocarbon, with the inner monolayer having more expanded hydrocarbon space and a more contracted one for the outer monolayer. SUVs are intrinsically unstable because ofthese curvature effects. To distinguish the two kinds of curvatures of monolayers, the curva-ture of the type found in the outer monolayer is called positive curvature and that of the innermonolayer, negative curvature. The definitions of positive and negative are arbitrary, but theydo reflect the opposite curvatures for the two kinds of bending.

2.3. Cubic Phases

The term cubic phase refers to the symmetry elements in the arrangement of the unit cells.There are many types of cubic phases. Cubic phases can be divided into two general classes:inverted micellar and bicontinuous. In an inverted micellar cubic phase the lipids are packedinto spherical aggregates, with each sphere representing a unit cell that packs together withother unit cells in a cubic array. Each of the micelles is “inverted” in the sense that the polarhead groups are pointing inward, toward the center of the sphere, whereas the surface ofthe sphere is hydrophobic. Because of their hydrophobic exterior, inverted micelles in waterare more likely to form ordered aggregates of cubic phase, when compared with normal phasemicelles; these will disperse in water as single micelles.

The other type of cubic phase is the bicontinuous cubic phase. The term bicontinuousrefers to the fact that both the lipid phase and the water channels are continuous in space (i.e.,one can move along the aqueous channels from one location in the structure to another with-out having to cross over a lipid barrier; similarly, one can move along with lipid for largedistances without having to enter the water phase). This bicontinuous arrangement is accom-plished with several different morphologies, all of which have cubic symmetry but belong todifferent space groups (i.e., they have different symmetry elements). An excellent anddetailed discussion of the various types of cubic phases can be found in the book by Hyde et al.(15). This book also discusses the possible biological relevance of the cubic phase, as doseveral review articles (16–18).

2.4. Hexagonal Phase

In the hexagonal phase, lipids are packed together to form hollow cylinders. The mostcommon kind of hexagonal phase formed in water is the inverted-hexagonal phase, referredto as the HII phase. In this phase, the methyl ends of the acyl chains form the exterior of thelipid cylinders, and the headgroups face the central core of the cylinder, which is filled withwater. These cylinders are packed together with hexagonal symmetry in which each cylinderis surrounded by six other cylinders, hence the name hexagonal phase. Being a type IIinverted phase structure that includes a bundle of cylinders, the exterior of the bundle wouldbe hydrophobic and in contact with water. In addition, a hexagonal array of straight cylinderswould have the entire acyl chains exposed to water at the ends of the cylinders.Nevertheless, HII phases are stable in excess water. Three factors may contribute to this. First,the cylinders may not be straight but have sufficient bending so that they can form a toroidalstructure, thus avoiding the exposure of blunt ends. There is some evidence from electron

Membrane Lipid Polymorphism 17

microscopy for this type of structure. The second factor is that these are very large aggregates,so that the surface-to-volume ratio is small. Thus, destabilizing interactions between waterand the exterior of the hexagonal phase aggregate may be compensated for by favorable inter-actions within the hexagonal phase structure. Finally, there may be a monolayer of lipidsurrounding the HII aggregate and exposing the headgroups of the monolayer. Although thereis no evidence for such a monolayer, its existence would be difficult to detect because itwould represent a very small fraction of the total lipid in the structure.

3. Factors Determining Phase PreferenceAlthough it was mentioned that the bilayer was the most common type of organization of

lipid molecules in biological systems, many of the major lipid components of biologicalmembranes, in isolated purified form, will form nonlamellar phases under ordinary condi-tions of room temperature and excess water. For example, phosphatidylethanolamine (PE)from natural sources will spontaneously form an HII phase in excess water (19,20). Normalmicelles have detergent-like properties and are formed by bile salts, which function biologi-cally as detergents, as well as by lysolipids and gangliosides. Some of the factors that deter-mine what phase will form under a particular set of conditions are considered.

3.1. Lipid Structure

The molecular structure and properties of the lipid molecule will be a major factor in deter-mining the type of phase a lipid will form. This is determined in part by steric factors. Stericinteractions among headgroups as well as the acyl chains are important. For example, PE andphosphatidylcholine (PC) are both zwitterionic lipids with similar chemical structures. Theadditional three methyl groups on the nitrogen of PC contribute a steric component to pre-vent this lipid from forming an inverted phase. The acyl chains also have repulsive stericinteractions that are greater for unsaturated than for saturated lipids, and also greater forlonger chain length or branched acyl chains. These factors can become sufficiently large thateven a lipid such as PC, which normally favors a bilayer arrangement, will readily convertinto a hexagonal phase (21).

Acyl chain double bonds can exist as one of two geometric isomers; either the cis, com-monly found in nature, or the trans-isomer, with its relation to health risks, as mentioned inthe introduction. The presence of a cis-double bond will cause the acyl chain to have a kinkat that position, resulting in increased steric repulsion toward the methyl terminus; thisincreased repulsion can be relieved, at least in part, by converting the bilayer into a hexago-nal phase. The HII phase will be more stable because its curved monolayer will have a largercross-sectional area on the outer, hydrophobic surface, and can therefore accommodate thesteric repulsion caused by the presence of a cis-double bond. Such an increase in the stericrepulsion toward the methyl terminus occurs to a much smaller degree with trans-doublebonds in the acyl chain because this geometry causes less change in the direction of the acylchain. These findings have given rise to the shape concept of lipid polymorphism, with cone-shaped lipids forming structures with positive curvature, such as micelles, whereas invertedcone-shaped lipids would form inverted phases, such as the HII phase.

However, steric factors are not the only properties that will determine the interactionforces among adjacent lipid molecules. In addition, attractive interactions, such as hydro-gen bonding among headgroups, can affect the phase preference. This is likely to be an

18 Epand

additional factor in the preference of PE for inverted phases. Charge repulsion amongheadgroups will have the opposite effect and will inhibit the formation of inverted phases.There are many examples of biological lipids that are anionic. In general, these lipids donot form inverted phases because of electrostatic repulsion among headgroups. However,addition of a cation, such as Ca2+ or lowering the pH to protonate the lipid, in many casescauses the lipid to convert from the lamellar to the hexagonal phase. A prime example ofthis is cardiolipin, a lipid with four acyl chains that readily forms inverted phases, yet onlyafter the negative charge of the headgroup is neutralized, for example, by bindingcations (22).

3.2. Hydration

Intermolecular hydrogen bonding between lipid headgroups competes with hydrogenbonding between a lipid headgroup and water. Thus, in cases where there is more bondingbetween headgroups, such as PE, there is less hydrogen bonding with water and, therefore alower degree of hydration of the lipid. Lower hydration of the lipid will promote invertedphase formation. One way to lower the effective hydration is with salting-out salts thatpromote hexagonal phase formation (23). Another way to reduce the hydration is simply todecrease the amount of water added to the lipid sample. However, there is an additionalfactor; there has to be sufficient water to fill the core of the hexagonal phase cylinders. Thisleads to the “re-entrant phenomenon:” as the hydration is lowered the hexagonal phasebecomes the more stable phase until a point is reached (at even lower hydration) at which thelamellar phase reappears (24).

3.3. Temperature

Temperature is an important factor in regulating lipid polymorphism. This arises as aconsequence of acyl chain splay. Carbon–carbon single bonds between adjacent methylenegroups in the acyl chains can undergo trans- to gauche-isomerization. This is different fromthe interconversion between trans- and cis-double bonds that require rearrangement ofcovalent bonds. Conversion of a trans- to gauche-form simply requires free rotation arounda single bond. The trans-rotamer is more stable because the extensions of the acyl chaincoming off at opposite sides of the C–C bond result in less steric repulsion. At low temper-ature, most of the C–C bonds are trans and the acyl chain is fully extended, approximatinga linear rod. As the temperature increases, entropy drives the conversion of some trans-bonds to gauche. This will result in a deviation of the acyl chain from its linear direction,with the result that the methyl terminal end of the acyl chain will have a larger deviationfrom the bilayer normal. As lipid polymorphism is usually studied in the presence of water,to be more biologically relevant, this usually restricts the range of temperatures to roughly0–100°C. Within this temperature range, some lipids form only one phase and do not exhibitthermotropic phase transitions. However, for those lipids that do exhibit thermotropic phasetransitions, the lamellar phase is favored at lower temperatures and the hexagonal phase athigher temperatures.

The cubic phase is often thought to be an intermediate between the lamellar and the HII phase(25). However, the cubic phase exhibits very slow kinetics, both in formation and conversion toother phases. Therefore, it is often uncertain what the stability of the cubic phase is, relativeto other phases, under any particular condition. It has been suggested that the cubic phase is

Membrane Lipid Polymorphism 19

formed by a pathway different from that of the HII phase (26), rather than as an intermediatein the pathway for formation of the hexagonal phase.

4. Lipid Polymorphism and Membrane PropertiesThere are few examples of nonlamellar structures forming in biological systems;

there are examples of lipid micelles that perform important biological functions, suchas micelles formed by bile salts functioning to disperse triglycerides in the intestine.There also have been some reports about the presence of cubic phases in cells (16,27).The formation of cubic phases in cells would be particularly intriguing because it couldprovide directed paths and barriers for translocating substances that could quickly formand dissipate. In addition to these limited, and in the case of cubic phases, speculativeroles for nonlamellar phases, there is also the modulation of biological functions result-ing from the presence of nonlamellar-forming lipids. The mechanism(s) by whichbilayer physical properties are modulated by the presence of such lipids is discussed inSubheading 4.1.

4.1. Curvature Strain

It is useful to consider the curvature properties of a lipid monolayer. A bilayer in a LUVor MLV will have almost no physical curvature on a molecular scale (i.e., locally, thebilayer is flat). However, the constituent monolayers of the bilayer may have an intrinsiccurvature that is not flat. Despite the fact that its physical shape is essentially flat, if eachmonolayer could bend to its preferred shape, without a change in the polarity of the envi-ronment of any of its groups, it would then achieve its intrinsic curvature. This cannot hap-pen in a symmetrical bilayer because each monolayer would bend in opposite directions,leaving a void between the ends of the acyl chains. However, the arrangement of lipid inthe HII phase can approximate that of a monolayer that has attained its intrinsic curvature.The extent of this curvature can readily be calculated from the lattice spacing of the HIIphase measured by diffraction. To be accurate, this measured intrinsic curvature requires acorrection, which results from the fact that the HII phase does not fill all space if it remainsas perfectly rounded cylinders. There will be voids between the cylinders that have to befilled by gauche to trans-isomerization of the acyl chains that point toward these voids.This will require energy, and as a consequence, the HII cylinder diameter will decrease tolower the extent of this hydrocarbon packing problem. There are experimental ways tocorrect for this factor, and thus an accurate value of the intrinsic curvature can still beobtained. This curvature is usually expressed as an intrinsic radius of curvature, which isdefined as the distance from the center of the cylinder to the pivotal plane; the pivotal planeis the position in the lipid structure whose cross-sectional area does not change when themonolayer bends (28).

A monolayer organized in a structure in which its physical curvature is equal to its intrin-sic curvature will not possess any curvature strain. It will not have any driving force to changeshape. Lipids that spontaneously form highly curved monolayers will have instability becauseof curvature strain when they form a planar bilayer. The curvature energy associated witheach of the monolayers depends on two factors: the intrinsic curvature and the elastic-bending modulus. In other words, the curvature strain of a monolayer will be equal to theenergy required to unbend it from the form in which it has achieved its intrinsic curvature tothe flat structure of the bilayer. This energy per unit area of interface is given by:

20 Epand

where Kc is the elastic bending modulus of the lipid monolayer and R0 is the lipid mono-layer’s spontaneous radius of curvature in excess water. The elastic-bending modulus is ameasure of the stiffness of the monolayer. The easier it is to bend the monolayer, the less cur-vature strain will be acquired by forming a structure whose curvature is different from theintrinsic curvature. The earlier discussion, to be more precise, refers to the mean curvature.There is a second curvature modulus called the Gaussian curvature. This refers to an averageof the sum of positive and negative curvatures. Thus, there can be morphologies, such as“saddle points” (structures shaped like a saddle) that have zero mean curvature but they haveGaussian curvature, which is the sum of negatively and positively curved surfaces, irrespec-tive of their sign. The importance of Gaussian curvature in the stability of membrane fusionintermediates has been recognized (29).

4.2. Lateral Pressure Profile

An alternative formulation of curvature-related instability has been proposed by Cantor(30,31). This approach focuses on the variation of lateral pressure as a function of the positionin the bilayer. Often lateral pressure profiles and curvature strain are alternative ways of describ-ing changes in bilayer properties resulting from membrane curvature strain. If there is a lateralpressure at the methyl terminus of the acyl chains higher than in that present in other regions ofthe bilayer, the scenario is equivalent to the bilayer having negative curvature strain.

Each of the two formulations has their own advantages. Curvature strain is a simpleridea and is more amenable to direct experimental measurement. However, the concept oflateral pressure profile provides a more detailed molecular description and can be moreinformative to interface, providing information about the structure and location of substanceswithin the membrane (32). One of the lines of evidence supporting the concept of the lateralpressure profile is that it correctly predicts the variation of anesthetic potency as a functionof chain length for different classes of molecules (33,34).

4.3. Tilt Modulus

In addition to monolayer bending and lateral pressure at a particular depth in the mem-brane, lipid monolayers can also be subjected to changes in acyl chain tilt (35). It has beencalculated that the tilt modulus involves two major contributions. One contribution arisesfrom the stretching of the hydrocarbon chains on tilt deformation, which also results in lossof chain conformational flexibility. The second contribution is purely entropic, arising fromthe constraints imposed by tilt deformation. The two factors have comparable energies. Thisformulation represents an alternative way of taking into account the factors that lead tocurvature strain.

5. Biological Roles of Membrane CurvatureThere are many specific interactions as well as bulk physical properties that modulate biolog-

ical function. Some bulk properties are intrinsic monolayer curvature, motional properties withinthe membrane (as reflected, e.g., by order parameter gradients), and properties of the membraneinterface, including polarity, penetration of water, and charge. In addition, there is domain for-mation, which reflects the nonuniform distribution of molecules in the plane of the membrane.

0.5K

Rc

02

Membrane Lipid Polymorphism 21

In this regard, there is an interesting example in which bending of a lipid monolayer causesdemixing of membrane lipid components (36). This section focuses specifically on the role ofnonlamellar-forming lipids in modulating certain membrane-related functions.

5.1. Homeostasis

Nonbilayer-forming lipids have been suggested to play an important role in biologicalmembranes by establishing an environment with an optimal balance between stability andplasticity (37). In particular, studies with bacteria have demonstrated that changing thegrowth temperature of the organism leads to an alteration in the lipid composition of themembrane, both in Escherichia coli (38) as well as in Acholeplasma laidlawii (39), to main-tain a constant curvature instability. For the latter organism, it has been suggested that thereis a specific enzyme, diglucosyldiacylglycerol transferase, whose activity is sensitive to thebalance between lamellar and nonlamellar lipids (40). This is likely to be a factor in explain-ing the origin of curvature instability under a variety of conditions. However, the fact thathomeostasis of curvature instability is maintained suggests that this physical property hasimportant biological consequences.

5.2. Membrane Fusion

Membrane fusion is a process in which two planar bilayers must undergo rearrangement intoa nonlamellar structure. Intermediates that have been proposed in membrane fusion involve dif-ferent changes in membrane curvature for the joining of opposing monolayers (41,42). Changesin membrane curvature required to form fusion intermediates can be facilitated by the insertionof protein segments. There has been much interest in the so-called “fusion peptides,” which aresegments of proteins that promote membrane fusion. Several examples of fusion peptidesare known from studies of the fusion of enveloped viruses to target membranes. Several of thesepeptides have been shown to promote membrane fusion by themselves and also increase thenegative curvature strain of membranes (43). Another contributing factor for the tendency ofviral fusion peptides to promote nonlamellar phases is suggested to be their partitioning intohydrocarbon voids and thereby stabilizing highly curved fusion intermediates (44).

Regarding lipid polymorphism, the formation of cubic phases has attracted interestbecause of its similarities to the formation of a membrane fusion pore (45). The lipid-linedaqueous channel that connects one unit cell with another in a bicontinuous cubic phase hasthe same structure as a fusion pore. A lowering of the rupture tension of the membrane byhydrophobic peptides can accelerate the conversion of an intermediate to the fusion pore aswell as to a cubic phase (46). Of course, the bicontinuous cubic phase is a three-dimensionallipid phase with many unit cells, whereas a fusion pore is formed as an isolated structure.Nevertheless, similar changes in membrane properties may promote both processes. In addi-tion, recent evidence proposes to determine the structure of the initial hemifusion intermedi-ate using diffraction methods (47).

5.3. Activity of Membrane-Bound Enzymes

The activity of several integral membrane and amphitropic proteins (proteins that exchangebetween membrane and aqueous compartments) has been shown to be sensitive to membranecurvature strain. These proteins include rhodopsin (48), G proteins (49–51), the proapoptoticBcl-2 proteins, t-Bid (52), and Bax (53) among others. In addition, the assembly of lactosepermease has been shown to be dependent on the presence of PE, a lipid imparting negative

22 Epand

curvature stress (54). More specifically, curvature stress has been suggested to modulate thefree energy and folding of integral membrane proteins (55). The focus will be on the proper-ties of two amphitropic enzymes whose activity is modulated by membrane curvature, yetapparently through different mechanisms. These enzymes are protein kinase C (PKC) andphosphocholine cytidylyltransferase (CT).

The activities of both PKC (56) and CT (57) are enhanced by increasing the negative cur-vature strain of the membrane. However, in the case of PKC there are two lines of evidencethat indicate that there is not a direct relationship between curvature strain and enzyme activ-ity (58). One indication comes from studies with a series of PEs containing 18:1 acyl chainsbut differing in the position of the C–C double bond. The curvature properties of this series oflipids have been determined (59), and the activity of PKC does not correlate with the curva-ture strain measured with membranes containing different members of this homologous seriesof lipids (60). However, the activity of PKC does correlate with properties of an interfacial flu-orescent probe. The situation with CT is different, and indicates that a change in curvaturestrain is the mechanism by which nonlamellar-forming lipids modulate the activity of thisenzyme (61,62). The other indication that the activity of PKC is not directly modulated bymembrane curvature strain comes from studies of the activity of PKC in the presence of lipidsin the cubic phase (63). Spontaneous conversion of bilayers to the cubic phase will result inthe relief of negative curvature strain. In order to compare the activity of PKC in the cubicphase with the activity in the lamellar phase, the cubic phase of mono-olein was converted toa lamellar phase by the addition of progressively larger amounts of phosphatidylserine. Inaddition, the activity of PKC using bilayer membranes made up of dielaidoyl PE that wereconverted into a bicontinuous cubic phase was compared with the addition of a small amountof alamethicin (64). In both systems, the activity of PKC was greater in the cubic phase thanin the lamellar phase. Furthermore, it was shown that this difference was not because of thesmall change in membrane composition but rather to the change of phase. Hence, despite therelief of negative curvature strain, the cubic phase is more potent in activating PKC. Therefore,it is concluded that although the activity of CT appears to be directly coupled withmembrane curvature, the activity of PKC is modulated by nonlamellar-forming lipids by a lessdirect mechanism.

6. SummaryThere is substantial evidence that the presence of nonlamellar-forming lipids is important

for the functioning of cells. There are a number of processes including membrane fusion andthe activity of membrane-bound enzymes that are affected by the presence of these lipids. Themechanism by which these lipids alter membrane properties appears to differ for differentsystems. In addition, other factors such as “fluidity” and lipid domain formation are alsoimportant for some membrane functions.

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26 Epand