Molecular Medicine ProgramΙατρικη Σχολη Πανεπιστημιου ΚρητηςDiomedes E. LogothetisMembrane Structure and ProteinsLigand-gated ChannelsLecture 2November 14, 2007

The first part of the lecture will consider the membrane environment that ion channel proteins reside examining the lipid composition and forces that contribute to the thermodynamic stability of the lipid bilayer. A brief general introduction to membrane proteins and methods used to study them is included. The second part of the lecture will present ion channels that are gated by intracellular molecules (e.g. ATP, G proteins, cyclic nucleotides) as well as ion channels that are gated by extracellular molecules (e.g. the neurotransmitters acetylcholine and glutamate).

Learning Objectives

1. Know the major classes of natural lipids, their structural characteristics and the properties of head groups and hydrocarbon chains

2. Understand the thermodynamic basis of lipid and detergent assembly: the hydrophobic effect

3. Understand the physical connection between the shape of lipid molecules and that of the aggregates they form: critical packing parameter

4. Understand the connection between the configuration of acyl chains, their packing and the properties of the bilayer

5. Know the connection between bilayer structure and dynamics and translational diffusion of lipids and proteins. Experimental approach to the measurement of diffusion of membrane components: Fluorescence Recovery After Photobleaching

6. Understand the characteristics of the fluid phase bilayer as shown by diffraction experiments and computer simulations of lipid dynamics

7. Modes of protein-membrane interaction.8. Prediction of membrane protein structure and topology: Hydropathy analysis and the

‘positive-inside’ rule.9. Lipid-modifications of proteins: hydrophobicity and membrane-binding affinity.10. Modulation of reversible protein-membrane binding: the myristoyl switch.11. Know the mechanism by which the metabolic state of the cell is coupled to membrane

electrical events, such as those leading to secretion of insulin.12. Know the mechanism of activation of G protein-gated K channels, as an example of a

membrane-delimited pathway of regulating the activity of intracellular ligand-gated ion channels.

13. Modulation of Ion channels by soluble second messengers14. Sensory transduction: Know the role of CNG channels in phototransduction. Understand

how the balance of CNG and K channels gives rise to the "dark current", which is inhibited during a light flash.

15. Know the subunit composition of nicotinic ACh channels and general topology of the a subunits.

16. Know the general activation mechanism for NMDA and non-NMDA channels and role in LTP.

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Readings:

Notes Alberts B., Johnson A., Lewis J., Raff M., Roberts K., Walter P. Molecular Biology of the

Cell. Fourth Edition, Garland Science. pp. 583-614 and pp. 631-657. R. B. Gennis. Biomembranes: Molecular Structure and Function, Springer-Verlag, 1989 Dr. Stephen H. White’s lab, at University of California at Irvine, maintains an interesting

Web site you should visit: http://blanco.biomol.uci.edu

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Membrane Structure

Biological membranes are formed by two layers, or leaflets, of lipids. The outer

surfaces of this bilayer are hydrophilic and exposed to water, whereas the interior of the

bilayer is shielded from water and forms the hydrophobic core of the membrane. The

main function of biological membranes is to act as permeability barriers, defining the

inside and outside of the cell and delimiting functionally different compartments. This

function alone, however, would not require the existence of the huge diversity of lipidic

constituents found in nature. If the only function of membranes were to act as

permeability barriers, it is reasonable to believe that just a few lipid species would be

sufficient.

Indeed, it has become evident that lipids perform a variety of functions in the

physiology of the cell, some of which will be briefly discussed below.

Lipids: chemical structures and classification

Two features common to all lipids are a polar portion, or head group, which is

exposed to the aqueous medium, and a non-polar portion, which is buried within the

interior of the bilayer.

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The major classes of lipids are:

Glycerophospholipids are the most abundant lipids, in which glycerol forms the

common backbone of the molecule, with one of its hydroxyls linked to a phosphate

via a phosphoester bond. The most common nomenclature

used to describe the chemical structure of these lipids is the

stereospecific numbering (sn) system.

If the glycerol is drawn in a Fisher projection with the middle

hydroxyl on the left, the three carbons are numbered as

shown in the figure, and indicated as sn-1 for C(1), etc.

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Glycerophospholipids include several subclasses:

- 1,2-diacylphosphoglycerides or phospholipids, in which two of the glycerol

hydroxyls (sn-1 and sn-2) are linked via ester bonds to fatty acid chains and the

phosphate is at the sn-3 position. These are the most abundant lipids in

eukaryotic and prokaryotic cells, excluding Archaebacteria, in which the

phosphate is at the sn-1 position and the chains are linked to the glycerol via

ether rather than ester bonds.

- Lysophospholipids are phospholipids from which one of the acyl chains is

missing.

- Plasmalogens are phosphoglycerides in which one of the hydrocarbon chains is

linked to glycerol via a vinyl-ether bond. Plasmalogens with an ethanolamine

head group are abundant in myelin and in cardiac sarcoplasmic reticulum.

- Cardiolipids are formed by the linkage of two phospholipids via phosphoester

bonds to the two outer hydroxyls of a glycerol moiety, hence the alternative name

of diphosphatidylglycerols.

They are found in significant

amounts in the inner membrane

of mitochondria and in

chloroplasts, but are rare in

other membranes.

Glycoglycerolipids are lipids in which the sn-3 position of glycerol is linked to a

carbohydrate via a glycosidic bond, rather than forming a phosphoester bond.

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Monogalactosyldiacylglycerol has been named “the most abundant lipid in nature”,

since it constitutes half of the thylakoid membrane in plant chloroplasts (the R

groups in the figure represent the hydrocarbon chains of fatty acids). However, they

are rare in animals.

Phosphosphingolipids, represented by sphingomyelin in the figure on p. 3, contain a

backbone formed by sphingosine, rather than by glycerol. The details of the core

structure of sphingolipids are illustrated in the diagram of the ganglioside GM1.

Sphingosine is an

amino alcohol in

which the sn-2 OH

is substituted by an

amino group and a

hydrocarbon chain

is attached via a

vinyl bond to the sn-1 carbon, leaving the sn-1 hydroxyl unreacted. A fatty acyl

chain is linked to the sn-2 amino group via an amide bond, forming ceramide. As in

glycerophospholipids, a phosphate bearing the head group is attached to the sn-3

position via a phosphoester bond. The amide and the hydroxyl group give these

lipids the ability to form intermolecular hydrogen bonds, which may be significant in

establishing interactions with proteins or in the formation of specialized membrane

regions, or microdomains.

Glycosphingolipids are sphingolipids in which the sn-1 hydroxyl of ceramide is linked

to a carbohydrate via a glycosidic bond. The carbohydrates, which constitute the

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head groups of these lipids, vary from a single sugar to very complex polymers.

Monogalactosyl ceramide is the most abundant component of the myelin sheath in

nerve. Gangliosides contain oligosaccharides with one or more molecules of anionic

sialic acid, while neutral oligosaccharides are contained in globosides.

Glycosphingolipids are usually minor components of the outer leaflet of plasma

membranes of animal cells, but they are significant in epithelial cells. The blood

group antigens consist of glycosphingolipids on the surface of erythrocytes.

Sterols, represented by cholesterol in the figure above, are structurally separate

from the previous lipid classes. The membrane-embedded portion of the molecule

consists of a stiff ring structure and a short side chain, while the surface exposed

portion is limited to a single OH group. The stiffness of the ring structure leads to

changes in the dynamics and packing of the surrounding lipids. Cholesterol is found

in animal cells, where it can contribute as much as 30% of the mass of lipid

membranes, whereas plants contain other sterols.

Head groups

In addition to the carbohydrates already mentioned, a variety of polar head

groups, differing in size, charge and chemical properties, are found in lipids. The

structures of the most common head groups are shown in the context of a typical

phospholipid, where the phosphate group is common to all head groups.

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An unmodified phosphate represents the smallest head group, such as in phosphatidic

acid, which carries a double negative charge at neutral pH. Addition of choline or

ethanolamine, both positively charged at physiological pH, result in formation of the

neutral head groups of phosphatidylcholine (PC) and phosphatidylethanolamine (PE),

two of the most common lipids. Additional negatively charged head groups are formed

by modification of the phosphate with the zwitterionic amino acid serine, to give

phosphatidylserine (PS), or with the uncharged glycerol or inositol moieties, yielding

phosphatidylglycerol (PG) and phosphatidylinositol (PI), respectively. The individual

charges contributed by the various lipid head groups are the major determinant of the

overall electrostatic properties of biological membranes. Although only a minor

constituent of cell membranes, phosphatidylinositol is the precursor of many important

signal mediators generated by phosphorylation of several hydroxyls on the inositol by

specific kinases.

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Hydrocarbon chains

An even greater variety exists in the types of hydrocarbon chains that are

attached to lipids. The table below lists some of the more abundant acyl chains,

including their common names and a useful condensed nomenclature.

The first number indicates the number of carbons, or methylene units, in the

chain, whereas the number on the right of the colon indicates its degree of unsaturation,

i.e. the number of C=C bonds in the chain. For unsaturated chains, the isomeric form of

the double bond, whether cis or trans, may also be given, as well as the number of the

first carbon at which the double bond is located, sometimes preceded by a greek letter

. Acyl chains with even numbers of carbons are more abundant than those with odd

numbers of carbons. The most common lengths are C16, C18 and C20, while the most

common unsaturated chains are 18:1, 18:2, 18:3 and 20:4. The double bonds are

usually in the cis configuration and in multiple unsaturated chains they are not

conjugated, i.e. they are separated by at least two C-C bonds. A large fraction of

phospholipids have one saturated and one unsaturated chain, the latter usually linked to

the sn-2 position of glycerol in animal cells.

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Thus, the number of possible combinations of head groups and acyl chains gives

rise to a huge variety of lipid structures. As mentioned above, membranes within an

organism do not contain uniform mixtures of these various lipids. Not only are cells in

certain tissues enriched in specific types of lipids, but also different cellular organelles

contain membranes formed by unique mixtures of lipids. Mitochondria, for example, are

rich in cardiolipin and, indeed, the activity of cytochrome c oxidase, the component of

the respiratory electron transfer chain responsible for the final step of oxygen reduction,

is dependent on the presence of this lipid.

Such fine-tuning of lipid composition extends even further, as demonstrated in

the plasma membrane of human erythrocytes, in which the inner leaflet contains a

different lipid mixture than the outer leaflet.

Thus, with respect to neutral

phospholipids, the extracellular leaflet is

enriched in phosphatidylcholine and

sphingomyelin, both bearing a

phosphorylcholine head group, whereas

phosphatidylethanolamine is localized

preferentially in the inner leaflet. Most

striking, though, is the absolute exclusion

from the outer leaflet of the anionic phospholipid phosphatidylserine. Such asymmetric

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distribution of amino-phospholipids is believed to be maintained by ATP-requiring

enzymes, called translocases, which catalyze the transbilayer transport of these lipids.

The physiological function of the surface expression of phosphatidylserine is related to

the clearance of aged red cells. After release into the bloodstream, a red cell remains in

circulation for 120 days on average. During this time, oxidative stress progressively

deteriorates the biochemical machinery of the cell, including the enzymatic activities

responsible for the segregation of this lipid. Eventually, phosphatidylserine appears on

the outer surface of the erythrocyte, and this signal is recognized by phagocytic cells

within the spleen, which clear the aged cell from circulation.

Platelets constitute another system in which the asymmetric phosphatidylserine

distribution is physiologically significant. In this case, the anionic lipid, which is normally

absent from the surface, becomes expressed on the outer membrane leaflet upon

activation of platelets at a site of injury. Together with calcium, phosphatidylserine is an

essential activator of blood coagulation factors.

Under physiological conditions, as well as in culture, cells may undergo

apoptosis, a controlled process of suicide. Thus, during development of the immune

system, self-recognizing thymocytes are eliminated in order to prevent autoimmune

reactions. One of the signatures of an apoptotic cell, in addition to DNA degradation, is

the expression of phosphatidylserine on the extracellular surface. This observation has

been used to develop an apoptosis test, which makes use of fluorescence-labeled

recombinant annexin, a normally cytoplasmic protein that binds specifically to

phosphatidylserine via calcium bridges.

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Phospholipases and lipids as precursors of second messengers

Another essential function of lipids is as precursors of many second messengers

that participate in a variety of signaling pathways. Each of these molecules is

generated by cleavage of a lipid precursor at a specific bond. For this purpose, several

classes of enzymes, named phospholipases, exist.

Phospholipase A2 enzymes hydrolyze the ester bond

between glycerol and the sn-2 acyl chain of

phospholipids, thus generating a lysophospholipid

and a free fatty acid. Among the released fatty acids

is arachidonic acid, which is oxidized to other active

metabolites, such as prostaglandins, which are

involved in inflammation and other patho-

physiological processes. Analogous enzymes, called phospholipase A1, hydrolyze the

ester bond at the sn-1 position, but they are not yet well characterized.

Phospholipase C catalyzes the hydrolysis of the phosphoester bond, releasing a

soluble phosphorylated head group and a diacylglycerol. Of widespread significance is

the cleavage of phosphatidylinositol(4,5)-bisphosphate (PIP2), which generates two

second messengers: the water-soluble inositol(1,4,5) trisphosphate (IP3), which causes

the release of calcium from the endoplasmic reticulum by binding to the IP3 receptor

found in the membrane of this organelle, and the membrane-bound 1-stearyl-2-

arachidonyl diacylglycerol, which is responsible for activation of protein kinase C and

enhanced phosphorylation of downstream signaling proteins.

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Phospholipase D instead generates phosphatidic acid and a free head group.

Phosphatidic acid is becoming recognized as a second messenger, for example as an

activator of the NADPH oxidase responsible for the generation of reactive oxygen

products in neutrophils activated upon binding to immunoglobulin-coated bacteria.

Hydrophobicity and thermodynamics of lipid assembly

In this section, we will try to answer semi-quantitatively the following questions:

Which intermolecular forces are involved in the assembly of lipid membranes? What is

the thermodynamic basis for the formation and stability of the bilayer?

Three basic forces contribute to the stability of lipid aggregates:

The van der Waals force, which is a short-range electrostatic interaction

between instantaneous dipoles on adjacent molecules. This interaction develops as a

result of the formation of a dipolar charge in one molecule, due to fluctuations of its

electronic-nuclear distribution, and the instantaneous induction of a dipole of opposite

orientation on an adjacent molecule. This dipole-induced-dipole interaction stabilizes

the overall interaction between the two molecules and is proportional to the

intermolecular contact surface. These van der Waals interactions occur among all

types of molecules, between head groups at the water-bilayer interface as well as

between hydrocarbon chains in the interior of the membrane.

The electrostatic force is also responsible for the stronger ionic interaction

between charged groups and for the formation of hydrogen bonds between hydrogen-

bond donors, such as NH and OH, and acceptors, such as CO, on adjacent

sphingolipids. Hydrogen bonding is particularly extensive between water molecules

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and is the origin of many of its solvent properties, such as its dielectric constant.

Attractive intermolecular ionic interactions form between the head groups of zwitterionic

lipids, such as phosphaditylcholine, whereby the negatively charged phosphate on one

head group interacts with the positively charged ammonium ion of the choline on a

nearby head group.

However, the greatest contribution to the stability of the membrane bilayer comes

from the hydrophobic force, or hydrophobic effect. Its physical origin can be

understood as follows. A hydrophobic molecule, such as a hydrocarbon chain, placed

in water must be surrounded by water molecules interacting with it. However, whereas

water-water interactions are stabilized by intermolecular hydrogen bonds, these

favorable polar interactions cannot be established between water and a molecule that

does not have hydrogen-bond donor or acceptor groups. Therefore, although bound

water molecules still maintain most of their hydrogen bonds with free water molecules,

some favorable electrostatic interactions are lost when water binds to the hydrocarbon

chain. This loss of hydrogen bonds represents an energetic, or enthalpic, penalty for

the system. However, the entropic cost of immobilizing water molecules on the surface

of the hydrocarbon chain is much more significant than this enthalpic penalty. The

water molecules bound to the hydrophobic surface lose the rotational and translational

degrees of freedom they had in pure liquid water. Because the free energy of the

system, G, is given by

G = H T S

where H and S represent the contributions of enthalpy and entropy, respectively, the

reduction in the S term due to immobilization of water on the hydrophobic surface

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leads to an increase in the G of the system. For analogous reasons, placing water

molecules within the hydrophobic core of the membrane is also very unfavorable.

However, if separate hydrocarbon chains in water aggregate and pack tightly, the water

molecules immobilized on their surfaces can be released back into the bulk, thus

regaining their lost degrees of freedom. This leads to an overall increase in the S term

and a reduction in the G of the system.

Thus, the thermodynamic basis for the stability of the lipid bilayer lies in the

entropy difference between a system consisting of isolated hydrocarbon chains coated

with immobilized water molecules and a system in which aggregation of hydrocarbon

chains and formation of a hydrophobic bilayer phase lead to release of solvation water

back to the bulk aqueous phase. Additional stabilization arises from the enthalpic

contributions of van der Waals interactions between lipid chains as well as ionic and

H-bonding interactions between lipid head groups.

The hydrophobicity of a molecule can be determined quantitatively by measuring

its distribution, or partition, in a solvent system composed of water and an immiscible

hydrocarbon phase, such as hexane. The equilibrium constant, or partition coefficient,

K of the solute in this system is defined as:

where the [X] values represent the solute concentration in mole fraction units in water

and in the hydrocarbon phase, respectively. The partition coefficient K is related to the

standard state free energy of transfer, G0trans, of the solute from water to the

hydrocarbon phase

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where the 0 values represent the standard chemical potentials of the solute in water

and in the hydrocarbon phase, respectively, R is the gas constant (1.987 cal °K-1 mol-1)

and T is the absolute temperature (°K).

It turns out that the hydrophobicity, as measured by G0trans, is proportional to the

surface of contact between the hydrophobic solute and water, which determines the

number of water molecules that would be constrained. The proportionality constant for

transfer of an alkane chain from water into a hydrocarbon phase is found to be

G0trans  25 cal / Å2. Based on surface area, each additional methylene (CH2) group

contributes ~ 800 cal/mol to the hydrophobicity of the chain. At 25°C, this contribution

increases the partition coefficient K of the alkane chain by a factor of ~4 in favor of the

hydrocarbon solvent. The partition coefficient of water in hexadecane (C16) indicates

that the water concentration within the membrane is in the millimolar range,

corresponding to about 1 water molecule per 1,000 phospholipid.

Detergents and micelle formation

A short-chain alkane, at very low concentration, can be dissolved in pure water.

However, as more alkane is added, a concentration is reached at which a separate

phase forms, into which any additional alkane partitions. This critical concentration is

called the solubility limit.

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Detergents and lipids, however, are amphiphilic molecules. For example, sodium

dodecyl sulfate (SDS), a detergent commonly used as a denaturant for protein gel

electrophoresis, has a 12-carbon long chain terminated by a charged sulfate group. Up

to a critical concentration of  1-2 mM (in 0.1M Na+), SDS dissolves in monomeric form.

Upon reaching the critical concentration, however, it forms a new phase composed of

spherical aggregates called micelles. Each SDS micelle contains 100 monomers,

whose hydrophilic head groups delimit the water-exposed surface while their methylene

chains form the “oily” interior. Additional SDS increases the concentration of micelles,

while the concentration of monomers in solution remains constant. The critical

concentration in this case is called the critical micellar concentration (CMC). Since the

CMC is a measure of hydrophobicity, its value is a function of the chemical structure of

the amphiphile. For example, detergents with longer chains have lower CMCs, as

shown by decylmaltoside (10-C long, CMC=1.5mM) versus dodecylmaltoside (12-C

long, CMC=0.12mM).

Biological phospholipids, with two long methylene chains, have extremely low

CMCs, below 1010 M. For this reason, exchange of lipids between membranes is

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facilitated by water-soluble cytosolic proteins, called phospholipid transfer proteins,

which bind lipid monomers and carry out the one-for-one exchange.

Lipid structure, lipid shape and lipid aggregate shape

The high-resolution structures of several lipids have been determined by x-ray

diffraction, using lipid crystal containing very little hydration water. A few examples are

given in the figure below.

A few noticeable features of these structures are:

Within the crystals, the lipid are found in a lamellar arrangement, with the hydrophilic

and hydrophobic groups organized in stacked bilayers

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The acyl chains are fully extended, or in the all-trans configuration, with the

exception of the first 2 methylenes in the sn-2 chains of PC and PE, which are

oriented parallel to the crystallographic bilayer plane

The tilt of the acyl chains away from the normal to the bilayer surface increases from

PE, in which they are virtually perpendicular, to PC , where the angle is 12°, to the

cerebroside, where the angle is 41°.

The glycerol is oriented perpendicular to the bilayer plane, whereas the choline and

ethanolamine head groups are almost parallel to the plane. In fact, the amino group

of ethanolamine interacts with the unesterified oxygens of an adjacent molecule

A closer examination of the structural parameters of a PC molecule allows us to

understand the reason for the differences in packing and acyl chain orientations among

the various lipids.

The volume occupied by

the molecule can be divided

into two parts: a polar region,

which includes the head group

and the glycerol, and a

hydrophobic region, comprising

the two acyl chains, except the

first two carbons of the sn-2

chain. Each region also defines

an area given by its projection onto the plane of the membrane: the head group cross-

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section area, S, and the acyl chain area, 2, where is the cross-section area of each

acyl chain. For saturated chains in the all-trans configuration, 19Å2. The relationship

between these two areas determines the degree of tilt of the acyl chains in the crystals,

as well as the shape of the aggregates formed by the lipid in aqueous solution. If

2  S, the chains will tilt to maximize the intermolecular van der Waals contacts and

accommodate the larger head group. This occurs in PC, where choline occupies an

area S50Å2. On the other hand, in PE S39Å2, so that 2  S and the chains can

attain optimal packing without tilting.

These qualitative considerations can be formulated in a more quantitative, albeit

empirical, formalism that allows us to predict the shape of the aggregate that each lipid

forms in solution. This formulation is based on the concept of the critical packing

parameter, which is defined as v  lSo, where v is the volume of the hydrocarbon

portion of the molecule, l is the maximum length of the acyl chain, and So is the optimal

surface area occupied by the molecule at the interface between the aggregate and

water. So is determined by the balance of repulsive and attractive interactions between

head groups, and is sensitive to solution conditions, such as ionic strength, divalent

cations and pH. The ratio v/l is analogous to the cross-sectional area of the

hydrocarbon portion (2). The molecular shapes of amphiphilic molecules

corresponding to each value of the critical packing parameter are illustrated in the figure

below, together with the shape of the aggregate they form in solution.

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Phospholipids, in general, have a good match between the areas of the two

molecular regions, and therefore they tend to naturally assemble in planar membranes,

forming a so-called bilayer phase. Under certain conditions, however, some of them

tend to form non-bilayer structures, consisting of tubular assemblies in which the head

groups face the interior lumen filled with water while the acyl chains, oriented outwards,

contact the chains from nearby cylinders. This macroscopic structure is called an

inverted hexagonal phase, HII, because the hydrophobic cylinders pack in a hexagonal

pattern. Lysophospholipids and most detergents, in which the single hydrocarbon chain

has a much smaller cross-sectional area than their head groups, form spherical

assemblies and give rise to a micellar phase. Both of these lipid phases are thought to

form locally at sites of membrane fusion.

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Fluid-bilayer structure

X–ray diffraction structures of lipid crystals show the bilayers as planar and well-

ordered structures, with all-trans acyl chains. Similarly, lower resolution TEM images

convey the impression that biological membranes are flat slabs, in which the lipid head

groups form smooth, planar surfaces. However, fluid-phase lipid bilayers are very

different from these pictures. The ‘structure’ of fluid bilayers has been determined by x-

ray and neutron diffraction methods using multilamellar stacks of bilayers, which present

a periodic order in the direction perpendicular to the membrane plane. This one-

dimensional order allows the measurement of the distribution of matter along the bilayer

normal. Thus, the ‘structure’ of a fluid bilayer represents the time-averaged spatial

distribution of structural groups of

the lipid (carbonyls, phosphates,

double bonds, etc.) projected onto

the axis perpendicular to the

bilayer plane. These distributions

give the probability of finding a

particular structural group at a

specific location along the axis,

which represents the distance

from the center of the hydrophobic

core of the bilayer.

The structure of a fluid

bilayer of dioleoylphosphatidyl-

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choline (DOPC, di-C18:1 cis-9) is shown in panel (b). The gaussian peaks give an

accurate representation of the true thermal motion of the molecules, which is a

fundamental property of fluid bilayers that plays a critical role in the interaction of

peptides and proteins with lipid membranes.

Several features of the fluid DOPC bilayer are important. First, the great amount

of thermal disorder is revealed by the widths of the probability density peaks, as

illustrated by the 10Å widths of the PO4 in the head groups and of the C=C acyl chain

group. The width of the C=C group is a vivid graphical representation of the effect of

the random configurational rearrangements occurring within the acyl chains, which

originate from thermally activated trans-gauche bond isomerizations. Second, the

overall thermal thickness of the interfacial region, defined by the distribution of the water

of hydration, is 30 Å, equal to that of the hydrocarbon core of the bilayer. As

illustrated by the end view diagram in panel (b) of the figure above, a peptide in an

a-helical conformation has a cross-sectional diameter of ~10 Å and can be easily

accommodated within the 15 Å thickness of the interface. Third, the physico-chemical

environment in the interface region is highly heterogeneous, presenting a steep but not

abrupt change from the solvation properties of water to those of the membrane

hydrocarbon core. Many membrane-supported reactions and protein-protein or lipid-

protein interactions occur in this heterogeneous environment. As illustrated in panel (a)

of the figure, a transmembrane a-helical peptide of twenty amino acids can be

completely accommodated within the hydrophobic core of the bilayer, whereas longer

peptides will have their ends protruding into the interfacial region. This imposes

constraints on the allowed amino acid sequences of the peptide, as discussed later.

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These conclusions drawn from the two-dimensional representation of the fluid

bilayer structure are fully confirmed by the three-dimensional pictures of bilayers

obtained by computer simulations of the molecular dynamics of lipid bilayers. A

snapshot of the transversal cross section of the bilayer extracted from one of these

simulations is shown in the figure below.

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The acyl chains, represented by the gray lines, are very disordered, each with

several kinks introduced by gauche bond configurations and only short all-trans

segments. The head group atoms, shown in red, are distributed over a wide range of

depths, and the surface delimiting the head-group region from the hydrocarbon region is

very rough. In agreement with the neutron diffraction data, water molecules,

represented by the blue oxygen atoms and white hydrogen atoms, are found deep in

the interfacial region, indeed as far as the boundary of the hydrophobic core.

Despite the disorder and the high degree of conformational fluctuations, the acyl

chains maintain tight packing and good van der Waals contacts, in agreement with the

minor increase in specific volume at Tm. Intrachain motions and translational diffusion of

individual molecules occur via thermally induced structural and packing fluctuations of

the surrounding molecules. This dense and crowded hydrophobic environment is the

origin of the high performance of thin biological membranes as permeability barriers, as

well as of the high cooperativity of lipid phase transitions.

Based on these experimental and theoretical results, the common illustrations,

which depict bilayers as two smooth surfaces separating polar and apolar regions, must

be considered misleading.

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Membrane Proteins: Structure and Interactions

Prediction of protein topology

Identification of protein transmembrane domains – Sequence hydropathy analysis.

The difficulties of membrane protein crystallization and structure determination,

on the one hand, and the abundance of sequence information, on the other, have led to

efforts to develop theoretical methods for the prediction of the location of potential

transmembrane domains. Perhaps the most widely used of such methods is one based

on sequence hydropathy analysis.

The first step in such analysis is to evaluate the degree of hydrophilicity or

hydrophobicity of the protein along its amino acid sequence. Because of the

hydrophobic environment in the bilayer core, a membrane-spanning protein segment is

expected to contain a preponderance of apolar amino acids. The transmembrane

sequence should also be folded, either as an a helix or as a strand in a barrel, so

that peptide H-bonds are satisfied intramolecularly rather than by bringing bound water

into the hydrophobic bilayer core. Thus, the method attempts to find the location and

number of transmembrane segments based on the relative hydrophilic and hydrophobic

properties of contiguous stretches of the amino acid sequence.

For this purpose, hydropathy scales have been devised to rank the relative

hydrophilicity and hydrophobicity of the 20 amino acids. One of these scales, proposed

by Kyte and Doolittle in 1982 (J. Mol. Biol. 157: 105-132), is still in widespread use.

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This scale was defined using the water-vapor partition coefficient or standard

free energy of transfer, G0transfer, of the amino acid side chains as well as their degree of

surface exposure in proteins of known crystallographic structure. The hydropathy

indexes are normalized between 4.5 and 4.5, with positive values indicating that free

energy is required to transfer the side chain to water and the amino acid is considered

hydrophobic. Conversely, negative values indicate that free energy is released upon

transferring the side chain into water and the amino acid is hydrophilic.

Given an amino acid sequence, the residue letter code is substituted with its

corresponding hydropathy index to give the sequence hydropathy profile. A window of

odd length, usually 7–13 residues, is scanned along the sequence. The hydropathy

index values within the window are summed, and the average hydropathy value of the

segment is computed by dividing the sum by the size of the window, as shown below.

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E I T W I V G M V I Y L L M M G A

i = 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

| sliding window |

Hi= -3.5 4.5 -0.7 -0.9 4.5 4.2 -0.4 1.9 4.2 4.5 -1.3 3.8 3.8 1.9 1.9 -0.4 1.8

Using a window size w = 7, the first value of the sequence hydropathy profile, Dl,

corresponds to position 4. The general recursive formula for the computation of Dl is

D4 = (-3.5+4.5-0.7-0.9+4.5+4.2-0.4) / 7 = 1.1

D5 = (4.5-0.7-0.9+4.5+4.2-0.4+1.9) / 7 = 1.87

Finally, a threshold value for the segmental hydropathy must be selected to

decide when a stretch of amino acids may be considered hydrophobic enough to be a

potential transmembrane protein sequence. For the Kyte-Doolittle scale, this threshold

value is usually taken to be 1.0-1.25.

Since the thickness of the bilayer hydrophobic core is ~30Å and each amino acid

contributes 1.5Å to the length of the a helix, a potential transmembrane segment in this

conformation is expected to be at least 20-residue long. Thus, a putative a-helical

transmembrane segment should be identifiable by a consecutive stretch of ~20 values

of Dl above the hydropathy threshold value.

As an example, the figure below shows the plot of the hydropathy profile of

glycophorin A, as it appeared in the original Kyte and Doolittle paper.

28

A sliding window of 7 residues was used to compute each value of hydropathy

index. However, the authors reported the sum of the indexes for the residues within the

sliding window rather than the average value, as it is now common practice (i.e. the

hydropathy index in the figure is 7Dl). The single a-helical transmembrane segment is

easily identified in the region of residues 73-95.

The “positive inside” rule

Once the putative transmembrane segments have been identified, their

orientation in the membrane must be chosen. Statistical analysis of single- and multi-

spanning proteins indicated that their topological determinants may reside in the polar

and loop regions flanking the transmembrane segments. In particular, the distribution of

positively charged residues, arginine and lysine, was well correlated with the topology.

In a sample of bacterial inner membrane proteins, the frequency of Arg + Lys residues

was 4-fold higher in the cytoplasmic than in the periplasmic membrane flanking regions.

A similar bias, though not as strong, has been found to apply to proteins from higher

organisms as well.

Thus, the “positive inside” rule, first proposed by G. von Heijne, is thought to be a

fundamental determinant of the topology of most integral membrane proteins. Clusters

29

of positively charged amino acids found near one end of a predicted transmembrane a

helix, identify it as the cytoplasmic end.

Lipid-anchored proteins

Intrinsic proteins are anchored to membranes of eukaryotic cell not only via

transmembrane domains, but also via covalently attached hydrocarbon chains. Four

classes of lipid-anchored proteins are distinguished by the type of chain and linkage.

1. Glycosylphosphatidylinositol (GPI)-anchored proteins are a heterogeneous

family of proteins found on the exoplasmic surface of cell membranes. The GPI

anchor is formed by a phosphatidylinositol linked, by N-acetylglucosamine, to a

polymannose chain and a phosphoethanolamine. The linkage between protein and

anchor is always located at the carboxyl-terminal amino acid. The GPI anchor is

always added post-translationally after proteolytic removal of a peptide, 17-31

residues long, from the C-terminus of a protein precursor. GPI-anchored proteins

include hydrolytic enzymes, (alkaline phosphatase, acetylcholine esterase, 5’-

nucleotidase), adhesion proteins (neural cell adhesion molecule or N-CAM),

receptors (FcRIIIB, a neutrophil receptor for Fc region of immunoglobulin G).

GPI-anchored proteins can be released from the surface by treatment with

exogenous phosphatidylinositol-specific phospholipase C.

2. N-Myristoylated proteins are found anchored to the cytoplasmic face of the plasma

membrane and to the membranes of other organelles. The 14-carbon chain is

attached co-translationally via a stable amide linkage to an N-terminal glycine

residue. Examples of N-myristoylated proteins are the protein tyrosine kinase p60src

and the catalytic subunit of cAMP-dependent protein kinase, also known as PKA.

30

3. S-prenylated proteins are anchored to the cytoplasmic plasma membrane surface

by a 15-carbon farnesyl or a 20-carbon geranylgeranyl unsaturated chain. The thio-

ether linkage of the anchor to a cysteine residue in the protein is catalyzed by a

farnesyl-transferase. The cysteine is initially the fourth residue from the C-terminus

of the protein. However, after the chain is attached, the 3 C-terminal residues are

cleaved by a protease and the carboxyl group of the cysteine is methylated.

4. S-palmitoylated proteins are anchored to the plasma membrane by a 16-carbon

saturated palmitic chain via a thio-ester linkage. This modification may occur by

spontaneous reaction of palmitoyl-coenzyme A with a protein already associated

with the membrane or may be catalyzed by a membrane-associated palmitoyl-

transferase. S-palmitoylation can take place on exposed cysteines anywhere in the

protein. Contrary to thio-ether bonds, a thio-ester linkage is labile and is hydrolyzed

under mild basic conditions. Proteins known to be palmitoylated include p21ras, the

glycoprotein hemagglutinin of influenza virus, the mammalian transferrin receptor

31

and the visual receptor rhodopsin, which is a seven-helix transmembrane

protein.In many cases, doubly acylated proteins are generated first by

co-translational N-myristoylation and, after interacting with the membrane, by

S-palmitoylation.

The protein affinity for the membrane conferred by these lipid anchors has been

estimated by studies of the equilibrium binding of lipid-modified fluorescent peptides.

The results are summarized in the table below.

On the right column, the values of the effective dissociation constant, Kdeff, are

listed. The binding energy of fatty acids and acylated peptides to phospholipid bilayers

increases linearly with the number of carbons in the chain. The slope of 0.8 kcal mol-1

per -CH2 group is equal to that found for the partitioning of the neutral form of a fatty

acid from water into a bulk alkane phase. The values of Kdeff for myristoyl and palmitoyl

anchors reflect the increase in G0transfer upon addition of two methylenes. Thus, the

binding energy of acylated peptides and proteins to membranes is due to the classical

hydrophobic effect. The membrane affinities of whole proteins are estimated to be

about 10-fold lower than those for short peptides, in other words their Kdeff values are

about tenfold higher. Thus, a myristoylated protein is expected to have a Kdeff 0.8mM

32

while the concentration of lipids in a cell is approximately millimolar. Since the lipid

concentration is comparable to Kdeff, simple myristoylation provides barely enough

hydrophobic energy to attach a protein to a phospholipid bilayer. Other factors, such as

electrostatic interactions between amino acid side chains and phospholipid head

groups, may help in partitioning these proteins to the membrane. These relatively weak

membrane-protein affinities create the potential for dynamic and reversible membrane

localization of these proteins, which may be targets of metabolic modifications leading

to changes in their binding affinity and effective concentration at the membrane.

Modulation of reversible protein-membrane interactions: The myristoyl-

electrostatic switch

An example of an electrostatic charge-induced reversible membrane association

is given by the myristoylated protein MARCKS, an acronym for myristoyl alanine-rich C-

kinase substrate. MARCKS binds to Ca2+-calmodulin and actin and is thought to

integrate protein kinase C (PKC) and Ca2+-calmodulin signals that affect interactions of

actin with the cytoskeleton and membranes. Binding of calmodulin requires Ca2+ and is

prevented by PKC phosphorylation of MARCKS, whereas binding and crosslinking of

actin filaments by MARCKS is blocked by phosphorylation and by Ca2+-calmodulin.

MARCKS is a rod-shaped protein with at least two domains: an N-terminal

myristoylation domain and a basic effector domain that contains the PKC

phosphorylation sites as well as the calmodulin and actin-binding sites.

Three peptide fragments, corresponding to the basic domain of MARCKS, were

used to characterize the effect of changes in their electrostatic properties on membrane

affinity:

MARCKS 151-175: KKKKKRFSKKSFKLSGFSFKKNKK

Tetra-Asp MARCKS: KKKKKRFDKKDFKLDGFDFKKNKK

Phos-MARCKS: KKKKKRFS(P)KKS(P)FKLS(P)GFS(P)FKKNKK

33

The MARCKS peptide is strongly cationic, because of all the lysine residues,

whereas the positive charges in the Asp-substituted and the tetra-phosphorylated

peptides are progressively neutralized by the added negative groups. Binding of the

peptides was measured as a function of the fraction of phosphatidylserine in the bilayer.

As shown in the figure below, addition of negative charges to the peptide diminishes its

affinity for the membrane. Thus, a higher density of anionic head groups is required to

reach 50% binding to the bilayer. Phosphorylation is particularly effective.

Thus, in the presence of bilayers containing 10-20% acidic lipids, MARCKS

phosphorylation leads to almost complete desorption of the peptide from the membrane.

This effect was demonstrated in a kinetic experiment by using mixed bilayers

containing phosphatidylserine and

fluorescence-labeled tracer lipids. Under the

experimental conditions, the fluorescence

intensity was proportional to the degree of

peptide binding to the membrane. Addition of

MARCKS 151-175 peptide produced an

increase in the fluorescence of the labeled

lipids. Upon addition of PKC in the presence

of ATP, serine phosphorylation caused the

immediate desorption of the peptide from the

34

membrane, which was completed after ~1 min, as judged by the decrease of the

fluorescence signal back to the level measured in the absence of peptide.

The binding of full-length MARCKS to membranes requires the contribution of

both the hydrophobic interactions of the myristoyl anchor with the bilayer core and the

electrostatic interactions between the basic domain and anionic lipid head groups.

Phosphorylation by PKC reduces the electrostatic binding energy and leads to

desorption of MARCKS, until dephosphorylation by protein phosphatases restores the

full interaction energy. Thus, the myristoyl anchor facilitates the initial transfer of the

protein to the membrane, which then increases the chance that the basic domain will

associate electrostatically with the anionic lipids. This mechanism for reversible protein-

membrane binding is known as the myristoyl-electrostatic switch.

Membrane anchoring of peripheral proteins via non-covalently bound lipids

Protein targeting to specific cellular compartments in response to external stimuli

is a fundamental component of signal transduction mechanisms in eukaryotic cells.

Such localization can be achieved by means of protein-protein interaction domains,

such as src-homology 2 (SH2) and src-homology 3 (SH3) domains, which recognize

specific phosphotyrosine motifs and proline-rich sequences, respectively, in the protein

binding partners (e.g. activated receptors).

Alternatively, localization can be carried out by protein domains that bind

specifically to lipids embedded in cell membranes. The best known members of this

class of domains are the protein kinase C (PKC) homology-1 (C1) and PKC homology–

2 (C2) domains, the FYVE domain, and the pleckstrin homology (PH) domain. Some

C1, C2, and PH domains interact with proteins in addition to or instead of lipids.

Fluorescence microscopy and fusion proteins derived from green fluorescent

protein (GFP) and several of these domains have allowed detailed studies of the

kinetics of spatial redistribution (e.g. from cytosol to membranes or vice versa) during

signaling.

35

A model of the membrane-docked structure of a representative domain of each

type is shown in the figure below.

Panel A: structure of the complex between the C1 domain of PKC and phorbol ester is

shown with the model of a myristoyl chain. Panel B: structure of the Ca2+-bound C2

domain of cPLA2 interacting with the membrane. Panel C: structure of the FYVE

domain of Vps27p with a model of phosphatidylinositol 3-phosphate (PI3P). Panel D:

structure of the PH domain of PLC1 complexed with Ins(1,4,5)P3 and with a model of

36

the two myristoyl chains. The secondary structure and molecular surface of each

domain are shown. The surface colors indicate the nature of the residues, green for

hydrophobic and blue for basic. Some specific and nonspecific contact residues are

also indicated. The two Zn2+ in the C1 and FYVE domains are shown as cyan circles,

while the two Ca2+ in the C2 domain are shown as blue circles. The domains are

positioned so that known membrane-interacting residues penetrate the membrane and

patches of basic residues are near the membrane surface. The membrane leaflet,

drawn to scale, is divided into an interfacial zone and a hydrophobic core, each ~15 Å

thick.

C1 domains

Originally discovered as a conserved region responsible for the activation of

PKCs by diacylglycerol or phorbol esters, C1 domains have been found in >200 other

proteins. The C1 domain is a compact motif of ~50 amino acid residues, containing two

small sheets and short C-terminal a helix that are built around two 3-Cys-1-His Zn2+-

binding clusters, with the two ions integral to the overall structure. One entire end of the

C1 domain surrounding the diacylglycerol-binding groove is very hydrophobic.

Membrane binding of C1 domains occurs by strong synergism between the

stereospecific interaction of diacylglycerol with its binding site and the nonspecific

hydrophobic interaction between the membrane and the C1 domain surface surrounding

the binding site.

In most PKCs, C1 domains occur in pairs. C1 domains from PKC have been

observed to translocate from the cytosol to the plasma membrane within a few seconds

after addition of diacylglycerol. In the inactive cytosolic form of PKC, the

diacylglycerol-binding sites are obstructed. Opening of these sites may require binding

of the enzyme to the membrane via Ca2+-mediated C2 domain-phospholipids

interactions. Diacylglycerol binding to the C1 domain is also believed to lead to

allosteric activation of the enzyme by a conformational change that alters the

interactions of the C1 domains with the catalytic domain of the enzyme.

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C2 domains

C2 domains consist of ~120 residues folded in a sandwich structure related to

that of immunoglobulin. Originally discovered as a conserved motif in Ca2+-dependent

PKCs, ~600 C2 domains have now been found in >400 proteins involved not only in

signal transduction, but also in inflammation, synaptic vesicle trafficking and fusion, and

many other processes. The properties of C2 domains vary, with some of them binding

to phospholipid membranes in a Ca2+-dependent manner, while others bind

constitutively. Other C2 domains exhibit both Ca2+-dependent and -independent binding

to proteins rather that membranes. The structures of C2 domains from synaptotagmin,

PKC- and -, and phospholipases A2 (cPLA2) and C-1 (PLC1) have been

determined. The Ca2+-binding sites are formed by three loops at one tip of the structure.

Most Ca2+-dependent C2 domains bind acidic phospholipids, but that of cPLA2 seems to

prefer neutral lipids, especially phosphatidylcholine (PC).

The subcellular localization of C2 domains correlates with their phospholipid

specificity. Thus, when the free Ca2+ concentration increases in response to a stimulus,

C2 domains from PKCa and PKC translocate to the plasma membrane, rich in the

acidic phosphatidylserine lipid, whereas cPLA2 translocates to the PC-rich nuclear

envelope and endoplasmic reticulum.

FYVE domains

FYVE domains have been found in ~60 proteins and consist of 70-80 residues

containing 8 Cys or 7 Cys and 1 His that coordinate two Zn2+. FYVE domains are

specific for phosphatidylinositol-3-phosphate (PI3P), whose concentration in the cell

increases following activation of phosphatidylinositol 3-kinases (PI3-kinase). As

illustrated in the figure above, FYVE domains bind to PIP3-containing membranes so

that the tip of the N-terminal loop, which contains hydrophobic residues, penetrates into

the bilayer.

Proteins containing FYVE domains localize to endosomal membranes containing

PI3P, and this localization is blocked by inhibition of PI3-kinase.

38

PH domains

Found in >500 proteins, PH domains bind various phosphorylated phosphatidyl-

inositols (phosphoinositides) with different affinities and thus respond sensitively to the

activities of phosphatidylinositol kinases, phosphatases and phospholipases. Of

particular interest, signaling through PI3-kinases depends on PH domain-containing

effectors, in addition to those containing FYVE domains. Structures are known for PH

domain of several proteins, among which spectrin, PLC1, -adrenergic receptor kinase

(ARK) and insulin receptor substrate 1 (IRS-1). The PH domain structure contains two

orthogonal antiparallel sheets of three and four strands, followed by a C-terminal a

helix. The sheets fold into a barrel-like structure, one end of which is capped by the

ahelix. The loops connecting the strands are involved in ligand binding and vary

substantially in sequence and structure between PH domains.

Based on the binding to different phosphoinosite polyphosphates and inositol

polyphosphates, PH domains have been classified into four groups.

Group1 contains proteins such as Bruton’s tyrosine kinase (Btk), whose PH

domains bind phosphatidylinositol 3,4,5-trisphosphate, PI(3,4,5)P3 with high specificity.

Group 2 includes proteins such as PLC1 and ARK, whose PH domains have

high affinity for PI(4,5)P2 and PI(3,4,5)P3 in vitro. In vivo, preferential binding to

PI(4,5)P2 may occur as a consequence of the higher abundance of this lipid rather than

discrimination against the 3-phosphorylated PI.

Group 3 includes proteins such as Akt, also known as protein kinase B (PKB),

whose PH domains bind preferentially PI(3,4)P2 and PI(3,4,5)P3.

Group 4 is a heterogeneous group that includes proteins with relatively low

affinity for all ligands mentioned above. The PH domain of PLC- binds

3-phosphoinositides, including PI3P, while the PH domains of PLC1 and PLC2 bind

nonspecifically and with low affinity to neutral and acidic phospholipids.

In addition to binding to membrane-bound phosphoinositides, PH domains

display variable affinity for soluble inositol phosphates. For examples, the PH domain of

PLC1 binds to PI(4,5)P2 in vesicles with micromolar affinity and to the soluble

Ins(1,4,5)P3 with Kd = 210 nM. The higher affinity for the latter may be important in

39

product inhibition of the enzyme. Stimulation of PLC causes repartitioning of a fusion

protein consisting of green fluorescent protein and PLC1 PH domain from the plasma

membrane to the cytosol concomitant with the hydrolysis of PI(4,5)P2 in the membrane

and formation of soluble Ins(1,4,5)P3. PH domains that bind 3-phosphorylated

phosphoinositides, including those of Btk and Akt, have similarly been observed to

translocate to the plasma membrane upon activation of PI3-kinases.

Many of the interactions described for these membrane-targeting domains are of

relatively low affinity and thus their physiological importance may be questioned.

However, many important interactions appear to be weak “by design”, so that

membrane binding of certain proteins may require the simultaneous presence of more

than one ligand, the coincident activation of more than one signaling pathway. This is

exemplified by PKC, which contains both diacylglycerol-binding C1 and

Ca2+-phospholipid-binding C2 domains, and requires both the production of

diacylglycerol and an increase in the concentration of free Ca2+ for full activation.

40

Intracellular Ligand-Gated Channels

Regulation of ion channel activity can modulate many physiological processes, such as electrical excitability, secretion, and salt transport across epithelia. Most channel proteins are post translationally modified (e.g. through phosphorylation or through interactions with intracellular signaling molecules) but certain channels depend on such interactions in order to be gated open or closed. Here we will consider three examples of intracellular ligand-gated channels: a K channels that is inhibited by the ligand ATP, a K channel that is activated by G proteins and a non-selective cationic channel that is activated by cyclic nucleotides.

ATP-sensitive K channels: This inwardly rectifying K channel is inhibited by cytosolic adenosine triphosphate (ATP), thus coupling the metabolic state of the cell to membrane electrical events. These channels are found in all types of muscle cells (skeletal, cardiac and smooth), in neurons, in renal tubular cells and in the cells of the pancreas, where their physiologic role is best understood. In pancreatic cells they regulate insulin secretion in response to glucose. These channels are normally active at rest and cells are thus kept at negative resting potentials. After a meal, when glucose is metabolized and ATP is produced (actually the channel senses the increased ATP/ADP ratio) the channel is inhibited the cell depolarizes, fires action potentials, allowing entry of Ca and secretion of insulin. These channels are associated with a member of the larger ATP-binding cassette proteins, called the sulphonylurea receptor or SUR (other members of these transmembrane proteins include: P-glycoprotein or multidrug resistance protein, the cystic fibrosis transmembrane regulator or CFTR, etc.- see lecture 12) (Fig. 1).

The association of the SUR and the K channel produces a functional channel that is blocked by sulphonylureas, drugs that constitute the principal treatment for adult onset diabetes. Similarly, the SUR association causes the K channel to be activated by SUR-binding drugs called potassium “channel openers” (e.g. diazoxide, pinacidil), and by nucleoside diphosphates (e.g. ADP) that bind SURs at the nucleotide binding folds (NBFs – Fig. 1). It has been established that there is an inverse relationship between ATP sensitivity and PIP2 levels and that the channel activity depends on the presence of PIP2. However, the details of the mechanism by which ATP inhibition or SUR-channel

41

Figure 1. The inward rectifier Kir6.2 combines with the sulfonylurea receptor (SUR) to generate ATP-sensitive K currents.

interactions relate to channel gating by PIP2 are unclear but are the subject of an active area of research. Moreover, the physiological importance of regulation of PIP2 levels on the activity of this K channel is not clear. It is long known for example that at glucose concentrations below the threshold for stimulation of insulin secretion and electrical activity, muscarinic stimulation that leads to PLC activation initiates electrical activity and insulin release. Whether, this effect is mediated by reduction of PIP2 levels and enhanced sensitivity to ATP inhibition of the K channels remains to be shown.

G protein-gated K channels: Using the cell-attached mode of the patch clamp technique it can be shown that extracellular application of ACh is effective in stimulating channel activity in an atrial patch, when perfused through the pipette but not through the bath (Fig 2). In their isolated patch, external signaling was limited to ACh in the patch pipette (since the membrane patch seems to be physically isolated at the sites of the gigaseal between the glass electrode and the plasma membrane from substances in the bath and from membrane molecules other than those within the patch) whereas internally, soluble second messengers (e.g. GTP) do have access to the cytoplasmic surface of the patch. This result has been used as evidence for the membrane delimited nature of the action of ACh. Using the whole-cell mode of the patch-clamp technique, it has been shown that the ACh effect proceeded via a pertussis toxin (PTX) sensitive G protein and that non-hydrolyzable GTP analogs could bypass signaling through the receptor and cause persistent stimulation of channel activity. Experiments with inside-out patches provided further evidence for the membrane delimited nature of the ACh signaling and the involvement of G proteins. Perfusion of inside-out patches with purified G subunits caused persistent

stimulation

of K current activity in a Mg-independent manner. The G activation of this K current provided the first example of the effector function of the G complex in any system. It has been shown

42

Figure 2. Unitary inward K currents measured on a rabbit atrial cell with an on-cell patch pipette containing isotonic KCl. The control trace is before ACh additions. The second trace is after perfusion of 100 nM ACh in the bath, and the third trace is after washing ACh out of the bath and perfusing 10 nM ACh into the pipette. Em = -90 mV.

that G binds directly to both the carboxy- and amino- cytoplasmic segments of the channel protein. It is thought that G binding stabilizes channel-PIP2 interactions that serve to open the channel gate. The details of this mechanism are an active area of research. The atrial (or nodal) K+ channel that is activated by acetylcholine through muscarinic m2 type receptors has served as the prototypical G protein-gated K channel. Pertussis toxin-sensitive, neurotransmitter-activated inwardly rectifying K+ currents have also been reported in the central nervous system and in other peripheral tissues such as the pancreas and the pituitary. Direct activation of potassium (K+) channels by G proteins is involved in the rapid inhibition of membrane excitability, such as in the slowing of heart rate by the vagus nerve or the autoinhibitory release of dopamine by midbrain neurons. Thus these K channels couple G protein-coupled receptor signaling to membrane excitability.

Figure 4 shows the crystal structure of the cytoplasmic domains of one of the G protein sensitive channels GIRK1. On this structure we have mapped mutations that affect

43

Figure 3. In the hypothesis of membrane-delimited signaling, drawn here for muscarinic modulation of a K(ACh) channel, only three macromolecules are used in the signaling cascade: receptor (M), G protein (Gk), and channel. They remain in the membrane throughout.

Figure 4. Crystal structure of the cytosolic domains of GIRK1. Mutation sites have been mapped onto the structure revealing that PIP2

and G interacting sites of the channel are in close proximity to the lipid (Nishida and MacKinnon, 12/27 2002, Cell 111:958-965)

channel-G and channel-PIP2 interactions. Most of these mutations seem to come together to a region that is in close proxility to the lipid bilayer.

Cyclic nucleotide-gated channels involved in phototransduction:

Cyclic nucleotide-gated channels (CNG) are composed of two subunits in a tetrameric arrangement, two a subunits and two subunits. The a subunits can produce functional channels when expressed in heterologous expression systems. The subunits do not express by themselves, but when co-expressed with their corresponding a subunit, they produce channels with altered permeation, pharmacology, and/or cyclic nucleotide selectivity. The primary structure of each CNG channel subunit is a six-membrane spanning segment protein resembling that of voltage-gated K channels (we will examine those in the next lecture). CNG channels are found in all sensory organs and possess a cyclic nucleotide-binding domain. This is a highly conserved stretch of approximately 120 amino acids that is homologous to similar domains of other proteins, including the cAMP- and cGMP-dependent protein kinases and the catabolite-activating protein (CAP), a bacterial transcription factor. Although the retinal and olfactory CNG channels exhibit a high degree of sequence similarity (over 80% amino acid identity) in the putative binding region, the native channels exhibit different cyclic nucleotide selectivities. For the retinal channel, cGMP is a much more potent and effective agonist than cAMP. For the native olfactory channel cAMP and cGMP have very similar effects.

Let us consider the role of retinal CNG channels in phototransduction. In the

retina of vertebrates phototransduction is accomplished by sensory cells (the rods and the cones), connected by interneurons (bipolar, horizontal and amacrine cells) to ganglion cells that transmit signals to the optic nerve and the brain (Fig. 5). Rods and cones have different sensitivities and respond to different frequencies of light. The

44

Figure 5. The retina has five major classes of neurons arranged into three nublear layers: photoreceptors (rods and cones), bipolar cells, horizontal cells, amacrine cells, and ganglion cells. Photoreceptors, bipolar, and horizontal cells make synaptic connections with each other in the outer plexiform layer. The bipolar, amacrine, and ganglion cells make contact in the inner plexiform layer. Bipolar cells bridge the two layers. Information flows vertically from photoreceptors to bipolar cells to ganglion cells. Information also flows laterally, mediated by horizontal cells in the outer plexiform layer and amacrine cells in the inner plexiform layer.

cones cells can further be subdivided into cells that preferentially sense different colors. However, it is rods, from a variety of species, that have been the favored cell type for studying visual transduction. Figure 6a shows the structure of a rod photoreceptor.

The cell has two parts. The rod outer segment is elongated and contains a stack of flattened disks made from internal membranes. This is connected by a thin bridge to the remainder of the cell, the inner segment that contains the nucleus, the mitochondria, and the presynaptic terminal that synapses onto other neurons in the retina. It is the outer segment that is the business end for visual transduction. Within the internal membranous disks is found the light-sensitive protein rhodopsin. This is made up of an opsin protein, bound to a light-sensitive molecule or chromophore termed retinal. The later molecule may exist in a number of different forms, of which 11-cis-retinal and all-trans-retinal are the two major isomers. On its own, neither opsin nor retinal absorbs visible light. In combination, however, absorption of a photon of light causes and isomerization of retinal from the 11-cis form to the all-trans form. The light-dependent

45

Figure 6a. A photoreceptor. The drawing (left) is of an entire rod photoreceptor. The micrograph (right) shows only the outer segment of a salamander cone.

Figure 6b. The dark current (left). In the dark, current flows through sodium channels in the outer segment of a rod photoreceptor. A pulse of light (right) closes these channels, resulting in hyperpolarization of the rod.

isomerization of retinal then causes a structural rearrangement of the protein. Rhodopsin that has been activated in this way is termed meta-rhodopsin. For all of the subsequent steps in visual transduction, it is useful to think of this molecule as analogous to a receptor that has just bound its neurotransmitter. In fact, the structure of the opsin protein is similar to G-protein coupled receptors. Not surprisingly, therefore, the steps that follow the production of meta-rhodopsin involve the production of a second messenger through the action of a G-protein. This G-protein is called transducin. When meta-rhodopsin binds to transducin, GDP is replaced by GTP, and the aT-subunit of transducin is liberated from its complex with the subunit. The target of the newly liberated aT is an enzyme in the membranous disks, a phosphodiesterase, that cleaves the second messenger cGMP to 5 ' GMP. Even in the dark, the levels of cGMP in the outer segments are maintained by a balance between its rate of synthesis through guanylate cyclase and degradation by the phosphodiesterase. The action of aT, formed after exposure to light, is to stimulate the phosphodiesterase, producing a drop in the levels of cGMP. This drop occurs within about 100 ms of the onset of a light flash, sufficiently fast to account for a visual response. In many respects, photoreceptors are built backwards. When excited by light, they respond by dropping, rather than raising, their concentration of the second messenger cGMP.

The dominant type of ion channel in the plasma membrane of the outer segments is the CNG channel that allows sodium and calcium to enter the cell. Because of the abundance of sodium ions in the extracellular fluid, the major ion that enters the outer segments through these channels is sodium. We would expect a cell with a preponderance of such CNG channels to have a very positive resting potential. The effect of the rod CNG channels is, however, counterbalanced by potassium channels. The interesting thing about these potassium channels is that they are found in a very different part of the cell, the membrane of the inner segment that includes the nucleus and synaptic terminal. Because there is good electrical continuity between the inner and outer segments, the mean membrane potential is kept fairly negative as a result of the open potassium channels. This spatial distribution of channels, however, creates a circulating current, termed the dark current (Fig 6b), which flows in through the outer segment CNG channels, through the bridge into the inner segment and out through the potassium channels. The effect of shining light on a rod is to shut down many of the CNG channels in the outer segment. This produces a marked decrease in the dark current. As a result the potassium channels, which remain open in the inner segment, hyperpolarize the cell toward EK, reducing the spontaneous release of neurotransmitter from the synaptic terminal. The closure of the CNG channels can be attributed directly to the drop in cGMP in the cytoplasm of the outer segment. The CNG channels normally bind cGMP directly, and remain open only when cGMP is bound. This can be demonstrated by making inside-out patch recording on membrane from the outer segments. When cGMP is added to the cytoplasmic face of the patch a large increase in conductance, attributable to the opening of the CNG channels can be measured. One interesting feature of the CNG channels is that, under normal conditions, the conductance of a single channel is extremely low. The reason for this is that calcium and magnesium ions, which are normally present in physiological solutions, partially block these channels. This block is relieved when calcium and magnesium are omitted from the solutions, and individual openings of the channel are much larger.

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This cascade of reactions that follows the formation of meta-rhodopsin produces a very significant amplification of the signal generated by light. It has been estimated that a single molecule of meta-rhodopsin, which is formed by the action of a single photon of light, diffuses in the membrane and activates several hundred transducin molecules before it is rendered inactive. The subsequent stimulation of the phosphodiesterase by aT provides further amplification such that a single photon of light can lead to the destruction of more than 100,000 molecules of cGMP.

The analogies between visual transduction and neurotransmitter action can be taken further, when one considers how the response to a flash of light is terminated. A protein called rhodopsin kinase phosphorylates meta-rhodopsin making it relatively ineffective at activating transducin, and thus terminating the light response. After phosphorylation, the all-trans-retinal dissociates from rhodopsin, leaving the opsin protein, which must bind another 11-cis-retinal before it can again be activated by light. Similarly, the -adrenergic receptor (stimulated by isoproterenol or norepinephrine) is phosphorylated by the -adrenergic receptor kinase (ARK) to make it ineffective in stimulating Gs.

Neurotransmitter Receptors

The diversity of neurotransmitters is extensive, but their receptors can be grouped into two broad classes: ligand-gated ion channels and G protein-coupled receptors. In this section, we describe two important receptors that are also ligand-gated ion channels. By far the most-studied receptor is the muscle nicotinic acetylcholine receptor, the first ligand-gated ion channel to be purified, cloned, and characterized at the molecular level. The structure and mechanism of this receptor are understood in considerable detail, and it provides a paradigm for other neurotransmitter-gated ion channels. When activated, these receptors induce rapid changes, within a few milliseconds, in the permeability and potential of the postsynaptic membrane. In contrast, the postsynaptic responses triggered by activation of G protein-coupled receptors occur much more slowly, over seconds or minutes, because these receptors regulate opening and closing of ion channels indirectly.

Opening of Acetylcholine-Gated Cation Channels Leads to Muscle Contraction

The nicotinic acetylcholine receptor, a ligand-gated cation channel, admits both K+ and Na+. Although found in some neurons, this receptor is best known for its role in synapses between motor neurons and skeletal muscle cells. Patch-clamping studies on isolated outside-out patches of muscle plasma membranes have shown that acetylcholine causes opening of a cation channel in the receptor capable of transmitting 15,000-30,000 Na+ or K+ ions a millisecond.

Since the resting potential of the muscle plasma membrane is near Ek, the potassium equilibrium potential, opening of acetylcholine receptor channels causes little increase in the efflux of K+ ions; Na+ ions, on the other hand, flow into the muscle cell. The simultaneous increase in permeability to Na+ and K+ ions produces a net depolarization to about –15mV from the muscle resting potential of –85 to –90 mV. This depolarization of the muscle membrane generates an action potential, which – like an action potential in a neuron – is conducted along the membrane surface via voltage-gated Na+ channels. When the membrane depolarization reaches a specialized region, it triggers Ca2+ movement from its intracellular store, the

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sarcoplasmic reticulum, into the cytosol; the resultant rise in cytosolic Ca2+ induces muscle contraction.

Two factors greatly assisted in the characterization of the nicotinic acetylcholine receptor. First, this receptor can be rather easily purified from the electric organs of electric eels and electric rays; these organs are derived from stacks of muscle cells (minus the contractile proteins) and thus are richly endowed with this receptor. (In contrast, this receptor constitutes a minute fraction of the total membrane protein in most nerve and muscle tissues). Second, a-bungarotoxin, a neurotoxin present in snake venom, binds specifically and irreversibly to nicotinic acetylcholine receptors. This toxin can be used in purifying the receptor by affinity chromatography and in localizing it. For instance, in autoradiographs of muscle-cell sections exposed to radioactive a-bungarotoxin, the toxin is localized in the plasma membrane of postsynaptic striated muscle cells immediately adjacent to the terminals of presynaptic neurons.

Careful monitoring of the membrane potential of the muscle membrane at a synapse with a cholinergic motor neuron has demonstrated spontaneous, intermittent, and random ~2-ms depolarizations for about 0.5-1.0 mV in the absence of stimulation of the motor neuron. Each of these depolarizations is caused by the spontaneous release of acetylcholine from a single synaptic vesicle. Indeed, demonstration of such spontaneous small depolarizations led to the notion of the quantal release of acetylcholine (later applied to other neurotransmitters) and thereby led to the hypothesis of vesicle exocytosis at synapses. The release of one acetylcholine-containing synaptic vesicle results in the opening of about 3000 ion channels in the postsynaptic membrane, far short of the number need to reach the threshold depolarization that induces an action potential. Clearly, stimulation of muscle contraction by a motor neuron requires the nearly simultaneous release of acetylcholine from numerous synaptic vesicles.

All Five Subunit in the Nicotinic Acetylcholine Receptor Contribute to the Ion

Channel

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The acetylcholine receptor from skeletal muscle is a pentameric protein with a subunit composition of a2. Each molecule has a diameter of about 9nm and protrudes about 6nm into the extracellular space and about 2nm into the cytosol (Figure 11-36). The a, , , and subunits have considerable sequence homology; on average, about 35-40 percent of the residues in any two subunits are similar. The complete receptor has a five-fold symmetry, and the actual cation channel is a tapered central pore, with a maximum diameter of 2.5 nm, formed by segments from each of the five subunits (Figure 11-36).

The channel opens when the receptor cooperatively binds two acetylcholine molecules to sites located at the interfaces of the a and a subunits. Once acetylcholine is bound to a receptor, the channel is opened virtually instantaneously, probably within a few microseconds. Studies measuring the permeability of different small cations suggest that the open ion channels is, at its narrowest, about 0.65-0.80 nm in diameter, in agreement with estimates from electron micrographs. This would be sufficient to allow passage of both Na+ and K+ ions with their bound shell of water molecules.

Although the structure of the central ion channel is not known in molecular detail, much evidence indicates that it is lined by five transmembrane M2 a helices, one from each of the five subunits. The M2 helices are composed largely of hydrophobic or uncharged polar amino acids, but negatively charged aspartate or glutamate residues are located at each end, near the membrane faces and several serine or threonine residues are near the middle. If a single negatively charged glutamate or aspartate in one subunit is mutated to a positively charged lysine, and the mutant mRNA is injected together with mRNAs for the other three wild-type subunits into frog oocytes, a functional channel is expressed, but its ion conductivity – the number of ions that can cross it during the open state – is reduced. The greater the number of glutamate or aspartate residues mutated (in one or multiple subunits), the greater that reduction in conductivity. These findings suggest that aspartate and glutamate residues – one residue

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from each of the five chains – form a ring of negative charges on the external surface of the pore that help to screen out anions and attract Na+ or K+ ions as they enter the channel (see Figure 11-36). A similar ring of negative charges lining the cytosolic pore surface also helps select cations for passage.

The two acetylcholine-binding sites in the extracellular domain of the receptor lie ~4 to 5 nm from the center of the pore. Binding of acetylcholine thus must trigger conformational changes in the receptor subunits that can cause channel opening at some distance from the binding sites. Receptors in isolated postsynaptic membranes can be trapped in the open or closed state by rapid freezing in liquid nitrogen. Images of such preparations suggests that the five M2 helices rotate relative to the vertical axis of the channel during opening and closing.

Two Types of Glutamate-Gated Cation Channels May Function in a Type of “Cellular Memory”

The hippocampus is the region of the mammalian brain associated with many types of short-term memory. Certain types of hippocampal neurons, here simply called postsynaptic cells, receive inputs from hundreds of presynaptic cells. In long-term potentiation a burst of stimulation of a post-synaptic neuron makes it more responsive to subsequent stimulation by presynaptic neurons. For example, stimulation of a hippocampal presynaptic nerve with 100 depolarizations acting over only 200 milliseconds causes an increased sensitivity of the postsynaptic neuron that lasts hours to days. Changes in the responses of postsynaptic cells may underlie certain types of memory.

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Two types of glutamate-gated cation channels in the postsynaptic neuron participate in long-term potentiation. Unlike other neurotranmitter-gated ion channels, both glutamate receptors have four subunits, each containing a pore-lining M2 helix; both are excitatory receptors, causing depolarization of the plasma membrane when activated. Because the two receptors were initially distinguished by their ability to be activated by the non-natural amino acid N-methyl-D-aspartate (NMDA), they are called NMDA glutamate receptors and non-NMDA glutamate receptors.

As illustrated in Figure 11-41, non-NMDA receptors are “conventional” in that binding of glutamate, released from the presynaptic cell, triggers their opening. NMDA glutamate receptors are different in two key respects. First, they allow influx of Ca2+ as well as Na+. Second, and more important, two conditions must be fulfilled for the ion channel to open: glutamate must be bound and the membrane must be partly depolarized. In this way, the NMDA receptor functions as a coincidence detector; that is, it integrates activity of the postsynaptic cell – reflected in its depolarized plasma membrane – with release of neurotransmitter from the presynaptic cell, generating a cellular response greater than that caused by glutamate release alone. Once a post-synaptic cell becomes “sensitized”, it takes fewer action potentials in the presynaptic neurons to induce a given depolarization in the postsynaptic neuron; in other words, the synapse “learns” to have an enhanced response to signals from the presynaptic cells.

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Opening of NMDA receptors depends on membrane depolarization because of the voltage-sensitive blocking of the ion channel by a Mg2+ ion from the extracellular solution. A small depolarization of the membrane causes the Mg2+ ion to dissociate from the receptor, thereby making it possible for glutamate binding to open the channel. Mutagenesis of a single asparagine residue in the pore-lining M2 helix of the NMDA receptor abolishes the effect of Mg2+, indicating that Mg2+ binds in the channel.

Since activation of a single synapse, even at high frequency, generally causes only a small depolarization of the membrane of the postsynaptic cell, long-term potentiation is induced only when many synapses simultaneously stimulate a single postsynaptic neuron. Thus, the requirements for membrane depolarization explains why long-term potentiation depends on the simultaneous activation of a large number of synapses on the postsynaptic cell.

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