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CHAPTER4

THESTRUCTUREANDFUNCTIONOFTHEPLASMAMEMBRANE

OBJECTIVES

Describe the functions of cellular membranes.

Elucidate the chemical components of cell membranes and review their chemical properties.

Describe the development of the models of membrane structure from the first suggestion of lipid

composition to the Fluid-Mosaic Model.

Explain carbohydrate involvement in membrane structure, their possible functions and location.

Describe the types of proteins found in membranes and the roles they play in membrane function.

Stress the importance of membrane fluidity to living cells and the mechanisms by which cells

maintain an appropriate level of fluidity.

Describe biological membrane asymmetry and the dynamic nature of membrane structure and

function.

Outline research techniques employed to determine the extent of cellular membrane fluidity.

Describe the different mechanisms employed by cells to transport materials across membranes:

simple and facilitated diffusion, channel proteins, active transport.

Summarize the properties of the well-studied red blood cell membrane as an example of the protein

and lipid composition of cellular membranes.

Outline the methods employed in the study of red blood cells as an example of membrane research

strategies.

Explain the process involved in generating an action potential, propagating the signal and getting it

across the synapse to the postsynaptic cell, thus demonstrating the ways in which cellular

membranes can function as part of a coordinated process.

CHAPTER4 OUTLINE

First Detection of Cell Membrane

I. Cells are separated from the world by a thin, fragile structure, the plasma membrane 5 10 nm thick

A. ~10,000plasma membranes stacked one on top of another would equal the thickness of a book's page

B. No hintof a plasma membrane is detected in a thin section under a light microscope since it is so thin

C. Finally, in the late 1950s, techniques for preparing & staining tissues had progressed to the point where

they could be visualized in the electron microscope

1. J. D. Robertson (Duke Univ.) portrayed plasma membrane as three-layered structure, consisting ofdarkly staining inner & outer layers & a lightly staining middle layer

2. All membranes examined closely (plasma, nuclear or cytoplasmic) or from plants, animals or

microorganisms had the same ultrastructure

3. The pictures touched off a vigorous debate as to the molecular composition of the various layers of

the membrane

4. The 2 dark-staining layers in the electron micrographs correspond primarily to the inner & outer

polar surfaces of the bilayer

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Summary of Membrane Functions and Overview of Membrane Structure

I. Compartmentalization - membranes enclose entire cell or diverse intracellular spaces in which occur

specialized activities that proceed with little outside interference & are regulated independently

A. They are continuous, unbroken sheets

B. A cell's various membrane-bound compartments have markedly different contentsII. Scaffold for biochemical activities membranes are also distinct compartments themselves

A. They provide cell with extensive framework (scaffolding) within which components can be ordered

for effective interaction

III. Provide selectively permeable barrier membrane can be compared to moat around a castle; a general

barrier that has gated "bridges" that allow desirable things to enter & leave space they surround

A. They prevent the unrestricted exchange of molecules from one side to the other - control what gets

into & out of cell; H2O moves easily

B. They also provide the means of communication between the compartments they separate

IV. Transporting solutes they have transport machinery to move substances from one side to the otherA. Can transport substances (ions, sugars, amino acids, etc.) up or down concentration gradient; sugars &

amino acids taken up since they are needed to fuel metabolism & build macromolecules

B. Can establish ionic gradients across itself (critical for nerves, muscles, maybe helps all cells respond

to their environment) by transporting specific ions

V. Response to externalsignals(signal transduction) plays critical role in response to external stimuli

(hormones, growth factors,neurotransmitters)

A. Receptors in membrane combine with specific molecules (ligands) having complementary structure

& then initiate response

1. Different cells have different receptors; can therefore recognize & respond to different ligands in

environment

B. Interaction of receptor with external ligand causes generation of new signal (second messenger);

stimulates or inhibits internal cell activities like:

1. Making more glycogen, preparing for cell division, concentrating particular compounds, releasing

calcium from internal stores, even committing suicide

VI. Intercellular interactions - allows cells to recognize & signal one another, adhere when appropriate &

exchange materials & information; mediates interactions between cells of multicellular organisms

VII. Energy transduction intimately involved in processes by which one type of energy is converted to

another type (energy transduction); done by membranes of chloroplasts & mitochondria

A. Photosynthesis & electron transport site in chloroplasts & mitochondria

1. Chloroplasts absorb sunlight energy in membrane-bound pigments & convert it into the chemical

energy of carbohydrates

2. Mitochondrial membranes transfer chemical energy from carbohydrates & fats to ATP

B. Allows storage of energy in gradients

A Brief History of Studies on the Structure of the Plasma Membrane

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I. Ernest Overton, University of Zurich (1890s) - knew that nonpolar solutes dissolve more readily in nonpolar

solvents than in polar ones & that polar solutes are most soluble in polar solvents

A. Since he realized that to enter a cell, a solute must pass first through the membrane, he used root hairs

with hundreds of different solutes & found that more lipid-soluble solutes enter root hair cells faster

B. Concluded dissolving power of outer cell boundary matched that of fatty oil

II. Gorter & Grendel (Dutch scientists, 1925) first proposed lipid bilayer; extracted lipids from red blood cell

(RBC) membrane, measured their surface area on H2O & compared it to estimated RBC surface area

A. Found surface area covered about twice (ratio was between 1.8 & 2.2) the surface area of RBCs

1. Used mammal RBCs since they lack both nuclei & cytoplasmic organelles; no other membranes

2. Plasma membrane is the only lipid-containing structure in cell; all lipids extracted from cells can be

assumed to have resided in plasma membrane

B. Propose lipid bilayer (bimolecular layerof lipids) with hydrophilicheadspointed out on bothsides

1. Thermodynamically favored arrangement; polar lipid head groups interact with surrounding H2O

molecules & hydrophobic fatty acid (acyl) tails protected from aqueous environment

2. Polar heads face cytoplasm on one side & blood plasma on the other; polar heads of each leaflet were

directed outward toward aqueous environment

C. Got right answer, but used several miscalculations; however, mistakes compensated for each otherIII. 1920s & 1930s - evidence accrued that there must be more to membranes than lipid bilayer

A. Lipid solubility was not the sole determinant of what can pass through membrane

B. Surface tensions of membranes were much lower than those of pure lipid structures

1. Protein film over artificial lipid membrane lowered its surface tension

2. Presence of protein could explain difference in surface tension

C. Artificial membranes can be formed spontaneously & studied

1. Natural & artificial membranes very similar (even in EM in more recent years)

2. Differences between natural & artificial membranes are due to proteins (especially permeability &

electrical resistance); cell membranes are about 5 10 nm wide

IV. Hugh Davson & James Danielli (1935) - proposed that membrane was composed of lipid bilayer linedon inner & outer surfaces by layer of globular proteins

A. Revisedversion(1954) added penetration of bilayer by protein-lined pores; allow polar solutes/ions

through membrane to account for selective permeability

1. Provide conduits for that allow these solutes to enter & exit the cell

B. In mid to late 1950s, with techniques for preparing & staining tissue, J. D. Robertson & others were

able to resolve cell membranes in the electron microscope (see above)

V. S. Jonathan Singer & Garth Nicolson (UC-San Diego, 1972) - proposed the Fluid-Mosaic Model, the

central dogma of membrane biology for >3 decades

A. Lipid bilayer remains core of membrane, but it is not frozen & immobile, but fluid; individual lipids

move laterally within plane of membrane

B. Proteins distributed differently - mosaic of discontinuous particles that penetrate into or through

membrane or contact its polar heads without penetrating the membrane

C. Membranes are dynamic structures whose components are mobile & able to engage in a variety of

transient or semipermanent interactions

The Chemical Comosition of Membranes

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I. Membranes - lipid-protein assemblies held together in thin sheet by noncovalent bonds

A. Lipid bilayer is structural backbone of membrane & barrier preventing random movements of water-

soluble materials into & out of cell

B. Proteins of membrane carry out most of its specific functions

C. Often include carbohydrates attached to membrane lipids & proteins

II. Lipid:protein ratio varies greatly depending on membrane type (cell membrane vs. ER vs. Golgi),

organism (prokaryote vs. plant vs. animal) & cell (cartilage vs. muscle vs. liver)

A. These differences largely relate to the particular functions of the membranes, e.g., inner mitochondrial

vs. myelin sheath

B. Example: inner mitochondrial membrane very high protein:lipid ratio relative to RBCs that are high

relative to myelin sheath (multilayered wrapping around nerve cell axon)

1. Inner mitochondrial membrane has protein carriers of electron transport chain; lipid reduced

2. Myelin sheath is primary electrical insulation for nerve cell it encloses; this function is best carried

out by a thick lipid layer of high electrical resistance with a minimal protein content

III. Membrane lipids wide diversity of amphipathic lipids with both hydrophobic & hydrophilic portions

A. Most have phosphate groups & are phospholipids (except cholesterol, glycolipids)B. 3 types of membrane lipids: phosphoglycerides, sphingolipids, cholesterol

IV. Phosphoglycerides (see drawing in Ch. 2) - most membrane lipids contain phosphate groups so they are

called phospholipids

A. Mostmembrane phospholipidsare built on a glycerol backbone& are thus called phosphoglycerides

1. In phosphoglycerides, glycerol is esterified to 1 phosphate group & 2 fatty acids (diglycerides)

2. With justphosphate&2 fattyacids it is phosphatidicacid(virtuallyabsent inmostmembranes)

B. Extra polar group usually added to phosphate (usually choline, ethanolamine, serine, inositol) to form

polar head group; called phosphatidylcholine (PC), phosphatidylethanolamine (PE), etc.

1. These groups are small & hydrophilic & combined with phosphate form hydrophilic domain at one

end (polar head group)2. At physiological pH, phosphatidylserine (PS) & phosphatidylinositol (PI) head groups have an

overall negative charge; those of phosphatidylcholine & phosphatidylethanolamine are neutral

C. Fatty (acyl) acid chains hydrophobic, unbranched hydrocarbons ~16 22 Cs long

1. Fatty acid tails may be polyunsaturated (>1 double bond), monounsaturated (1 double bond),

saturated (no double bonds)

2. Phosphoglycerides often contain 1 saturated + 1 unsaturated fatty acyl chains

D. Recent interest has focused on the apparent health benefits of 2 highly unsaturated fatty acids (EPA &

DHA) found at high concentration in fish oil

1. EPA & DHA contain 5 & 6 double bonds, respectively, & are incorporated primarily into PE & PC

molecules of certain membranes, most notably in the brain & retina

2. EPA & DHA are described as omega-3 fatty acids, because their last double bond is situated 3

carbons from the omega (CH3) end of the fatty acyl chain

E. With fatty acid chains at one end & a polar head groupat the other end, all of the phosphoglycerideshave

amphipathic character

V. Sphingolipids - less abundant class of membrane lipids; derivatives of sphingosine

A. Sphingosine (an amino alcohol with long hydrocarbon chain) + fatty acid (attached to amino group; R in

figure) = a ceramide; all are amphipathic & similar in structure to phosphoglycerides

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B. The various sphingosine-based lipids have additional groups esterified to the terminal alcohol of

sphingosine moiety

1. Attach phosphorylcholine to terminal alcohol of sphingosine = sphingomyelin (the only membrane

phospholipid not built with a glycerol backbone)

2. Attach carbohydrate at terminal alcohol = glycolipid; if carbohydrate is simple sugar =

cerebroside; if carbohydrate is a small cluster of sugars = ganglioside

3. Since all sphingolipids have 2 long hydrophobic chains on one end & hydrophilic region on the

other end, they are amphipathic & similar in structure to phosphoglycerides

C. Glycolipids relatively little known about them, but evidence suggests that they play crucial roles in cell

function; particularly prominent in membranes of nervous system

1. Ex.: galactocerebroside (galactose + ceramide) - mice without the enzyme that adds galactose to

ceramide have severe muscular tremors & eventual paralysis

2. Similarly, humans who are unable to synthesize a particular ganglioside (GM3) suffer from a

serious neurological disease characterized by severe seizures & blindness

D. Glycolipids also play role in certain infectious diseases; cholera & botulism toxins enter their target

cell by first binding to cell-surface gangliosides, as does influenza virus

C

H

OH

CH

CH

(CH )2 12

CH3C

NH

CO R

H

C

H

H

OP

O

O

O-

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H

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C

H

H

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(CH )2 2

HC3

Sphingomyelin (a sphingolipid)

VI. Cholesterol a sterol that can be up to 50% of animal membrane lipids; it is missing from most plant & all

bacteria cell membranes

A. Small hydrophilic hydroxyl group is oriented toward membrane surface; the rest is embedded in thelipid bilayer

B. Cholesterol rings are flat & rigid; interfere with movement of phospholipid fatty acid tails

The !ature and "mortance of the #iid Bilayer

I. Each type of cell membrane has its own characteristic lipid composition

A. Differfromeachotherintypesoflipids,natureofheadgroups&particularspecies of fattyacyl chain(s)

B. Some biological membranes may contain hundreds of chemically distinct species of phospholipids;

the role of this remarkable diversity of lipid species remains the subject of interest & speculations

C. The percentages of some major types of lipids vary from membrane to membrane

II. Lipid composition can influence biological properties of membrane; not just structural elements

A. Can influence activity of particular membrane proteins

B. Can determine physical state of membrane

C. Can play role in health & disease - Tay-Sachs disease (fatal inherited condition caused by build-up of

particular lipid, a ganglioside, in brain cells)

D. Provide precursors for highly active chemical messengers that regulate cellular function

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III. Combined fatty acyl chains of lipid bilayer span width of ~30 & each row ofhead groups (with itsadjacent shell of water molecules) adds another15 ; thus entire bilayeris only ~60 (6 nm) thick

IV. Presence in membranes of this thin film of amphipathic lipid molecules has remarkable consequences

for cell structure & function

A. Due to thermodynamic considerations, the hydrocarbon chains of the lipid bilayer are never exposed

to the surrounding aqueous solution

1. Thus, membranes never seen to have a free edge due to cohesion & spontaneous formation (they

are closed bimolecular sheets)

2. They are always continuous, unbroken structures & thus form extensive interconnected networks

within cell

B. Due to flexibility of lipid bilayer, membranes are deformable & can change their overall shape (as in

locomotion & cell division)

C. Bilayer facilitates regulated fusionor buddingof membranes events of secretion (cytoplasmic vesicles

fuse to plasma membrane; exocytosis), endocytosis or fertilization (2 cells fuse to form single cell)

1. Both involve processes in which 2 separate membranes come together to become a continuous sheet

D. Membrane is also important in maintaining proper internal composition of cell & in separating electric

charges across plasma membranes & many other cell activities

V. Membrane can self-assemble in aqueous solutions (demonstrated easily within test tube)

A. If a small amount of phosphatidylcholine is dispersed in aqueous solution, the phospholipid molecules

assemble spontaneously to form the walls of liposomes (fluid-filled spherical vesicles)

1. Their walls made of single continuous lipid bilayer organized in same way as natural membrane

2. Valuable in membrane studies - insert membrane proteins, study their function in simpler environment

than that of a natural membrane

B. Liposomes have been developed as vehicles to deliver drugs or DNA to specific target cells in body; can

be linked to liposome walls or placed at high concentrations in liposome central cavity (lumen)

1. Build liposome walls to contain specific proteins (antibodies, hormones)

2. The proteins allow liposomes to bind selectively to surfaces of particular target cells where drug orDNA is supposed to go

3. When first tried, immune system phagocytes removed them - now stealth liposomes (e.g., Caelyx)

are given synthetic polymer outer coating that protects them from immune destruction

VI. Asymmetry of membrane lipids lipid bilayer consists of 2 distinct leaflets that have distinctly different

lipid composition

A. Experiments that have led to this conclusion take advantage of fact that lipid-digesting enzymes cannot

penetrate plasma membrane &, thus, can only digest lipids residing in membrane outer leaflet

1. If treat intact human RBCs with lipid-digesting phospholipases (only affect outer leaflet lipids since

they cannot penetrate membrane)..

a. ~80% of the membrane phosphatidylcholine (PC) is hydrolyzed, but only ~20% of membrane

phosphatidylethanolamine (PE) &

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C. All glycolipids of plasma membrane are in outer leaflet, where they probably serve as receptors for

extracellular ligands

1. Phosphatidylethanolamine, which is concentrated in inner leaflet, tends to promote the curvature of

the membrane, which is important in membrane budding & fusion

2. PS is also concentrated in inner leaflet it has a net"-" charge at physiological pH, which makes it a

candidate for binding positively-charged lysine & arginine residues

a. Such arginine & lysine residues are adjacent to the membrane-spanning -helix of glycophorin A

& PS may bind them

b. PS appears on the outer surface of aging lymphocytes & thus marks them for destruction by

macrophages

c. PS's appearance on outer surface of platelets leads to blood coagulation

3. Phosphatidylinositol, which is concentrated in inner leaflet, plays a key role in the transfer of

stimuli from the plasma membrane to the cytoplasm

Membrane Carbohydrates

I. Eukaryotic cell plasma membranes also contain carbohydrate

A. Depending on species & cell type, carbohydrate content of plasma membrane ranges between 2 - 10%

by weight, e.g., RBC membrane - ~52% protein, 40% lipid, 8% carbohydrate

B. 90% of

membrane carbohydrates are covalently linked to protein to form glycoprotein

II. All membrane carbohydrates face toward outside of cells into extracellular space or toward organelle

interior (carbohydrates of internal cellular membranes); in both cases, they face away from cytosol

A. Phosphatidylinositol of membrane does not count even though it contains sugar group

B. Composition&structureofoligosaccharidesattached to membrane proteins & lipids vary considerably

C. Provides for their specificity in interactions with each other & other molecules

III. Glycoproteins - carbohydrates are short, branched oligosaccharides with < ~15 sugars per chain; addition

of these sugars or glycosylation is most complex of protein modifications occurring in cellA. Oligosaccharides vary considerably in composition & structure; sialic acid usually on end giving

negative charge

B. Attach to several different amino acids by two major types of linkages

C. Play an important role in mediating interactions of cell with other cells & nonliving environment &

sorting of membrane proteins to different cell compartments

IV. Glycolipids - short, branched oligosaccharide chains

A. On RBCs, glycolipids determine ABO blood type (have different enzymes that add sugars to ends of

carbohydrate chains)

1. Person with blood type A has enzyme that adds N-acetylgalactosamine to end of chain

2. Person with blood type B has enzyme that adds galactose to chain terminus

3. The 2 enzymes are encoded by alternate versions of the same gene, yet they recognize differentsubstrates

4. AB people possess both enzymes; people with type O blood lack enzymes capable of attaching

either terminal sugar

5. The function of the ABO blood-group antigens remains a mystery

B. May play roles in certain infectious diseases (cholera toxin & influenza virus bind to gangliosides); this

suggests that they probably serve as some kind of receptor in normal cell function

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Structure and Function of Membrane Proteins$ Overview

I. Membranes may contain hundreds of different proteins depending on cell type or particular organelle

A. Each membraneproteinhas defined orientation relative to cytoplasm; thus, the properties of 1 membrane

surface are very different from those of other surface - this asymmetry referred to as membrane sidedness

B. All proteins are asymmetrically situated according to function - properties of one membrane surface arevery different from those of other surface

C. Parts of proteins interacting with extracellular substances, other cells, extracellular matrix elements face

out; those interacting with cytoplasmic molecules face inward

II. Three classes of membrane proteins distinguished by intimacy of their relationship to lipid bilayer

A. Integral proteins - penetrate into lipid bilayer; they pass entirely through bilayer (transmembrane)

1. Have domains that protrude from both sides of membrane (extracellular & cytoplasmic)

2. Some have only one membrane-spanning segment; others are multispanning

3. Genome-sequencing studies suggest that integral membrane proteins constitute 20 -30% of all

encoded proteins

B. Peripheral proteins located entirely outside of bilayer on either the extracellular or cytoplasmic side;

associated with membrane surface by noncovalent bonds

C. Lipid-anchored proteins located outside bilayer on either extracellular or cytoplasmic side, but they are

covalently linked to membrane lipid situated within bilayer

Structure and Function of Membrane Proteins$ "nte%ral Membrane Proteins

I. Most integral membrane proteins function in the following capacities:

A. As receptors that bind specific substances at the membrane surface

B. As channels or transporters involved in the movement of ions & solutes across the membrane or

C. As agents that transfer electrons during the processes of photosynthesis & respiration

II. Integral membrane proteins - amphipathic; hydrophobic parts contact fatty acids in bilayer & seal proteinsinto membrane lipid "wall"; hydrophilic parts on outside or coating aqueous channel through it

A. Amino acid residues in transmembrane domains for van der Waals interactions with fatty acyl chains of

bilayer

1. Intimate contact of membrane & integral proteins preserves permeability barrier & protein is brought

into direct contact with surrounding lipid molecules

2. Lipid molecules that are closely associated with a membrane protein might play an important role in

the protein's activity

3. However, the degree to which a particular protein requires specific interactions with particular lipid

molecules remains unclear

B. Thoseportions of an integral membrane protein that project into cytoplasmor extracellularspace tend to

be morelike globular proteins1. These nonembedded domains tend to have hydrophilic surfaces that interact with water-soluble

substances (low MW substrates, hormones, other proteins) at the edge of the membrane

C. Several large families of membrane proteins have an interior channel that provides an aqueous

passageway through the lipid bilayer

1. The linings of these channels typically contain key hydrophilic residues at strategic locations

D. Integral proteins need not be fixed structures but may move laterally within membrane

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II. Freeze-fracture replication analysis showed that proteins can penetrate through membranes

A. Procedure tissue is frozen solid & then struck with knife blade, fracturing the block into 2 pieces

1. A favored path of the fracture plane is between the 2 leaflets of bilayer so the membrane is split

2. Metals are deposited on exposed membrane surfaces to form shadowed replica & viewed in EM

3. Looks like road strewn with pebbles (called membrane-associated particles)

4. Since fracture plane passes through bilayer center, particles correspond to integral proteins that

extend at least halfway through lipid core of bilayer

5. When fracture plane reaches a given particle, it goes around it rather than cracking it in half so each

protein (particle) separates with one half of membrane leaving a pit in the other half

B. Allows an investigation of the microheterogeneity of membrane so one can see localized differences in

parts of membrane

1. Biochemical observations average out such individualities; microscopic observations do not & thus

allow such individualities to be appreciated

III. Studying structure & properties of integral membrane proteins difficult to isolate in soluble form due to

their hydrophobic transmembrane domains

A. Extraction from membrane normally requires the use of detergents

1. Ionic (charged) detergents like SDS, which denatures proteins2. Nonionic (uncharged) like Triton X-100, which usually does not alter protein tertiary structure

B. These detergents are amphipathic (polar end, nonpolar hydrocarbon chain) so they can substitute for

phospholipids in stabilizing the proteins, while solubilizing them in aqueous solution

1. Once solubilized, various analytical procedures can be carried out to determine protein's amino acid

composition, molecular mass, amino acid sequence, etc.

C. Hard to get crystals of most integral proteins for X-ray crystallography

1. In fact, 95,000 different conditions for crystallization

2. Despite such success, researchers still rely largely on indirect approaches for determining 3D

organization of most membrane proteins

F. Many integral membrane proteins have substantial portion present in cytoplasm or extracellular space

sometimes this soluble portion has been cleaved from the transmembrane domain

1. It is then crystallized & its tertiary structure determined

2. Provides valuable data about protein, but fails to provide information about the protein's orientation

within the membrane

IV. Identifying transmembrane domains which segments are embedded in membrane?

A. A great deal can be learned about the structure of a membrane protein & its orientation within the

lipid bilayer from a computer-based (computational) analysis of its amino acid sequence

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1. This is readily deduced from the nucleotide sequence of an isolated gene

2. Segments embedded within membrane (called transmembrane domains) have a simple structure &

consist of string of 2030 predominantly nonpolar amino acids that span bilayer as an -helix

a. Ex.:glycophorinA,majorerythrocytecell membraneintegralprotein of the 20 amino acids of its

lone -helix (amino acids 73 92)

b. All but 3 have hydrophobic R groups (or an H atom in the case of glycine residues); theexceptions are serine & threonine, which are non-charged, polar residues

c. Hydroxyl groups of threonine residue side chains can form H bond with one of the oxygen atoms

of the peptide backbone

d. Fully charged residues may also appear in transmembrane helices, but they tend to be

accommodated in ways that allow them to fit into their hydrophobic environment

e. As example, if helix contains a pair of charged residues, the side chains can reach out & interact

with the innermost polar regions of membrane, even if it requires distorting the helix to do so

f. Aromatic ring on tyrosine can be oriented parallel with hydrocarbon chains with which it has

become integrated

3. The maximum number of H bonds between neighboring amino acids allowed by -helix creates a

highly stable (low-energy) configuration

4. This is important for a membrane-spanning polypeptide that is surrounded by fatty acyl chains and

is thus unable to form H bonds with an aqueous solvent anyhow

5. Since each amino acid occupies 1.5 of polypeptide length & the hydrophobic core of bilayer is 30wide, it takes at least 20 amino acids to span the hydrophobic part of membrane

6. A few integral membrane proteins have been found to contain loops or helices that penetrate but do

not span the bilayer

B. Transmembrane segments usually identified using hydropathy plot; each site along polypeptide is

assigned value giving a measure of the hydrophobicity of amino acid at that site & its neighbors

1. Gives a running average of hydrophobicity of short sections of polypeptide & guarantees that one or

a few polar amino acids in a sequence do not alter the profile of the whole stretch

2. Hydrophobicity is determined by various criteria: lipid solubility or energy required to transfer them

from an aqueous into a lipid medium3. Transmembrane segments usually identified as a jagged peak extending well into hydrophobic side of

spectrum

4. Reliable predictions concerning transmembrane segment orientation within bilayer can usually be made

by examining flanking amino acid residues

5. Those parts of the polypeptide at the cytoplasmic flank of a transmembrane segment tend to be more

positively charged than those at the extracellular flank

C. Not all integral membrane proteins have hydrophobic transmembrane -helices

1. A number of membrane proteins contain a relatively large channel positioned within circle of

membrane-spanning -strands organized into a barrel

2. To date, aqueous channels constructed of -barrels have only been found in the outer membranes of

bacteria, mitochondria & chloroplasts

V. Determining spatial relationships between amino acids within integral membrane proteins

A. Use of site-directed mutagenesis - you have isolatedagene for an integral membrane protein,which

based on its sequence, predicts protein has 4 apparent membrane-spanning -helices

1. How are they oriented & which amino acids face lipids? start by introducing specific changes into

gene that codes for protein

2. Replace aminos in neighboring helices with cysteine residues that may then form disulfide bond

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3. If disulfide bond forms, then the helices must reside in very close proximity

4. Helix VII of bacterial lactose permease, a sugar-transporting protein in bacterial cell membranes, was

found to be close to both helices I & II using this method

B. Can also clarify dynamic events occurring as protein functions introduce chemical groups whose

properties are sensitive to distance between them; shows distance between selected protein residues

1. Nitroxides are chemical groups that contain unpaired electron (produces characteristic spectrum

when monitored by technique called electron paramagnetic resonance [EPR] spectroscopy)

2. Can introduce nitroxide at any site in protein by first mutating that site to cysteine & then attaching

nitroxide to SH group of cysteine

C. Example: used to detect conformational changes in protein as its channel is activated in response to

changes in medium pH; the bacterial K+channel (tetramer made of 4 identical subunits)

1. Cytoplasmic opening to channel is bounded by 4 transmembrane helices, one from each subunit

2. Introduce nitroxide near cytoplasmic end of each transmembrane helix > EPR spectra change when

pH is 6.5 (channel closed) & pH is 3.5 (channel open)

3. Shape of each line depends on proximity of nitroxides to one another spectrum broader at pH 6.5

since nitroxide groups on 4 subunits closer together at this pH (lowers EPR signal intensity)

4. Suggests that channel activation is accompanied by increased separation between the labeled residues

of the 4 subunits5. Increase in channel opening diameter allows cytoplasmic ions to reach actual permeation pathway in

channel allowing only the passage of K+ions

Structure and Function of Membrane Proteins$ Periheral Membrane

Proteins

I. Peripheral membrane proteins - attach by noncovalent (weak electrostatic) bonds to hydrophilic head groups

of lipids or to hydrophilic portions of integral proteins protruding from bilayer

A. Can usually be solubilized by extraction with aqueous salt solutions

B. In multisubunit proteins, some subunits may be peripheral & others integral (blurs distinction between

integral & peripheral proteins)

II. Best-studied peripheral proteins are located on cytosolic membrane surface where they form fibrillar

network that acts as membrane skeleton

A. These proteins give mechanical support to membrane & function as an anchor for integral proteins

B. Other peripheral proteins on internal membrane surface function as enzymes, specialized coats or

factors that transmit transmembrane signals

III. Typically have dynamic relationship with membrane, being recruited to or released from membrane

depending on prevailing conditions

Structure and Function of Membrane Proteins$ #iid&Anchored MembraneProteins

I. Lipid-anchoredproteins- 2 kinds marked by lipid anchor types & surface on which they are exposed

A. GPI-anchored proteins - on external face of plasma membrane; bound to membrane by short

oligosaccharide linkedto molecule of glycophosphatidylinositol (GPI) in membrane outer leaflet;

1. Discovered when certain membrane proteins were found that are released by a phospholipase that

specifically recognized & cleaved inositol-containing phospholipids

2. Include various receptors, enzymes, cell-adhesion proteins, PrPC(normal cellular scrapie protein)

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3. Rare anemia (paroxysmal nocturnal hemoglobinuria) results from GPI synthesis deficiency that

makes RBCs susceptible to lysis

B. Another group on cytoplasmic side of membrane is anchored to membrane by long hydrocarbon chains

embedded in bilayer inner leaflet

1. At least two, Src & Ras, are implicated in transformation of a normal cell to a malignant state

Membrane #iids and Membrane Fluidity

I. Physical state of membrane lipids described by fluidity (or viscosity) fluidity (measure of ease of flow)

& viscosity (measure of resistance to flow) are inversely related

II. Lipids exist in 2 states (solid & liquid phase of varying viscosity depending on temperature); ex. artificial

bilayer with phosphatidylcholine & phosphatidylethanolamine (largely unsaturated fatty acids)

A. At warm temperatures (37C), lipid in relatively fluid, liquidlike state; a 2 dimensional liquid crystal1. Molecules retain a specified orientation as in crystal; molecule long axes stay essentially parallel

2. Yet individual molecules can rotate around axis or move laterally within bilayer plane

B. At colder temperatures, forms frozen crystalline gel in which phospholipid fatty acid chain movement

is greatly restricted1. If temperature is lowered slowly, a point is reached where bilayer distinctly changes

2. Temperature at which this change occurs is called transition temperature

III. Transition temperature - temperature at which membrane goes from fluid state to crystalline gel

A. Transition temperature depends on ability of lipid molecules to be packed together which depends in turn

on the particular lipids of which the membrane is constructed

1. Saturated chains pack more closely & are less fluid since they have shape of straight, flexiblerod

2. Cis-unsaturated fatty acids have crooks in chain since carbons sharing double bond cannot rotate;

crooks in chains cause unsaturated lipids to pack together less tightly

3. Thus, phospholipids with saturated chains pack together more tightly than those with unsaturated

chains4. Higher degree of bilayer fatty acid unsaturation > lower temperature before bilayer gels

5. Introduce one double bond into stearic acid > lowers melting temperature almost 60C6. Plant oils highly unsaturated (polyunsaturated) & liquid; animal fats highly saturated & solid

B. Fatty acid chain length can affect membrane fluidity & transition temperature: the shorter the fatty acyl

chains the lower its melting temperature

C. Different lipids undergo their phase change over a very wide temperature range

1. Various phosphatidylcholines can be made & used to build bilayers whose transition temperatures

range from below 0C to >60CD. Cholesterol also affects membrane physical state; interacts with membrane phospholipid fatty acid

chains & alters the way the fatty acids pack together

1. Because of their orientation within the bilayer, cholesterol disrupts the close packing of fatty acyl

chains & interferes with their mobility

2. It tends to abolish sharp transition temperatures & creates a condition of intermediate fluidity

3. In physiological terms, it tends to raise membrane durability & lower membrane permeability

V. Importance of membrane fluidity

A. Membrane fluidity provides a perfect compromise between rigid, ordered structure & a totally fluid,

nonviscous liquid

1. In the rigid structure - mobility would be absent

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2. In the totally fluid structure - components could not be oriented; structural organization & mechanical

support would be lacking

B. Moderate fluidity also allows interactions to take place within membrane; clusters of membrane proteins

can assemble at particular sites within membrane & form specialized structures

1. Among the specialized structures: intercellular junctions, light-capturing photosynthetic complexes,

synapses

2. Molecules that interact can come together, carry out necessary reaction & move apart

C. Membranes arise only from preexisting membranes their growth is accomplished by the insertion of

lipid & protein components into the fluid matrix of the membranous sheet

D. Many of the most basic cell processes (cell movement, growth & division; intercellular junction

formation, secretion, endocytosis/exocytosis) depend on membrane component movement

1. These processes would probably not be possible if membranes were rigid, nonfluid structures

VI. Maintaining membrane fluidity - cells respond to environmental changes in temperature by regulating

membrane fluidity,exceptfor birds & mammals,which are warm-blooded;homeostasis at cellular level

A. Since correct degree of fluidity is essential for many activities, cells alter membrane phospholipids to

maintain fluidity when temperature changes

1. Cell membrane physical properties are matched to prevailing environmentB. Lower cell culture temperature > cells respond metabolically with initial response handled by

enzymes that remodel membranes to make them more cold resistant

1. Fatty acyl chain single bonds are desaturated forming double bonds; catalyzed by desaturases

2. Chains are reshuffled between different phospholipids to produce those with 2 unsaturated chains;

this greatly lowers the bilayer's melting temperature

a. Reshuffling done by phospholipases (split fatty acids from glycerol backbone)

b. Acyltransferases transfer the fatty acid chains to a different phospholipid

3. Cell also changes the types of phospholipids synthesized to those containing more unsaturated

fatty acids

C. The above strategies are seen in hibernating mammals, pond-dwelling fish (temperature changes

markedly from day to night), cold-resistant plants, bacteria living in hot water springs

VII. Lipid rafts community of cell biologists is split into believers & nonbelievers

A. When membrane lipids are extracted from cells & used to prepare artificial lipid bilayers, cholesterol

& sphingolipids tend to self-assemble into microdomains

1. These microdomains are more gelated & highly ordered than surrounding regions consisting

primarily of phosphoglycerides

2. Due to distinctive physical properties, microdomains tend to float within more fluid, disordered

artificial membrane environment; thus, these cholesterol/sphingolipid patches are called lipid rafts

3. When added to artificial bilayers, certain proteins tend to become concentrated in lipid rafts, whereas

others tend to remain outside their boundaries

4. GPI-anchored proteins show a particular fondness for the ordered regions of the bilayer

B. Controversy arises over whether similar types of cholesterol-rich lipid rafts exist within living cells

1. Most evidence in favor of lipid rafts is derived from studies that employ detergent extraction or

cholesterol depletion, which makes the results difficult to interpret

2. Attempts to demonstrate presence of lipid rafts in living cells have generally been unsuccessful,

3. This either means that lipid rafts do not exist or that they are so small (5 25 nm in dia) & short-

lived as to be difficult to detect with current techniques

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C. The concept of lipid rafts is very appealing because it provides a means to introduce order into a

seemingly random sea of lipid molecules

D. Lipid rafts are postulated to serve as floating platforms that concentrate particular proteins, thereby

organizing the membrane into functional compartments

1. They may provide a favorable local environment for cell-surface receptors to interact with other

membrane proteins that transmit signals from the extracellular space to the cell interior

The Dynamic !ature of the Plasma Membrane$ #iid and Protein Mobility

I. Lipid bilayer is relatively fluid - polar lipid heads linked to gold particles or fluorescent compounds are

seen to move in the microscope

A. Phospholipids move laterally within the same leaflet with considerable ease - it has been estimated

that a phospholipid can diffuse from one end of bacterium to the other end in 1 2 sec

B. Lipids do not flip-flop to other leaflet very often (takes a matter of hours to days), since polar heads

movingthroughhydrophobicmembraneisthermodynamically unfavorable;mostrestrictedmovement

1. However, cells contain enzymes (flippases) that move certain phospholipids between leaflets

2. Flippases may play role in establishing lipid asymmetry or reverse the slow rate of passive

transmembrane movementC. Lipids provide the matrix in which integral proteins of membrane are embedded; thus the physical state

of lipids is an important determinant of integral protein mobility

1. Demonstrated movement of integral membrane proteins was a cornerstone in the formulation of the

fluid-mosaic model

II. Diffusionof membraneproteinsafter cell fusion Larry Frye & Michael Edidin, Johns Hopkins (1970)

A. Cell fusion is a technique in which 2 different cell types or cells from 2 different species can be fused

to produce one cell with a common cytoplasm & a single continuous membrane

1. Make cell membranes sticky to ease fusion by adding some inactivated viruses that attach to the

membrane surface, by a mild electric shock or by adding polyethylene glycol

2. These treatments make the plasma membranes adhere to one another

3. Cell fusion has played important role in cell biology & is now used as part of a technique to prepare

specific antibodies

B. Fused mouse & human cells > follow lateral movement of proteins with specific antibodies

covalently linked to fluorescent dyes (mouse proteins green dye; human proteins red dye)

1. Mouse & human protein location seen by viewing cells under a fluorescence light microscope

2. Right after fusion, cell is half green & half red; later, proteins move laterally into opposite halves

3. By 40 minutes, all proteins were uniformly distributed around the entire hybrid cell membrane

4. At lower temperatures, the mobility of the membrane proteins decreased because of the increased

viscosity (decreased fluidity) of the lipid bilayer

5. These early experiments suggested that integral membrane protein movements were virtually

unrestricted; later, membrane dynamics were found to be much more complex than first thought

III. Protein mobility patterns in living cell membranes are shown by 2 light microscopy methods; they are

excellent for measuring protein movement extent & rate

A. FRAP (fluorescence recovery after photobleaching) - fluorescently label integral membrane components

in cultured cells in a general or specific manner

1. Treat with fluorescein isothiocyanate, a nonspecific dye, [reacts with all exposed proteins] or label

with fluorescent antibodies or other specific probes

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2. Place under microscope & individually irradiate cells by sharply focused laser beam > irreversibly

bleachessmall circular area (~1 m dia)on cell > followfluorescence recovery rate3. If labeled proteins are mobile, their random movements cause fluorescence to reappear in circle

4. Rate of fluorescence recovery is measure of diffusion rate (D - diffusion coefficient); recovery

extent (percentage of original intensity) reflects percentage of labeled molecules free to diffuse

5. Early studies proteins move much more slowly in cell membranes than in artificial bilayers & a

significant fraction of membrane proteins (30-70%) was not free to diffuse back into circle

6. Drawbacks to technique - only follows large population of labeled molecules diffusing over a

relatively large distance (1m); cannot see individual protein paths7. Thus, it is hard to tell immobile proteins from those that diffuse only a limited distance in the time

allowed, so other techniques have been developed to compensate for these deficiencies

B. Single-particle tracking (SPT) - label individual membrane proteins with antibody-coated gold

particles (~40 nm in diameter) & track them with computer-enhanced video microscopy

1. Solves problems of FRAP with individual protein tracking & results often depend on the

particular protein being studied

2. Some proteins move randomly through membrane at rates lower than in artificial bilayers

a. If mobility is based strictly on physical parameters (lipid viscosity, protein size), one would

expect proteins to migrate with diffusion coefficients of ~10-8 10-9cm2/secb. The rates actually observed for these molecules are 10-10 10-12cm2/sec

c. The reasons for the reduced diffusion coefficient have been debated

3. Some proteins fail to move & are considered to be immobilized

4. Some proteins move in highly directed (nonrandom) manner toward one part of cell or other, e.g.,

one might move toward leading or trailing edge of a moving cell

5. Most proteins exhibit random (Brownian) movement in membrane at rates like free diffusion

(diffusion coefficients ~5 x 10-9cm2/sec

a. But these protein molecules are unable to move more than a few tenths of a micron

b. The membrane appears to contain barriers that prevent extended movement

IV. Restraints on protein mobility (factors affecting membrane protein diffusion) in summary, lipid matrixviscosity & protein mass are partially responsible, but other forces also restrain protein mobility

The Dynamic !ature of the Plasma Membrane$ Control of Membrane Motility

I. Interactions occurring within membrane itself & materials on outer surface control membrane motility

A. Some membranes are crowded with proteins; thus, a protein's movement is impeded by its neighbors

B. The strongest influences on an integral membrane protein are thought to be exerted from just beneath

the membrane on its cytoplasmic face

1. Membranes of many cells possess a fibrillar network, a membrane skeleton, consisting of peripheral

proteins situated on the cytoplasmic surface

2. Acertainproportionofintegralmembraneproteinsaretetheredtomembrane skeleton or otherwise

restricted by it; if not firmly anchored, skeleton may limit distance they can freely migrate

C. Optical tweezers have been used to trap integral proteins & drag them through the membrane with a

known force; yields information about the presence of membrane barriers

1. Takes advantage of the tiny optical forces generated by a focused laser beam

2. Tag subject integral proteins with antibody-coated beads (serve as handles gripped by laser field)

3. Usually, optical tweezers drag integral proteins a limited distance before they encounter a barrier that

causes their release; upon release, they typically spring backward suggesting elastic barriers

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D. Genetic modification of cells so that they produce altered membrane proteins can be used to study

factors that affect membrane protein mobility

1. Genetic deletion of the cytoplasmic portions of these proteins allows them to move much greater

distances than their intact counterparts

2. This indicates that barriers reside on the cytoplasmic side of the membrane, suggesting that the

membrane's underlying skeleton forms a network of "fences" around portions of the membrane

3. This creates compartments that restrict the distance an integral protein can travel

E. At times, proteins move across boundaries to different microdomain through breaks in fences

1. Breaks mayappear& disappearalong with dynamicdisassembly/reassemblyofmeshworkportions

2. Membrane compartments may function primarily to keep specific combinations of proteins in close

enough proximity to facilitate their interaction

F. External materials restrict movement - proteins engineered to lack the portion that normally projects

into the extracellular space move at much faster rate than the wild type version of protein

1. Suggests that extracellular materials entangle external parts of transmembrane proteins, slowing

them

II. Membrane lipid mobility phospholipids are small molecules that make up the very fabric of the lipid

bilayer & one would expect their movement to be unfettered, but they also seem to be restrictedA. Tag individual phospholipids, follow them by under microscope using ultra-high-speed cameras >

they are confined for very brief periods & then hop from one confined area to another

1. Individual phospholipid followed over period of 56 sec it diffuses freely within onecompartment before it jumps "fence" into neighboring compartment

2. It then jumps over another fence into an adjacent compartment, etc.

B. Treatment of membrane with agents that disrupt the underlying membrane skeleton also removes the

fences that restrict phospholipid diffusion

C. How does membrane skeleton interfere with phospholipid movement if it lies beneath bilayer?

1. Some conclude that the fences are made of rows of integral membrane proteins whose

cytoplasmic domains are attached to membrane skeleton (like cows confined by picket fence)

III. Membrane domains & cell polarity most studies of membrane dynamics were initially of the relatively

homogeneous upper or lower surface of cell on culture dish

A. Most membranes are not like this; they exhibit distinct variations in protein composition & mobility,

especially in cells whose various surfaces display distinct functions & must maintain order

B. Example: epithelial cells lining intestinal wall & microscopic kidney tubules (both highly polarized);

their different surfaces carry out different functions

1. Apical plasma membrane of epithelial cells (intestinal, kidney tubules) selectively absorbs

lumenal materials & has enzymes different from lateral surface

2. Lateral surfaces of epithelial cells interact with neighboring cells

3. Basal membrane sticks to underlying extracellular substrate (basement membrane)

4. Neurotransmitter receptors are concentrated in regions of membrane within synapses

5. Low density lipoprotein receptors are concentrated in patches of membrane specialized to

facilitate their internalization

The Plasma Membrane of the 'ed Blood Cell ('BC)

I. Why is RBC membrane so well studied & the best-understood membrane?

A. The cells are inexpensive to obtain and readily available in huge numbers from whole blood

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B. The cells are already present as single cells and need not be dissociated from a complex tissue

C. RBCs are simple in comparison to other cells; they have no contaminating internal cell membranes

D. One can obtain pure, intact RBC membranes (ghosts) by hypotonic lysis of cell (hemolysis)

1. One can do this by placing the cells in a dilute (hypotonic) salt solution

2. In response to this osmotic shock, cells expand, their cell membranes become leaky & contents

(mostly hemoglobin) flow out

II. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) used to separate (fractionate) membrane proteins

(see Ch. 18) after they have been solubilized; gives one an idea of membrane protein diversity

A. Extract proteins & denature with ionic detergent SDS (sodium dodecyl sulfate) that solubilizes &

coats integral proteins with negative charges allowing their migration in an electric field

B. The charge distribution of the proteins is equal despite protein size, since number of SDS molecules per

unit weight of protein is constant; thus, proteins separate on basis of molecular weight

C. The largest proteins move slowest & the smallest move fastest through molecular sieve of gel

D. Themajor erythrocytemembraneproteins are separated into ~12 bands by SDS-PAGE; they include:

1. A variety of enzymes: glyceraldehyde 3-phosphate (a glycolytic enzyme), transport proteins(for ions

& sugars), & skeletal proteins (like spectrin)

2. Various proteins can be correlated with the transport of oxygen & carbon dioxide & with the physicalstresses that the cells encounter as they circulate through the body

E. At the same time, the erythrocyte membrane is presumed to be simpler than that of most other cells

because it lacks proteins involved in cell signaling & cell-cell or cell-matrix interactions

III. Erythrocyte membrane integral proteins its most abundant integral proteins are 2 CHO-containing,

membrane-spanning proteins (band 3 & glycophorin A); freeze-fracture shows them at high density

A. Band 3 (third band on electrophoresis gel) - has a relatively small amount of carbohydrate (6-8% of

molecule's weight); each subunit spans the membrane at least 12 times

1. It is a homodimer meaning that it is a dimer composed of 2 identical subunits; each band 3 dimer has

a channel in the center that serves as a channel for passive anion exchange across membrane

2. As blood circulates through tissues, CO2dissolves in blood plasma (fluid of bloodstream) &undergoes the following reaction: H2O + CO2> H2CO3> HCO3

- + H+

3. Exchange HCO3- (bicarbonate) & Cl-ions; HCO3

-ions move in & Cl-ions move out of RBCs

4. In lungs where CO2is released, HCO3-ions leave RBCs & Cl -ions go into cells

B. Glycophorin A - its amino acid sequence was the first of a membrane protein to be determined; other

related glycophorins (B, C, D, & E) are also present at much lower concentrations

1. Like band 3, glycophorin A is also present in the membrane as a dimer

2. Unlike band 3, each glycophorin A subunit spans membrane only once; it contains a bushy

carbohydrate cover (16 oligosaccharide chains; together they make up ~60% of molecule's MW)

3. Primary function of the glycophorins may derive from the large number of "-" charges on the sugar

residue (sialic acid) on end of each carbohydrate chain

4. The "-" charges may allow RBCs to repel each other & prevent clumping as they circulate through

the body's tiny blood vessels

5. If peoplelack glycophorinsA & B > no ill effects; band 3 proteins are more heavily glycosylated

to compensate for missing negative charges needed to prevent cell-cell interactions

6. Glycophorin also is the receptor used by the protozoan that causes malaria; provides a path for

entry into RBC; without glycophorins A & B, a person may be protected against malaria

7. Differences in glycophorin amino acid sequence determines MM, MN or NN blood type

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IV. Erythrocyte fibrillar membrane skeleton made of peripheral membrane proteins on inner surface;

maintains biconcave RBC shape under punishment of circulation

A. The membrane skeleton can also establish domains within the membrane that enclose particular groups

of membrane proteins & may greatly restrict the movement of these proteins

B. Spectrin major component of the skeleton; long, flexible fibrous protein, 2 subunits (& ) that curl

around one another; a heterodimer (~100 long)1. Two such dimeric molecules are linked at head ends to form 200 -long filament; it is both elastic &

flexible

2. It is attached to the internal membrane surface by noncovalent bonds to ankyrin (also a peripheral

protein), which is linked noncovalently to the cytoplasmic domain of band 3 molecule

3. Spectrin filaments are organized into hexagonal or pentagonal arrays; this 2-dimensional network is

constructed by linking both ends of each spectrin filament to a cluster of proteins

4. The cluster of proteins includes a short filament of actin & tropomyosin, proteins typically involved

in contractile activities

C. Gene mutations that alter the structure & function of ankyrin & spectrin yield fragile, abnormally

shaped RBCs that lead to genetic diseases, specifically the hemolytic anemias

D. Remove peripheral proteins from RBC ghosts > the membrane fragments into small vesicles,

indicating that the inner protein network is required to maintain the membrane's integrity

V. Erythrocytes are circulating cells that are squeezed under pressure through microscopic capillaries whose

diameter is considerably less than that of the erythrocytes themselves

A. To traverse these narrow passageways day after day, RBCs must be highly deformable, durable &

capable of withstanding shearing forces that tend to pull them apart

B. The spectrin-actin network gives RBCs the strength, elasticity & pliability needed to carry out its

function

C. When first discovered, the erythrocyte membrane skeleton was thought to be a unique structure suited to

the unique & mechanical needs of this cell type

1. However, as other cell types were examined, similar types of membrane skeletons containing

members of the spectrin & ankyrin families have been revealed2. This indicates that inner membrane skeletons are widespread

D. Dystrophin, for example, is a member of the spectrin family that is found in the membrane skeleton of

muscle cells

1. Mutations in dystrophin are responsible for causing muscular dystrophy, a devastating disease that

cripples & kills children

2. As in the case of cystic fibrosis, the most debilitating mutations are ones that lead to a complete

absence of the protein in the cell

3. The plasma membranes of muscle cells lacking dystrophin are apparently destroyed as a consequence

of the mechanical stress exerted upon them as the muscle contracts

4. As a result, the muscle cells die & eventually are no longer replaced

The Movement of Substances Across Cell Membranes$ Diffusion and Osmosis

I. Membrane has dual function it retains dissolved materials of cell so they do not leak out into the

environment & it must allow the necessary exchange of materials into & out of the cell

A. Lipid bilayer is ideally suited to prevent loss of charged & polar solutes (ions, sugars, amino acids)

1. Must make special provisions for movement of nutrients, respiratory gases, hormones, wastes, etc.

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B. Membranes are selectively permeable barrier - how movement controlled? - two means for movement

both of which lead to net flux of ions/compounds (influx - into cell; efflux - out of cell)

1. Passively by diffusion

2. Actively by an energy-coupled transport process

C. Several different processes by which substances move across membranes

1. Simple diffusion through lipid bilayer

2. Simple diffusion through an aqueous, protein-lined channel

3. Facilitated diffusion via a protein transporter

4. Active transport via an energy-driven protein pump capable of moving substances against a

concentration gradient

II. Energetics of solute movement - depends on magnitude of concentration gradient

A. Diffusion is a spontaneous process in which substance moves from region of high concentration to

region of low concentration, eventually eliminating concentration difference between the 2 regions

B. Depends on random thermal motion of solutes; an exergonic process driven by entropy increase

III. When solute is a non-electrolyte, it will move down a concentration gradient (exergonic)

A. When nonelectrolyte (uncharged solute) diffuses across membrane, the free-energy change dependson magnitude of concentration gradient

B. The following formula describes the movement of nonelectrolyte into the cell:

G = RT ln([CI]/[Co]) or G = 2.303 RT log10([CI]/[Co]) where:

1. G = free energy change

2. R = gas constant

3. T = absolute temperature

4. [CI]/[Co] = ratio of the concentration of solute on inside (i) & outside (o) surfaces of membrane

C. At 25C, G = 1.4 kcal/mole x log10([CI]/[Co])1. If the ratio of [CI]/[Co] is less than 1.0, then the log of the ratio is negative, G is negative & the

net influx of solute is thermodynamically favored (exergonic)

2. If external solute concentration is 10 times the internal concentration, G = -1.4 kcal/mole3. Thus, maintenance of a ten-fold concentration gradient represents storage of 1.4 kcal/mole

4. As solute moves into the cell. the concentration gradient decreases, the stored energy is dissipated &

the G decreases, until, at equilibrium, G = 0

D. To calculate G for movement of a solute out of cell, the term for concentration ratios is [Co]/[Ci]

IV. If solute is an electrolyte (a charged species), the overall charge (potential difference, voltage) difference is

also important; bigger difference in charge, bigger difference in free energy

A. If charges of electrolyte & compartment to which it is moving are opposite, attraction results; if they

are the same, there is repulsion

1. Due to mutual repulsion of ions of like charges, it is thermodynamically unfavorable for an

electrolyte to move across membrane into a compartment having a net charge of the same sign2. If the electrolyte charge is opposite in sign to the compartment into which it is moving, the

process is thermodynamically favored

3. The greater the difference in charge (potential difference or voltage) between the 2 compartments,

the greater the difference in free energy

B. The tendency of an electrolyte to diffuse between 2 compartments depends upon 2 gradients:

1. A chemical gradient, determined by the concentration difference of the substance between the 2

compartments

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2. Electric potential gradient, determined by the difference in charge

C. Combination of the 2 gradients is electrochemical gradient & it can be very strong; the free energy

change for diffusion of an electrolyte into cell is G = RT ln([CI]/[Co]) + zFEmwhere

1. z is the charge of the solute

2. F is the Faraday constant (23.06 kcal/V.equivalent, where an equivalent is the amount of the

electrolyte having 1 mole of charge)

3. Emis the potential difference (in volts) between the 2 compartments

D. Suppose a concentration gradient consists of Na+ions present at tenfold higher concentration outside

the cell than inside

1. Voltage across a membrane is typically ~-70 mV, thus the free energy change for the movement

of a mole of Na+ions into the cell under these conditions can be calculated

2. G = -1.4 kcal/mole+zFEm = -1.4kcal/mole + (1)(23.06 kcal/vxmole)(-0.07V)= -3.1 kcal/mole

3. This compares to the G of 1.4 kcal/mole for a nonelectrolyte with a tenfold concentration difference

across a membrane at 25C4. Thus under the conditions described, the concentration difference & the electric potential make similar

contributions to the storage of free energy across a membrane

E. Example: interplay between concentration & potential differences seen in K+ion diffusion out of cell

1. Efflux of K+is favored by K+concentration gradient ([K+] is higher inside cell)

2. Effluxis hinderedby the electricalgradientthat its diffusioncreates(leaveshigher"-"chargeincell

III. Diffusion of substances through membrane

A. 2 qualifications must be met before nonelectrolyte can diffuse passively across a membrane

1. Substances must go down gradient (must be present at higher concentration on one side of

membrane than the other

2. Membrane must be permeable to the substance

B. Membranes are permeable to a given solute in two ways

1. Solute can pass directly through bilayer or

2. Solute can traverse an aqueous channel (pore) that spans the membrane & prevents the solute from

coming into contact with lipids of bliayer

IV. Factors that determine the ability of molecules to pass directly through membrane

A. Polarity of a solute a measure of polarity or nonpolarity is its partition coefficient

1. A solute's partition coefficientis the ratio of its solubility in a nonpolar solvent (octanol, vegetable

oil) to that in H2O under conditions where the nonpolar solvent & H2O are mixed together

2. Higher nonpolar solvent (e. g., oil) : water solubility ratio > solute more able to pass bilayer

(first clue that membrane has lipid layer)

3. In other words, greater lipid solubility leads to faster penetration of the membrane

B. Size - smaller molecules pass through membrane faster - small inorganic substances penetrate rapidly;

bigger polar molecules do not pass easily or at all

1. If two molecules have approximately equivalent partition coefficients, the smaller one tends topenetrate a lipid bilayer more rapidly than the larger one

2. Very small, uncharged (inorganic) molecules (O2, CO2NO, H2O) penetrate very rapidly through

membranes; these smaller molecules may slip between adjacent phospholipids

3. Larger polar molecules (sugars, amino acids, phosphorylated intermediates, etc.) cant penetrate

membrane

4. Thus, the lipid bilayer of cell membrane is an effective barrier that keeps these larger essential

metabolites from diffusing out of the cell

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5. Some of these molecules (sugars, amino acids) must enter cells from bloodstream, but cannot do

so by simple diffusion; special mechanisms must be available to allow their penetration

6. The use of such mechanisms allows a cell to regulate the movement of substances across its

surface barrier

V. Diffusion of H2O through membranes since H2O moves faster through membranes than solutes (dissolved

ions, small polarorganicsolutes; essentially nonpenetrating),membranescalled semipermeable

A. Osmosis ready movement of water through a semipermeable membrane from a region of lower

solute (high water) concentration to a region of higher solute (low water) concentration

1. Water moves toward hypertonic (higher solute concentration, hyperosmotic) environments &

away from hypotonic (lower solute concentration, hypoosmotic) environments

2. 2 solutions with equal solute concentrations - isotonic (no net water movement; isoosmotic)

B. Response of cells to nonisotonic environments

1. Animal cells in hypotonic environments take on water (swell) & eventually lyse (RBCs hemolyze)

2. Plant cells in hypotonic environment take in H2O; no lysis due to cell wall - internal pressure

(turgor) builds up; important for support for nonwoody plants & nonwoody plant parts3. Plant cells in hypertonic environment lose water (volume shrinks) - membrane pulls away from

cell wall (plasmolysis); without water, plants wilt

C. The above observations show that a cell's volume is controlled by the difference between the solute

concentration inside the cell & that in the extracellular medium

D. Swelling or shrinking of cells in slightly hypotonic or hypertonic media are usually temporary events;

within a few minutes, cells recover & return to original volume

1. In hypotonic medium, cells recover as they rid themselves of ions (primarily K+& Cl-)

2. In hypertonic medium, cells recover as they gain ions (mostly Na+& Cl-) from medium

3. Once [internal solute] (including a high concentration of dissolved proteins) equals [external

solute], external & internal fluids are isotonic (no net movement of H2O into or out of cells)

VI. Osmosis is important factor in multitude of bodily functions

A. Digestive tract secretes several liters of fluid daily; it is reabsorbed osmotically by cells lining intestine;

if it is not reabsorbed, as in cases of extreme diarrhea, can lead to rapid dehydration

B. Plant cells are usually hypertonic compared to fluid environment (unlike animal cells which are

isotonic); thus, water tends to enter cell causing internal (turgor) pressure to push against cell wall

1. In hypertonic medium, cell volume shrinks & membrane pulls away from cell wall (plasmolysis);

loss of water via plasmolysis causes plants to lose their support & wilt

2. Turgor pressure provides support for nonwoody plant parts (leaves) & nonwoody plants; loss of

water causes plants to lose support & wilt

VII. Many cells are much more permeable to water than can be explained by simple diffusion through lipid

bilayer

A. Peter Agee & colleagues (Johns Hopkins Univ., early 1990s) attempted to isolate & purify

membrane proteins responsible for Rh antigen on surface of RBCs

1. While trying this they identified a protein they thought might be the long-sought water channel of

the erythrocyte membrane

2. To test hypothesis, they engineered frog oocytes to incorporate the newly discovered protein into

their plasma membranes & then placed the oocytes in a hypotonic medium

3. As predicted, the oocytes swelled due to the water influx & eventually burst

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II. Bewildering variety of ion channels identified, each with integral protein(s) surrounding aqueous pore;

most are highly selective in allowing one particular type of ion to pass through pore

A. Diffusion is always downhill (from higher to lower energy state; higher to lower concentration)

1. Ion channels are bidirectional allowing ion passage in either direction; net flux depends on

electrochemical gradient

B. Comparisons of amino acid sequences of different types of ion channels in diverse organisms (bacteria,

plants, animals) show that they are all members of a small number of giant superfamilies

1. Members of given superfamily may have very different ion selectivities, but all are very similar in

amino acid sequence & overall structure

2. Suggests they are derived from a single protein present in a common ancestor alive over a billion

years ago

C. Most ion channels can exist in either an open or a closed conformation; said to be gated

1. Opening & closing of gates is subject to complex physiological regulation

2. Can be induced by a variety of factors depending on the particular channel

III. Three major categories of gated channels are distinguished

A. Voltage-gated channels conformational state depends on the difference in ionic charge on the 2 sides of

membraneB. Ligand-gated channels conformational state depends on binding of specific molecule (the ligand),

which is usually not the solute that passes through the channel

1. Some ligand-gated channels are opened (or closed) after binding of molecule to outer surface of

channel; others open (or close) after binding of ligand to inner surface of channel

2. Neurotransmitters (e.g., acetylcholine) act on outer surface of certain cation channels; cyclic

nucleotides (e.g., cAMP) act on inner surface of certain calcium ion channels

C. Mechano-gated channels conformational state depends on mechanical forces (e.g., stretch tension) that

are applied to the membrane

1. Members of one family of cation channels are opened by the movements of stereocilia on the hair

cells of the inner ear in response to sound or motions of the head

IV. Structure & function of K+ion channels Roderick MacKinnon et al., Rockefeller Univ. (1998) first

atomic-resolution image of an ion channel protein, a bacterial K+ion channel called KcsA

A. Figuringoutstructureled directlyto learningmechanismby which thechannelsselectoverwhelmingly K+

ions over Na+ions while allowing incredibly rapid K+ ion conductance through membrane

1. The mechanisms of ion selectivity & conductance in this bacterial channel are thought to be virtually

identical to those operating in the much larger mammalian channels

2. Evidently, the basic challenges in operating an ion channel were solved relatively early in evolution,

although many refinements appeared over the next billion or 2 years

B. KcsA channel consists of 4 subunits; each subunit contains 2 membrane-spanning helices (M1 & M2) &

a pore region (P) at the extracellular end of the channel

1. P consists of a short pore helix that extends about one-third of the width of the channel & a

nonhelical loop that forms the lining of a narrow selectivity filter

2. The selectivity filter acquires its name because of its role in allowing the passage of only K+ions

3. Selectivity filter lining contains highlyconserved pentapeptide(Gly-Tyr-Gly-Val-Thror GYGVT)

4. Mutations within this stretch of amino acids often destroy the channel's ability to discriminate

between K+& Na+ions

C. The X-ray crystal structure of the KcsA channel shows that the backbone carbonyl (C=O) groups from

the conserved pentapeptide create 5 successive rings of oxygen atoms

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1. 4 rings are made up of carbonyl oxygens from the polypeptide backbone & 1 ring consists of oxygen

atoms from threonine side chain

2. Each ring contains 4 oxygen atoms (one from each subunit) & has a diameter of ~3, which isslightly larger than the 2.7diameter of a K+ion that has lost its normal hydration shell

3. Thus, the electronegative O atoms that line the selectivity filter can substitute for the shell of water

molecules that are displaced as each K+ion enters the pore

4. While the selectivity filter is a precise fit for a dehydrated K+ion, it is much larger than the diameter

of a dehydrated Na+ion

5. Thus, a Na+ion cannot interact with the 4 carbonyl O atoms needed to stabilize its structure; thus the

smaller Na+ions cannot overcome the higher energy barrier required to penetrate the pore

D. KcsA structure has been determined at very high (2) resolution; it has allowed investigators tovisualize individual K+ions & water molecules

1. The selectivity filter in the models contains 4 potential K+ionbinding site, only 2 of which are

occupied at any given time

2. K+ ions are thought to move 2 at a time from one pair of binding sites to the next pair (sites 1 & 3 to

sites 2 & 4)

3. The entry of a third K+ ion into the selectivity filter creates an electrostatic repulsion that ejects the

ion bound at the opposite end of the line4. Studies indicate that there is virtually no energy barrier for an ion to move from one binding site to

the next, which accounts for the extremely rapid flow of ions across the membrane

E. TheKcsAchannelhas a gate,like eukaryoticchannels;its opening&closing is regulatedbymediumpH,

rather than by voltage across the membrane or binding of ligand; it opens in response to very low pH

1. It has been impossible to crystallize the KcsA channel in its open conformation

2. However, the structure of a homologous prokaryotic K+ channel (called MthK) in its open

conformation has been crystallized & its structure determined

F. Comparison of MthK open structure &the homologousproteinKcsAclosed structurestronglysuggested tha

their gating happens by conformational changes of cytoplasmic ends of inner (M2) helices

1. In closed conformation, the M2 helices are straight & cross over one another to form a "helix bundle"

that seals the cytoplasmic face of the pore2. Channel opens when M2 helices bend by ~30at specific hinge point where glycine residue is found

V. Example of eukaryotic voltage-gated channels: K+ion channels; genes encoding a variety of distinct

voltage-gated K+(or Kv) channels have been isolated & their molecular anatomy scrutinized

A. The Kv channels of plants play an important role in salt & water balance & in cell volume regulation; Kv

channels of animals are best known for their role in muscle & nerve function

1. These more complex eukaryotic versions are thought to perform in a manner similar to prokaryotic

channels

B. Members of this protein family have their N- & C-terminal domains situated on cytoplasmic side of

membrane

C. Eukaryotic Kv channels contain 6 membrane-associated helices (named S1S6) grouped into 2

functionally distinct domains

1. A pore domain has same basic architecture as that of entire bacterial channel & contains selectivity

filter that permits the selective passage of K+ions

a. Helices M1 & M2 & the P segment of the KcsA channel are homologous to helices S5 & S6 & the

P segment of the voltage-gated eukaryotic channels

b. Like the M2 helices of KcsA, the S6 helices line much of the pore & their configuration

determines whether the gate to the channel is open or closed

2. A voltage-sensor domain consists of helices S1-S4; senses the voltage across the plasma membrane

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VI. The 3D crystal structure of a complete eukaryotic Kv channel purified from rat brain was determined by

use of a mixture of detergent & lipid throughout the purification & crystallization process

A. The presence of negatively charged phospholipids is thought to be important in maintaining the native

structure of the membrane protein & promoting its function as a voltage-gated channel

B. Like KcsA channel, a single eukaryotic Kv channel consists of 4 homologous subunits arranged

symmetrically around the central ion-conducting pore

1. The selectivity filter, & thus the presumed mechanism of K+ion selection, is virtually identical in the

prokaryotic KcsA & eukaryotic Kv proteins

2. The gate leading into a Kv channel is formed by the inner ends of the S6 helices & is thought to open

& close in a manner roughly similar to that of the M2 helices of the bacterial channel

C. The S4 helix, which contains several positively charged amino acid residues spaced along the

polypeptide chain, acts as the key element of the voltage sensor

1. The voltage-sensing domain is seen to be connected to the pore domain by a short linker helix

denoted as S4-S5

2. Under resting conditions, the negative potential across the membrane keeps the gate closed

3. A change in potential to a more positive value (depolarization) exerts an electric force on the S4 helix

4. This force is thought to cause S4 helix to move in such a way that its positively charged residues shiftfrom a position exposing them to the cytoplasm to a new position exposing them the outside of cell

D. Voltage sensing is dynamic process; mechanism cannot be resolved by a single static view of protein

1. Several competing models describing the mechanism of action of voltage sensor are currently

debated

2. However it occurs, the S4 helix movement in response to membrane depolarization initiates a series

of conformational changes within the protein that opens the gate at the cytoplasmic end of channel

E. Once pore opened, >100K+pass throughchannel/msec (nearlyrate thatwouldoccur by freediffusion in

solution)

1. Due to large ion flux, opening of a relatively small number of K+channels has significant impact on

the membrane electrical properties

2. After channel is open a few msec, K+

ion movement is automatically stopped by a process known asinactivation

F. Eukaryotic Kv channels typically contain a large cytoplasmic structure whose composition varies among

different channels

1. Inactivation of the channel is accomplished by movement of a small inactivation peptide that dangles

from the cytoplasmic portion of the protein

2. The inactivation peptide is thought to gain access to the cytoplasmic mouth of the pore by snaking its

way through one of 4 "side windows"

3. When one of these dangling peptides moves up into the mouth of the pore, the passage of ions is

blocked & the channel is inactivated

4. At a later stage of the cycle, the inactivation peptide is released & the gate to the channel is closed

5. Thus the potassium channel can exist in 3 different states: open, inactivated & closed

VII. Potassium channels come in many different varieties C. elegans, a nematode worm with a body of ~1000

cells has >90 different genes that encode K+channels

A. A single cell, no matter the what kind of organism it is in, is likely to possess a variety of different K+

channels that open & close in response to different voltages

B. The voltage needed to open or close a particular K+channel can vary depending on whether or not the

channel protein is phosphorylated, which, in turn, is regulated by hormones & other factors

1. Ion channel function is under the control of a diverse & complex set of regulatory agents

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The Movement of Substances Across Cell Membranes$ Facilitated Diffusion

I. Facilitated diffusion - diffusion during which substance binds selectively to a membrane-spanning

protein (facilitative transporter), which facilitates diffusion process

A. The term facilitative transporter distinguishes these proteins from active transporters whose activity is

coupled to a process that releases energy1. Technically, transporter applies to membrane protein that can only bind a solute from one side of

membraneatatime&inwhichshapechangeismechanismfor solute movementacross membrane

2. Definition distinguishes transporters from channels, which, if open, can bind solutes from either side

of membrane at same time; distinction between them is becoming blurred as more learned

B. Solute binding on one side of membrane changes protein shape, exposin