CHAPTER 4

THE STRUCTURE AND FUNCTION OF THE PLASMA

MEMBRANE
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.
CHAPTER 4 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. ~10,000 plasma membranes stacked one on top of another would equal the thickness of a book's page
B. No hint of 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 of
darkly 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
127 127
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 contents
II. 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; H
2
O 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 other
A. 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 external signals (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
128 128
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 H
2
O & 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 layer of lipids) with hydrophilic heads pointed out on both sides
1. Thermodynamically favored arrangement; polar lipid head groups interact with surrounding H
2
O
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 other
III. 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 lined
on inner & outer surfaces by layer of globular proteins
A. Revised version (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
129 129
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. Most membrane phospholipids are 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 just phosphate & 2 fatty acids it is phosphatidic acid (virtually absent in most membranes)
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 (CH
3
) end of the fatty acyl chain
E. With fatty acid chains at one end & a polar head group at the other end, all of the phosphoglycerides have
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
130 130
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 (G
M3
) 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
C
H
C
H
(CH )
2 12
CH
3
C
NH
C O R
H
C
H
H
O P
O
O
O
-
C
H
H
C
H
H
N
+
(CH )
2 2
HC
3
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 the
lipid 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. Differ from each other in types of lipids, nature of head groups & particular species of fatty acyl 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
131 131
III. Combined fatty acyl chains of lipid bilayer span width of ~30 & each row of head groups (with its
adjacent shell of water molecules) adds another 15 ; thus entire bilayer is 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 fusion or budding of 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 or
DNA 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) & <10% of its phosphatidylserine (PS) are attacked
b. Thus, relative to inner leaflet, outer one has relatively high [PC] (& [sphingomyelin]), low [PS]
& [PE]
2. Thus, one can think of bilayer as 2 more-or-less stable, independent monolayers having different
physical & chemical properties; different classes of lipids in membrane also have different properties
B. Biological role of lipid asymmetry - not generally well understood; may lead to different charges on each
side of membrane
132 132
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. <10% of membrane carbohydrate is covalently linked to lipids to form glycolipids & >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 & structure of oligosaccharides attached 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 cell
A. 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 different
substrates
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
133 133
Structure and Function of Membrane Proteins$ Overview
I. Membranes may contain hundreds of different proteins depending on cell type or particular organelle
A. Each membrane protein has 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 are
very 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 proteins
into 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. Those portions of an integral membrane protein that project into cytoplasm or extracellular space tend to
be more like globular proteins
1. 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
134 134
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 proteins
2. 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, <1% of known high-resolution protein structures represent integral membrane proteins
2. Furthermore, most of these structures represent prokaryotic versions of a particular protein, which
are often smaller than their eukaryotic homologues & easier to obtain in large quantity
3. Once the structure of one member of membrane protein family is determined, researchers can usually
apply strategy called homology modeling to learn about structure & activity of other family members
a. Example solution of bacterial K
+
channel KcsA structure provided a wealth of data that could be
applied to structure & mechanism of action of the much more complex eukaryotic K
+
channels
D. One of the first membrane proteins whose entire 3D structure was determined by X-ray crystallography:
bacterial photosynthetic reaction center; it has 3 subunits with 11-membrane-spanning -helices
E. Some technical difficulties in preparing membrane protein crystals have been overcome with new
methodologies & laborious efforts
1. Researchers were able to get high-quality crystals of a bacterial transporter after testing & refining
>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
135 135
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.: glycophorin A, major erythrocyte cell membrane integral protein 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); the
exceptions 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 30
wide, 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 medium
3. 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 isolated a gene 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
136 136
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 subunits
5. 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 Membrane
Proteins
I. Lipid-anchored proteins - 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 linked to 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, PrP
C
(normal cellular scrapie protein)
137 137
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 crystal
1. 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 restricted
1. 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, flexible rod
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
chains
4. Higher degree of bilayer fatty acid unsaturation > lower temperature before bilayer gels
5. Introduce one double bond into stearic acid > lowers melting temperature almost 60C
6. 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 >60C
D. 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
138 138
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, except for 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 environment
B. 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
139 139
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
moving through hydrophobic membrane is thermodynamically unfavorable; most restricted movement
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 movement
C. 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. Diffusion of membrane proteins after 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
140 140
2. Place under microscope & individually irradiate cells by sharply focused laser beam > irreversibly
bleaches small circular area (~1 m dia) on cell > follow fluorescence recovery rate
3. 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 paths
7. 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
-9
cm
2
/sec
b. The rates actually observed for these molecules are 10
-10
10
-12
cm
2
/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
-9
cm
2
/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 matrix
viscosity & 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. A certain proportion of integral membrane proteins are tethered to membrane 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
141 141
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 may appear & disappear along with dynamic disassembly/reassembly of meshwork portions
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 restricted
A. 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 one
compartment 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
142 142
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. The major erythrocyte membrane proteins 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 physical
stresses 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, CO
2
dissolves in blood plasma (fluid of bloodstream) &
undergoes the following reaction: H
2
O + CO
2
> H
2
CO
3
> HCO
3
-
+ H
+
3. Exchange HCO
3
-
(bicarbonate) & Cl
-
ions; HCO
3
-
ions move in & Cl
-
ions move out of RBCs
4. In lungs where CO
2
is released, HCO
3
-
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 people lack glycophorins A & 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
143 143
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 revealed
2. 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.
144 144
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 depends
on magnitude of concentration gradient
B. The following formula describes the movement of nonelectrolyte into the cell:
G = RT ln([C
I
]/[C
o
]) or G = 2.303 RT log
10
([C
I
]/[C
o
]) where:
1. G = free energy change
2. R = gas constant
3. T = absolute temperature
4. [C
I
]/[C
o
] = ratio of the concentration of solute on inside (i) & outside (o) surfaces of membrane
C. At 25C, G = 1.4 kcal/mole x log
10
([C
I
]/[C
o
])
1. If the ratio of [C
I
]/[C
o
] 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/mole
3. 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 [C
o
]/[C
i
]
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 sign
2. 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
145 145
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([C
I
]/[C
o
]) + zFE
m
where
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. E
m
is 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 + zFE
m
= -1.4kcal/mole + (1)(23.06 kcal/v x mole)(-0.07 V) = -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 25C
4. 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. Efflux is hindered by the electrical gradient that its diffusion creates (leaves higher "-" charge in cell
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 coefficient is the ratio of its solubility in a nonpolar solvent (octanol, vegetable
oil) to that in H
2
O under conditions where the nonpolar solvent & H
2
O 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 to
penetrate a lipid bilayer more rapidly than the larger one
2. Very small, uncharged (inorganic) molecules (O
2
, CO
2
NO, H
2
O) 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
146 146
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 H
2
O through membranes since H
2
O moves faster through membranes than solutes (dissolved
ions, small polar organic solutes; essentially nonpenetrating), membranes called 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 H
2
O; no lysis due to cell wall - internal pressure
(turgor) builds up; important for support for nonwoody plants & nonwoody plant parts
3. 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 H
2
O 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
147 147
4. They had discovered a family of small integral proteins (aquaporins) that allow the passive
movement of water from one side of plasma membrane to the other
B. Each aquaporin subunit (in the 4-subunit protein) contains a central channel that is lined primarily by
hydrophobic amino acid residues & is highly specific for water molecules
1. A billion or so water molecules can pass in single file through each channel every second
2. At the same time, H
+
ions, which normally hop along a water molecule chain, are not able to
penetrate these open pores
C. The apparent mechanism by which these channels are able exclude protons has been suggested by a
combination of X-ray crystallographic studies & computer-based simulations
1. X-ray crystallography revealed the protein structure; computer simulations put the protein structure
into operation
2. Very near its narrowest point, aquaporin channel wall contains pair of precisely positioned "+"
charges (residues N203 & N68)
3. These charges attract O atom of each H
2
O molecule as it speeds through constriction in protein
4. This interaction reorients the central water molecule in a position that prevents it from maintaining
H bonds that normally link it to its neighboring water molecules
5. This removes the bridge that would normally allow protons to move from one water molecule to
the next
D. Aquaporins are particularly prominent in cells, like those of a kidney tubule or plant root, where the
passage of water plays a crucial role in the tissues' physiological activities
1. The hormone vasopressin, which stimulates water retention by the collecting ducts of the kidney,
acts by way of one of these proteins (AQP2)
2. Some cases of the inherited disorder congenital nephrogenic diabetes insipidus have been traced to
mutations in this aquaporin channel
3. Persons suffering from this disease excrete huge quantities of urine because their kidneys do not
respond to vasopressin
The Movement of Substances Across Cell Membranes$ Diffusion of "ons
I. Ions go through ion channels (integral proteins surrounding aqueous pores) since core of lipid bilayer is
highly impermeable to charged substances like Na
+
, K
+
, Ca
2+
, & Cl
-
A. Most channels are very selective allowing only one ion through in a downhill direction
B. Ion conductance (rapid movement across membranes) is critical in many cell activities like:
1. Nerve impulse formation & propagation, secretion of substance into extracellular space, muscle
contraction, cell volume regulation, opening of plant leaf stomatal pores
2. Mutations in genes encoding ion channels can lead to many serious diseases
C. Alan Hodgkin & Richard Keynes, Cambridge Univ. (1955) proposed that membranes contain ion
channels (openings in membranes permeable to specific ions); worked on squid giant nerve cells
D. Bertil Hille (Univ. of Washington) & Clay Armstrong (Univ. of Penna.), late 1960s & 1970s
obtained evidence for the existence of ion channels
E. Bert Sakmann & Erwin Neher, Max Planck Inst. in Germany (late 1970s & early 1980s) proof;
developed methods to monitor ionic current passing through a single ion channel
1. Used very fine micropipette-electrodes made of polished glass placed on outer cell surface &
sealed to membrane by suction
2. Voltage across the membrane can be maintained (clamped) at any particular value
3. Current originating in the small patch of membrane surrounded by pipette can be measured
4. These landmark studies were first successful studies into activities of individual protein molecules
148 148
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
membrane
B. 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. Figuring out structure led directly to learning mechanism by which the channels select overwhelmingly 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 highly conserved pentapeptide (Gly-Tyr-Gly-Val-Thr or 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
149 149
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 is
slightly larger than the 2.7 diameter 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 to
visualize individual K
+
ions & water molecules
1. The selectivity filter in the models contains 4 potential K
+
ion

binding 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 line
4. 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. The KcsA channel has a gate, like eukaryotic channels; its opening & closing is regulated by medium pH,
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 homologous protein KcsA closed structure strongly suggested that
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 pore
2. Channel opens when M2 helices bend by ~30 at 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
150 150
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 shift
from 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, >100 K
+
pass through channel/msec (nearly rate that would occur by free diffusion 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 as
inactivation
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
151 151
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 energy
1. Technically, transporter applies to membrane protein that can only bind a solute from one side of
membrane at a time & in which shape change is mechanism for solute movement across 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, exposing solute to other surface from
where it can diffuse down its concentration gradient
1. Since they are passive (not coupled to energy release), they mediate solute movement equally well
in both directions; net flux direction depends solely on gradient direction (down gradient)
C. Facilitated diffusion is similar in many ways to an enzyme-catalyzed reaction
1. Like enzymes, they are specific for the transported molecule; for example, they distinguish between
D & L stereoisomers; like enzymes, they exhibit saturation-type kinetics
2. Move hundreds to thousands of solutes/sec while channels can move millions of ions/sec
3. Like enzymes & ion channels, their activity can be regulated to fit present cell needs
4. Facilitated diffusion is very important in mediating the entry & exit of polar solutes (amino acids,
sugars) that cannot penetrate the lipid bilayer
II. Example: glucose transporter
A. Glucose is body's primary source of direct energy; most mammalian cells have a membrane protein
that facilitates the diffusion of glucose from the bloodstream into the cell
1. Gradient favoring glucose diffusion into cell from bloodstream is maintained by phosphorylating
glucose after it enters cytoplasm, thus lowering intracellular glucose concentration
2. Humans have at least 5 related transporters (isoforms; GLUT 1-5) that facilitate glucose diffusion
into cell; they are distinguished by tissue where found & kinetic & regulatory characteristic
B. Increase in blood glucose levels triggers insulin secretion by pancreas, which stimulates glucose
uptake into various target cells (most notably skeletal muscle, adipocytes or fat cells)
1. Insulin-responsive cells share a common isoform of the facilitative glucose transporter
(specifically GLUT 4)
2. When insulin levels are low, these cells contain relatively few glucose transporters on their plasma
membrane; instead, the transporters are present in membranes of cytoplasmic vesicles
3. Rising insulin levels act on target cells to stimulate the fusion of cytoplasmic vesicles to the
plasma membrane, which provides the transporters necessary to bring glucose into the cell
The Movement of Substances Across Cell Membranes$ Active Transort
I. Many ion gradients are very strong & are generated by the expenditure of energy (active transport)
A. [K
+
] inside a mammalian cell is at least 100 mM; outside - ~5 mM > K
+
ions "want" to leave cell
B. [Na
+
] - ~150 mM outside cell; inside cell - ~10 - 20 mM > Na
+
ions "want" to enter cell
C. [Ca
2+
] - cytosol concentration 10
-7
M; outside cell - 10,000 times higher than that inside cell; this is an
even greater difference than with Na
+
or K
+
152 152
II. Establishment of such steep gradients depends on integral proteins that selectively bind solute & move it
against a gradient (endergonic process); proteins that carry out active transport are often called pumps
A. Such steep gradients cannot occur by either simple or facilitated diffusion
B. Driven by conformational shift along with energy expenditure; coupled to exergonic reaction like ATP
hydrolysis, light absorption, electron transport, flow of other substances down their gradients
III. Coupling active transport to ATP hydrolysis: the Na
+
-K
+
pump; only in animals; primary means to
maintain cell volume & establish the steep gradients needed for nerve-muscle impulses
A. Jens Skou, Danish physiologist (1957) - found ATP-hydrolyzing enzyme in crab nerve cells
1. It was only active in the presence of Na
+
& K
+
ions; Mg
2+
acts as a cofactor by binding ATP
2. Proposed it to be Na
+
-K
+
pump that transported these ions (also called it Na
+
/K
+
-ATPase)
B. Pump is unidirectional & pumps 3 Na
+
ions out of cell for every 2 K
+
ions pumped in & is
electrogenic, contributing directly to the separation of charge across the membrane
1. It is a P-type ion pump that requires phosphorylation of pump during cycle
2. Phosphate is transferred from ATP after its hydrolysis to an aspartic acid residue of pump, causing an
essential conformational change within the protein
3. Changes alter pump's affinity for the 2 cations so it can pick up Na
+
& release K
+
& vice versa
4. To pick either ion up from a low concentration region, protein must bind them with relatively high
affinity; to release them into much higher concentration region, affinity for ion must drop
5. Thus, the affinity for each ion on the two sides of the membrane must be different
6. This is achieved by phosphorylation, which changes the protein's shape & also serves to expose the
ion binding sites to the different sides of the membrane
C. The Na
+
-K
+
pump cycle repeats - uses ~33% of most animal cells' energy (~67% in nerve cells)
1. Pump binds 3 Na
+
ions on inside of cell
2. A bound ATP molecule is hydrolyzed & phosphate transferred to an aspartate residue of pump
3. Pump's shape shifts from the E
1
to the E
2
conformation; it opens to outside, exposing Na
+
-binding sites
there; since E
2
conformation has reduced affinity for Na
+
ions, they are released outside cell
4. Once the 3 Na
+
ions are released, the pump now picks up 2 K
+
ions
5. Pump is then dephosphorylated
6. A molecule of ATP then binds to the protein, causing it to shift back to original E
1
conformation;
pump is now open to inside of cell, its affinity for the 2 K
+
ions is reduced & they are released
7. The cycle is then repeated
D. Digitalis (foxglove plant steroid), a good congestive heart failure treatment for 200 years, inhibits Na
+
-
K
+
pump & leads to chain of events that increases Ca
2+
availability inside heart muscle cells
1. This, in turn, strengthens heart contraction
E. The Na
+
/K
+
pump is found only in animal cells
1. It may have evolved in primitive animals as primary way to maintain cell volume
2. It may also have evolved as a mechanism to generate steep Na
+
& K
+
gradients that play a key role in
the formation of muscle & nerve cell impulses
F. Directionality of pump & its orientation in membrane are important
1. In resealed RBC ghosts, K
+
had to be outside, Na
+
and ATP inside & pump in membrane
2. Ouabain (inhibitor of cation transport similar to digitalis) inhibits pump only if outside ghosts
IV. Other ion transport systems the best-studied P-type pump is the Ca
2+
-ATPase whose 3D structure has
been determined at several stages of the pumping cycle
A. Ca
2+
-ATPase - P-type pump in ER membrane; it actively moves Ca
2+
ions out of cytosol into ER lumen
1. The Ca
2+
pump is accompanied by exceptionally large conformational changes
153 153
2. These changes couple ATP hydrolysis to changes in access & affinity of the ion binding sites
B. Plant cells have P-type transporter for H
+
- this cell membrane proton pump has key role in secondary
solute transport, control of cytosolic pH & as cell growth control by acidification of plant cell wall
C. Stomach epithelial lining H
+
/K
+
-ATPase - P-type pump secreting concentrated acid solution (up to 0.16
N HCl) into stomach chamber
1. At rest, found in cytoplasmic membranes of stomach lining parietal cells & are nonfunctional
2. With food, hormonal message transmitted to parietal cells that causes pump-containing membranes
to move to apical surface
3. They fuse with apical surface membrane & secrete acid (aids digestion but can lead to heartburn)
4. New acid-blocking medications (Zantac, Pepcid, Tagamet) do not inhibit H
+
/K
+
-ATPase directly; they
block parietal cell receptors & stop hormone activation of cells & thus halt H
+
/K
+
-ATPase
5. Another drug, Prilosec, prevents heartburn by inhibiting stomach's H
+
/K
+
-ATPase
D. V-type pumps use ATP energy without formation of phosphorylated protein intermediate
1. They actively pump H
+
ions across cytoplasmic organelle membranes & vacuoles (hence the name)
2. Found in membranes lining lysosomes, secretory granules & plant cell vacuoles, where they maintain
the low pH of the contents
3. Found in membranes of a variety of cells like kidney tubules - V-type pump helps maintain body's
acid-base balance (secretes protons into the forming urine)
4. V-type pumps are large multi-subunit complexes similar to that of ATP synthase
E. ATP-binding cassette (ABC) transporters diverse group of proteins that actively transport ions
1. All members of this superfamily share an homologous ATP binding domain; one is involved in
cystic fibrosis
V. Utilizing light energy for active ion transport - Halobacterium salinarium (or H. halobium), an
archaebacterium that lives in extremely salty environments (Great Salt Lake)
A. Its plasma membranes turn purple when conditions anaerobic, due to bacteriorhodopsin which contains
retinal (rhodopsin prosthetic group)
1. Rhodopsin is light-absorbing protein of vertebrate retina rods
B. Light energy absorption by retinal group induces series of conformational changes in protein, which
causes a proton to move from retinal group through a channel in protein to cell exterior
C. Pumped H
+
is replaced by one transferred to protein from cytoplasm; this generates a steep H
+

gradient across membrane
D. Gradient subsequently drives ATP synthesis by enzyme (similar to mitochondrial electron transport)
VI. Cotransport: Coupling active transport to existing ion gradients
A. Gradient establishment can be used to store free energy in cells; the potential energy stored in these
ionic gradients is then used in various ways to perform work, e. g. solute transport
B. Sodium ions involved in cotransport of glucose across intestinal epithelium & then into bloodstream after
high MW polysaccharides have been hydrolyzed into simple sugars within intestinal lumen
1. Movement of glucose across apical membrane of intestinal epithelial cells against a concentration
gradient occurs by cotransport with Na
+
ions
2. Primary transport of Na
+
out of cell done by Na
+
-K
+
ATPase in basal & lateral membranes; keeps
Na
+
concentration very low within cells (this is the primary active transport system)
3. Na
+
-K
+
ATPase pumps Na
+
ions out of cell against a concentration gradient
4. Na
+
ion tendency to diffuse back across apical membrane is tapped by epithelial cells to drive
glucose molecule cotransport into cell against their concentration gradient (2 active transport)
5. Na
+
moves glucose up its gradient as Na
+
flows down its gradient across apical membrane
C. Transport protein (Na
+
/glucose cotransporter) moves 2 Na
+
ions & 1 glucose molecule with each cycle
154 154
1. Once inside the cell, glucose diffuses through cell & moves across basal membrane by facilitated
diffusion
D. To appreciate the power of an ion gradient in accumulating other types of solutes in cells, one need only
look at the energetics of the Na
+
/glucose cotransporter
1. The Na
+
ion gradient is very strong; the cotransporter is capable of moving glucose into a cell against
a gradient greater than 20,000-fold
2. Free energy change for moving 1 mole of Na
+
ions into cell is equal to 3.1 kcal/mole & thus 6.2
kcal for 2 moles of Na
+
ions is available to move 1 mole of glucose up gradient into cell
3. Using equation for movement of nonelectrolyte like glucose across the membrane:
G = RT ln (C
i
/C
o
) = 2.303 RT log
10
(C
i
/C
o
)
4. Use equation to calculate how steep a concentration gradient of glucose (X) the cotransporter can
generate; at 25C, -6.2 kcal/mole = 1.4 kcal/mole x log
10
X > log
10
X = -4.43 > X =1/23,000
E. Plants use 2 active transport systems to move many nutrients (sucrose, amino acids, nitrate) into cell,
coupled to the downhill influx of H
+
ions instead of Na
+
ions
VII. Types of transport systems uniports vs. cotransport systems (symports, antiports)
A. Uniport system - transport one solute at a time
B. Cotransport systems - >1 solute moved across membrane at same time by a single transport molecule
1. 2 transported solutes move in same direction (symport)
2. If the transported solutes move in opposite directions (antiport)
a. Many cells maintain proper cytoplasmic pH by coupling downhill Na
+
ion movement into cell to
H
+
ion movement outward; proteins that act as antiports are usually called exchangers
Membrane Potentials and !erve "mulses
I. All organisms possess the ability to respond to external stimulation, a property referred to as irritability; it is a
function of the cell membrane; seen in nerve cells & amoebae, which can respond to stimuli
A. Poke amoeba with fine glass needle > it withdraws pseudopodia, rounds up, moves in new direction
B. Irritability in amoeba depends on same basic membrane properties that lead to formation & propagation
of nerve impulses
II. Nerve cells (neurons) are specialized for the collection, conduction & transmission of information, which is
coded in the form of fast-moving electrical impulses; the basic parts of a neuron are discussed below:
A. Neuron nucleus is located within expanded region called cell body, the metabolic center of cell & the site
where most of its material contents are manufactured
B. Extending from cell bodies of most neurons are a number of miniscule extensions (dendrites), which
receive incoming information from external sources, typically other neurons
C. Also emerging from cell body is a single, more prominent extension (an axon), which conducts outgoing
impulses away from cell body toward the target cell(s)
1. Impulses are generally initiated in axon hillock, the region where the cell body merges into axon
2. Some axons may be only a few micrometers in length; others extend for many meters in body of a
large vertebrate (giraffe, whale)
3. Most axons split near their ends into smaller processes, each ending in terminal knob, a specialized
site where impulses are transmitted from neuron to target cell
4. Many neurons in brain end in thousands of terminal knobs, allowing these brain cells to communicate
with thousands of potential targets
D. Most neurons in the vertebrate body are wrapped in lipid-rich myelin sheath
155 155
III. Voltage (electric) potential difference can exist between inside & outside of cell; a positive ion excess on
one side of membrane & a negative ion excess on the other
A. One can measure a voltage potential difference with fine glass electrodes (microelectrodes; one inserted
into cell cytoplasm, one placed in the extracellular fluid outside the cell)
1. The microelectrodes are then connected to a voltmeter, an instrument that measures a difference in
charge between two points
B. The potential difference of the squid giant axon was determined - difference is ~70 millivolts with the
cell interior being more negative with respect to the outside (-70 mV); not unique to neurons
1. Such potentials seen in all cell types (range from ~-15 to ~-100 mV); in nonexcitable cells (other than
neurons & muscle cells), it is called membrane potential
2. In nerve or muscle cells, it is called resting potential, since it can change dramatically
C. Voltage magnitude & direction across membrane is determined by ion concentration differences on
either side of membrane & their relative permeabilities
1. Na
+
/K
+
-ATPase sets up steep gradient of these ions: Na
+
ions high outside, K
+
ions high inside
2. Na
+
should leak in & K
+
should leak out through their respective ion channels because of the
directions of their gradients
3. But vast majority of open membrane ion channels in resting nerve cell are selective K
+
leak channels
that allow more K
+
to leak out than Na
+
leaking in; there is slight Na
+
& Cl
-
permeability
D. The nature of K
+
leak channels is still controversial
1. Recently, a new K
+
channel family (the KCNK family) that lacks S4 voltage sensor & seems not to
respond to changes in voltage has been found
2. K
+
leak channels appear to be members of this family (mammalian brain tissue studies)
3. These proteins are called twin-pore channels since each subunit contains 2 ion pores rather than the
single ion pore found in subunits of other K
+
channels
E. Since K
+
ions are the only charged species with significant permeability in the resting nerve cell, their
outflow through membrane leaves excess of negative charges on cytoplasmic side of membrane
1. While the K
+
gradient favors continued K
+
efflux, the electrical gradient (excess "-" charge inside
the cell) favors K
+
retention inside cell
2. If these 2 opposing forces are balanced, the system is at equilibrium & no more net K
+
movement
F. Using the Nernst equation, one can calculate membrane potential (V
m
) that would be measured at
equilibrium if the nerve cell membrane is only permeable to K
+
ions
1. Nernst equation derived by setting G = 0, which is the case when the ion movement is at
equilibrium; in this case, V
m
would be equal to potassium equilibrium potential (E
K
)
2. E
K
= 2.303 RT/zF x log
10
([K
+
o
]/[K
+
I
]); for a squid giant axon, the internal [K
+
I
] is ~350 mM while the
external [K
+
o
])

is ~10 mM
3. Thus, at 25C (298K) & z = +1 for the univalent K
+
ion, E
K
= 59Log
10
0.028 = -91 mV
4. A similar calculation of the Na
+
equilibrium potential (E
Na
) would produce a value of ~+55 mV
5. Since measurements of voltage across the resting nerve membrane are similar in sign & magnitude (-70
mV) to the potassium equilibrium potential (-91 mV) just calculated above
a. The movement of K
+
ions across the membrane is considered the most important factor in
determining resting potential
b. The difference between calculated K
+
equilibrium potential (-91 mV) & measured resting potential (-70
mV) is due to a slight membrane permeability to Na
+
through recently described Na
+
leak channel
6. Slight Na
+
& Cl
-
leakage accounts for difference between calculated & measured resting potentials
IV. Action Potential (AP) - our present knowledge of membrane potentials & nerve impulses rests on work on
giant squid axons in late 1940s & early 1950s by Alan Hodgkin, Andrew Huxley & Bernard Katz
156 156
A. Squid axon (~1 mm in dia) carries a high speed impulse, so squid can escape rapidly from predators
B. Stimulate squid (or other axon) - poke it with fine needle or jolt it with very small electric current
1. Some of its Na
+
channel gates open allowing a limited number of ions in; potential gets less negative
(drop in polarity between 2 sides of membrane); membrane less polarized (depolarization)
2. If stimulus is small & depolarization is slight (-70 mV to 60 mV) > potential returns rapidly to
resting potential when stimulus stops
C. Stronger stimulus depolarizes membrane more & if it gets to certain point (threshold; ~-50 mV), an AP
starts; causes voltage-gated Na
+
channels to open & Na
+
ions flow freely in
1. Na
+
ions move down both their concentration & electric gradients; membrane potential briefly
reverses itself becoming positive (~+40 mV); close to the Na
+
equilibrium potential
2. After ~1 msec, Na
+
channels spontaneously inactivate, blocking further Na
+
influx; results from
random diffusion of inactive peptide into channel pore opening (similar to that for K
+
channels)
3. The membrane potential change caused by Na
+
influx causes voltage-gated K
+
channels to open so K
+
ions diffuse freely out of cell & down their steep concentration gradient
4. The decreased Na
+
permeability

& increased K
+
permeability makes potential swing back to a
negative value again (~80 mV), approaching that of K
+
equilibrium potential
5. The large negative membrane potential causes voltage gated K
+
channels to close > returns cell to
resting potential
D. Collectively, the changes in membrane potential are called an action potential; the entire sequence of AP
changes takes ~5 msec in squid axon & less (<1msec) in myelinated mammalian nerve cell axon
E. Following an action potential, the membrane enters a brief refractory period following which the
membrane cannot be restimulated
1. Once negative resting potential is reestablished, the inactivated Na
+
ion channels close until they
are stimulated to open again by threshold depolarization
2. The refractory period occurs because the Na
+
channels that were inactivated during the initial
stage of the AP must close before they can be reopened in response to another stimulus
3. The transformation of the ion channel from the inactivated to the closed conformation can only
occur after the inactivating peptide has swung out of the opening of the pore
F. AP greatly changes membrane voltage, but only a minute percentage of ions on the two sides of the
membrane are involved in given AP
1. Dramatic changes not caused by changes in Na
+
& K
+
ion concentrations on two sides of
membrane, which are insignificant
2. Instead, they are caused by the movements of charge in one direction or the other that result from
the fleeting changes in permeability to these ions
3. Those ions that do cross membrane during AP are pumped back by Na
+
/K
+
ATPase
4. Even if Na
+
/K
+
ATPase is inactivated, a neuron can continue to fire thousands of impulses before the
ionic gradients established by the pump are dissipated
V. Further characteristics, vital statistics & information about APs
A. Certain local anesthetics (procaine & novocaine) act by closing ion channels in sensory cells & neurons
& prevent APs; as long as these channels stay closed > no APs in these cells
1. Affected cells are unable to inform the brain of events occurring at skin or teeth > no pain
B. All-or-None Law - if cell depolarizes to threshold, there is an automatic, full-blown response with no
further stimulation, there is no in-between response
1. A subthreshold depolarization is incapable of triggering AP
2. Stronger stimuli cannot produce "bigger" impulses than weaker stimuli
C. AP proceeds without more energy expenditure (energy is just needed to establish gradients) an AP
simply results from the flow of ions down their respective electrochemical gradients
157 157
VI. Propagation of APs as an impulse
A. Once AP is initiated, the impulse is propagated down the length of the cell axon (nerve impulse)
1. APs are normally initiated at axon hillock of neuron & move from there to opposite ends of cell (the
nerve terminals)
2. Stimulate axon in center as in experiment above, AP moves from stimulus site in both directions
B. AP at one site is large enough to cause depolarization above threshold at adjacent site causing sodium
channels to open, etc.
1. Large depolarization of AP draws positive ions toward that site on membrane outer surface
2. It also forces the positive ions away from the depolarization site on the inner membrane surface
3. This current flow causes the membrane in the region just ahead of AP to depolarize
4. Since the depolarization accompanying AP is very large, the adjacent membrane region is depolarized
to a level greater than threshold value
5. The Na
+
channel gates swing open in this adjacent region generating another AP
C. Thus, once triggered, a succession of APs travels down the entire length of the neuron to target cell
with no loss of intensity (strength); arrives at its target cell with the same strength as at its initiation
D. Differences in strengths of stimuli can be detected how if all impulses exhibit the same strength?
depends on several factors:
1. A stronger stimulus (scalding H
2
O) activates more nerve cells than weaker stimulus (warm H
2
O)
2. Stronger stimulus also activates high-threshold neurons that would not fire with weaker stimulus
3. Stimulus strength is also encoded in pattern & frequency by which APs are launched down
particular neuron (generally a stronger stimulus generates a larger number of impulses)
VII. Speed impulse can travel depends on:
A. Diameter of axon - if larger, there is less resistance to local current flow & AP is easier to propagate (an
AP at one site can activate adjacent membrane regions more rapidly)
1. Some invertebrates (squid, tube worms) evolved giant axons to escape danger; this approach has
limits, since conduction speed increases (slowly) as the square root of the increase in diameter
2. A 16X increase in diameter gives only a 4X rise in speed of conduction; an axon 480 m in diameter
can conduct AP only 4 times faster than one that is 30 m in diameter
B. During vertebrate evolution, an increase in conduction velocity was achieved when the axon became
wrapped in a myelin sheath
1. Myelin sheath is formed from nervous system accessory cells (oligodendrocytes in central NS,
Schwann cells in peripheral NS)
2. Since it is composed of many layers of lipid-containing membranes, the myelin sheath is ideally
suited to prevent the passage of ions across the cell membrane
3. Nearly all of the ion channels of a myelinated neuron reside in the unwrapped gaps (nodes of
Ranvier) between adjacent Schwann cells that make up the sheath
4. Thus, nodes of Ranvier are the only sites where APs can be generated; AP at one node triggers AP at
next node; impulse jumps from node to node without having to activate intervening membrane
5. Impulse propagation by this mechanism called saltatory conduction; conduction speed along
myelinated axon is nearly 120 meters/sec (>20X faster than unmyelinated axon of same diameter)
C. Importance of myelination is dramatically illustrated by multiple sclerosis, a disease associated with
deterioration of myelin sheath that surrounds axons in various parts of nervous system
1. Manifestations of disease usually begin in young adulthood
2. Victims experience weakness in their hands, difficulty in walking & problems with their vision
3. MS is characterized by progressive muscular dysfunction
158 158
VIII. Neurotransmission: Jumping the synaptic cleft
A. Neurons are linked with their target cells at specialized junctions (synapses) - no direct contact; they
are separated from each other by narrow gap of ~20 50 nm (gap called the synaptic cleft)
1. Presynaptic cell (PrSC; neuron, sensory cell) conducts impulses toward a synapse, which it must
cross to get to postsynaptic cell (PoSC; neuron, muscle, gland cell)
2. PoSC always lies on receiving side of synapse
3. Between axon & muscle cell, synapse is called neuromuscular junction or motor end plate
B. Studies done decades ago indicated that a chemical substance is involved in the transmission of an
impulse from one cell to another
1. Terminal knobs (tips) of axon branches have been seen in electron microscope to contain large
numbers of synaptic vesicles that serve as storage sites for chemical transmitters that act on PoSCs
2. They contain neurotransmitters (NTs), like two of the best-studied NTs, acetylcholine &
norepinephrine, which transmit impulses to skeletal & cardiac muscles
C. Sequence of initial events in neurotransmission (synaptic transmission)
1. Impulse reaches terminal knob & the accompanying depolarization induces the opening of a
number of voltage-gated Ca
2+
channels in this part of the PrSC plasma membrane
2. Ca
2+
ions diffuse from extracellular space into PrSC (Ca
2+
ions are usually present at a very low
concentration within neurons as

in all cells [~100 nM]); [Ca
2+
] rises >1000-fold near channels)
3. When gates open, Ca
2+
ions diffuse from extracellular fluid into terminal knob of neuron, causing
[Ca
2+
] to rise >1000-fold within localized microdomains near the channels
4. The elevated [Ca
2+
] triggers rapid synaptic vesicle fusion (one or a few) with PrSC membrane >
neurotransmitters released into synaptic cleft
5. After release from synaptic vesicles, NTs diffuse across narrow gap of cleft & bind selectively to
receptor molecules concentrated directly across synaptic cleft on PoSC membrane
D. Such NT molecules can have one of two opposite effects depending on the type of receptor on the target
cell membrane to which it binds:
1. Triggers cation-selective channel opening in PoSC membrane; leads to mainly Na
+
ion influx & less
"-" (more "+") potential; depolarization excites cell; more likely to respond with its own AP
2. Anion-selective channels in PoSC membrane open > mainly Cl
-
ion influx & more "-" potential
(hyperpolarized), inhibits cell; AP generation less likely (need >Na
+
influx to reach threshold)
E. There is also an important class of NT receptors that are not ion channels & thus do not directly affect
membrane voltage
1. This other group of receptors are members of a class of proteins called GPCRs
2. When a NT binds to one of these receptors, it can initiate a variety of responses, which often includes
the opening of ion channels by an indirect mechanism
F. Most nerve cells in brain receive both excitatory & inhibitory signals from many different PrSCs >
summation of these opposing influences determines if PoSC generates AP or not
1. All terminal knobs of a given neuron release the same NT(s)
2. However, a given NT may excite one particular postsynaptic membrane & inhibit another
159
H C C O CH CH N(CH )
2
Acetylcholine (ACh)
2 3 3 3
+
O
HO
HO C CH NH
2 3
+
OH
H
Norepinephrine
159
3. Example: acetylcholine inhibits heart contractility, but stimulates skeletal muscle contractility
G. In brain, glutamate is primary excitatory NT & gamma-aminobutyric acid (GABA) is primary inhibitory
NT
1. A number of general anesthetics, as well as valium & its derivatives, act by binding to GABA
receptor & enhancing the activity of the brain's primary "off" switch
IX. Actions of drugs on synapses - NTs must have short half-life (a few msec) after release from PrSC to
prevent extended effect, PoSC would not recover from this > thus impulse lasts only a few msec
A. NT is thus eliminated from synapse in 2 ways:
1. Enzymes destroy NT in synaptic cleft or
2. Proteins transport NTs back to the presynaptic terminals from which they were released (reuptake)
B. Interference with NT destruction or reuptake can have dramatic physiological & behavioral effects
C. Example: acetylcholinesterase (AChase) - located in synaptic cleft where it hydrolyzes acetylcholine
(ACh); inhibit AChase by nerve gas DFP exposure > muscles contract violently due to high [ACh]
1. Many drugs act by inhibiting the transporters that sweep NTs out of synaptic cleft
2. A number of widely prescribed antidepressants, including Prozac, inhibit the reuptake of serotonin, a
NT implicated in mood disorders
D. Example: dopamine (DOP) released by certain nerve cells in limbic system portion of brain, which
contains the brain's pleasure or reward centers
1. Sustained DOP presence in limbic system synaptic clefts produces short-lived euphoric feeling, as
well as a strong desire to repeat the activity
2. Cocaine interferes with dopamine reuptake in brain limbic system causing "high", then "crash"
3. With repeated use, the pleasurable effects of cocaine are increasingly reduced, but its addictive
properties are enhanced
E. Example: amphetamines also act on DOP-releasing neurons; they are thought to stimulate excessive DOP
release from presynaptic terminals & interfere with the reuptake of the NTs from synaptic cleft
1. Mice genetically engineered to lack the DOP transporter (DAT; protein responsible for DOP
reuptake) show same behavior as normal mice given doses of cocaine or amphetamines
2. Cocaine or amphetamine doses have no additional behavioral effects on mice lacking DAT gene
F. The active compound in marijuana (
9
-tetrahydrocannabinol) acts by a totally different mechanism
1. It binds to cannabinoid (CB1) receptors located on presynaptic terminals of certain brain neurons
2. The binding reduces the likelihood that that these neurons will release NTs
3. CB1 receptors normally interact with compounds called endocannabinoids, which are produced by
postsynaptic neurons after depolarization
4. These substances diffuse backwards across the synaptic cleft to the presynaptic membrane where they
bind to CB1 receptors, suppressing synaptic transmission
5. CB1 receptors located in many brain areas, including hippocampus, hypothalamus & cerebellum;
explains effects of marijuana on memory, motor coordination & appetite, respectively
6. If marijuana increases appetite by binding to CB1 receptors, it follows that blocking these receptors
might decrease appetite
7. This line of reasoning has led to development of a new CB1-blocking weight-loss drug (Acomplia)
that is likely to be available in the near future
G. Significant for Parkinsons disease studies (results from low DOP levels in certain parts of brain)
1. Drop in DOP levels results in motor dysfunction
2. If we develop drugs that block the DOP transporter, limited DOP released by neurons of Parkinson's
patients could be more effective in producing a proper response in PoSCs
160 160
X. Synaptic plasticity - synapses are more than just connecting sites between adjacent neurons; they are key
determinants in the routing of impulses through nervous system and their study is important
A. The human brain is thought to contain at least 100 trillion synapses; these synapses act like gates
stationed along the various pathways
1. They allow some pieces of information to pass from neuron to neuron or
2. They hold back other pieces of information or reroute them in another direction
B. Dynamic changes in synapse structure & activity (synaptic plasticity), a remarkable property, play a role
in learning & memory, even though synapses are often perceived as fixed, unchanging structures
1. Synaptic plasticity is particularly important during infancy & childhood when the neuronal
circuitry of the brain achieves its mature configuration
C. Synaptic plasticity is most readily seen in studies of neurons from the hippocampus (a part of the
brain vitally important in learning & short-term memory)
1. Repeated hippocampal neuron stimulation over short time period > synapses connecting them to
neighbors are strengthened by long-term potentiation (LTP); may last days, weeks, even longer
D. LTP research has focused on NMDA receptor, one of several receptor types that bind the excitatory
NT glutamate
1. Glutamate binding to a postsynaptic NMDA receptor, opens an internal cation channel within the
receptor that allows Ca
2+
ion influx into the postsynaptic neuron
2. The Ca
2+
influx triggers a cascade of biochemical changes that lead to synaptic strengthening
3. Synapses that have undergone LTP transmit weaker stimuli & evoke stronger responses in PoSCs
4. These changes are thought to play a major role as newly learned information or memories are
encoded in brain neural circuits
5. When lab animals are treated with drugs that inhibit LTP, like those that interfere with the activity of
the NMDA receptor, their ability to learn new information is greatly reduced
E. Synaptic malfunction or dysfunction may cause disease (myasthenia gravis, Parkinson's disease,
schizophrenia, & even depression)
EXPERIMENTAL PATHWAYS: THE ACETYLCHOLINE RECEPTOR
I. Claude Bernard (foremost physiologist of 19
th
century; once a pharmacist & aspiring playwright)
interested in elucidating the mechanism by which nerves stimulate skeletal muscle contraction
A. Used curare (highly toxic drug isolated from tropical plants & used by native S. American hunters to
make poisonous darts)
1. Found that curare paralyzed skeletal muscle without interfering with either the ability of nerves to
carry impulses to the muscle or the ability of the muscle to contract on direct stimulation
2. He concluded that curare somehow acted on the region of contact between the nerve & muscle
B. John Langley (physiologist, Cambridge Univ.) confirmed & extended Bernard's conclusion
1. Studied the ability of nicotine (also derived from plants) to stimulate contraction of isolated frog
skeletal muscles & the effect of curare in inhibiting nicotine action
2. 1906 Langley concluded that nerve impulse does not pass from nerve to muscle by electric
discharge but instead by secretion of a special substance on the end of the nerve
3. Proposed that the chemical transmitter was binding to a receptive substance on muscle cell surface at
the same site that bound nicotine & curare farsighted conclusions
C. Otto Loewi (Austrian-born physiologist, 1921) confirmed Langley's suggestion that nerve-muscle
stimulus was transmitted by a chemical with ingenious experiment that came to him in a dream
161 161
1. Vertebrate heart rate is regulated by input from 2 opposing (antagonistic) nerves; Loewi isolated a
frog heart with both nerves intact
2. Stimulated inhibitory (vagus) nerve > chemical released from heart prep into salt solution, which
drained into medium bathing second isolated heart > second heart's rate slowed dramatically
3. Second heart acted like its vagus (inhibitory) nerve was activated; called substance that inhibited
frog's heart "Vagusstoff"; soon showed its chemical/physiological properties matched acetylcholine
4. Concluded acetylcholine (ACh) was substance released by tips of nerve cells in vagus nerve
D. David Nachmansohn (neurophysiologist at the Sorbonne; 1937) visited Paris World's Fair & observed
several living electric fish of species Torpedo marmarota
1. These rays have electric organs that deliver strong shocks (40 60 volts) that can kill potential prey
2. Studied acetylcholinesterase (destroys ACh after its release from motor nerve tips) & realized electric
organs were derived from modified skeletal muscle tissue & asked for fish to study
3. He found that the electric organ was an extraordinarily rich source of acetylcholinesterase & also a
very rich source of nicotinic acetylcholine receptor (nAChR)
4. nAChR is present on postsynaptic membranes of skeletal muscle cells & binds ACh released from
motor nerve tips; nAChR is called nicotinic since it is activated by nicotine as well as by ACh
5. This contrasts with muscarinic ACh receptors of parasympathetic nerve synapses (activated by
muscarine, but not nicotine; inhibited by atropine, but not curare)
6. Smokers' bodies get accustomed to high nicotine levels & experience withdrawal symptoms when
they stop smoking, since postsynaptic neurons possessing nAChRs are not stimulated at usual level
7. The drug Chantix, which is marketed as an aid to stop smoking, acts by binding to a specific type of
brain nAChR (one with a42 subunits)
a. Once bound, the Chantix molecule partially stimulates the receptor while preventing binding of
nicotine
II. nAChR is integral membrane protein & wasn't isolated until 1970s, since isolation techniques were not
perfected until then
A. The ideal nAChR assay was a compound that bound selectively & tightly to it (found in 1963 by Chen-
Yuan Lee et al. at National Taiwan Univ.), -bungarotoxin (found in venom of a Taiwanese snake)
1. -bungarotoxin causes paralysis by binding tightly to nAChR on skeletal muscle cell postsynaptic
membrane, blocking response of muscle to ACh
2. Using labeled -bungarotoxin to assay for nAChR, electric organs as a source & a detergent to
solubilize membrane proteins, many researchers isolated acetylcholine receptors in the 1970s
B. In one study, membranes containing nAChR were isolated by homogenizing the electric organs in a
blender & centrifuging the suspension to pellet membrane fragments
1. Triton X-100, a nonionic detergent, gave the best results
2. It has a long hydrophobic portion that can replace the phospholipids that normally surround the
hydrophobic portions of membrane proteins & a hydrophilic end that is soluble in water
3. Unlike most detergents, which bear a negatively charged carboxyl group, Triton X-100 lacks a charge
(nonionic) & is thus less disruptive of protein structure
4. Proteins were extracted from membrane fragments with Triton X-100 & mixture was passed through
column containing tiny beads coated with synthetic compound, whose end looks like ACh
5. 2 proteins with binding sites for ACh, nAChR & acetylcholinesterase (AChE) stuck to the beads
6. The remaining 90% of protein in the extract failed to bind to the beads & simply passed through
7. Next, a solution of 10
-3
M flaxedil was passed through the column; it selectively removed nAChR
from beads leaving AChE behind
8. nAChR measured by -bungarotoxin was purified >150-fold in one step (affinity chromatography)
162 162
C. The structure of the nAChR receptor Arthur Karlin (Columbia Univ.) determined that nAChR is a
pentamer (a protein consisting of 5 subunits)
1. Each receptor contained 2 copies of a subunit called & one copy each of 3 other subunits
2. They could be distinguished by extracting membrane proteins in Triton, purifying nAChR by affinity
chromatography & then doing SDS-PAGE separating the proteins according to size
III. Purified receptor was found to act as both a site for ACh binding & a channel for the passage of cations
A. Jean-Pierre Changeux (Pasteur Inst., Paris) postulated years earlier that binding of ACh to receptor
caused conformational change that opened ion channel within protein
1. Na
+
ion influx through the channel could then lead to depolarization of membrane & activation of
muscle cell
2. During last half of 1970s, Changeux et al. incorporated purified nAChR molecules into artificial
lipid vesicles
3. Using vesicles containing various concentrations of labeled Na
+
& K
+
ions, they showed that
binding of ACh to receptors in lipid bilayer initiated a flux of cations through membrane
B. The pure ACh protein contains all the structural elements needed for chemical transmission of an
electrical signal an ACh binding site, an ion channel & a mechanism for coupling their activity
IV. During past decade, researchers have focused on determining nAChR structure & mechanism by which it
becomes concentrated within one small part of muscle membrane, the part adjacent to motor neuron tip
A. Scientists used purified genes, amino acid sequence determination & site-directed mutagenesis to identify
specific parts of polypeptides that span membrane, bind neurotransmitter or form ion channel
B. Electron microscopy was employed in one approach nAChR first seen in EM of electric organ
membranes
1. Receptors appeared ring-shaped with diameter of 8 nm & central channel of 2 nm diameter &
protruded out from lipid bilayer into external space
C. Nigel Unwin et al. (Med. Research Coun., England) used mathematical analysis of frozen electric organ
membrane electron micrographs; able to analyze nAChR structure as it is in its native lipid environment
1. He described the arrangement of the 5 subunits around central channel
2. Ion channel consists of narrow pore lined by a wall made of 5 inner (M2) -helices, one from each of
the surrounding subunits
3. The gate to the pore lies near the middle the membrane, where each of the M2 -helices bends inward
to form a kink in the unactivated receptor
4. The side chain of a leucine residue projects inward from each kink; the leucine residues from the 5
helices form a tight hydrophobic ring that prevents the ions from crossing the membrane
5. The gate opens after binding of 2 ACh molecules, one per subunit, each one binding to a site located
within a pocket of an subunit
D. Unwin then studied the changes in the nAChR during channel opening
1. nAChR-rich membrane preparations were applied to a support grid that was allowed to fall into a bath
liquid nitrogen cooled ethane, which freezes the membranes
2. ~5 msec before they reached the freezing bath surface, the grids were sprayed with a solution of ACh,
which bound to the receptors & triggered conformational change that was required to open channel
3. Compared electron micrographs of nAChRs trapped in open vs. closed state; found that ACh binding
triggers small shape change in receptor subunit extracellular domains near the 2 ACh binding sites
163 163
4. This conformational change is propagated down the protein, causing a small (15) rotation of the M2
helices that line the ion conduction pore
5. The rotation of these inner helices breaks apart the hydrophobic gate, which allows the Na
+
ions to
enter the cell
6. Other models for channel opening have been proposed by other labs
a. A more definitive statement about the mechanism of gating will probably require a high-resolution
X-ray crystal structure of the protein in both the open & closed states
164 164