Module 9 - Membranes:

Membrane Functions:

1) Separate cells from the external medium to create an unique intracellular environment
a) Have to create barriers
2) Allow selective transport of substrates in and out of the cell
a) Needs to uptake nutrients, get rid of wastes, and be aware of what is going
around it
3) Provide a location for specialized pathways and processes (e.g. energy conversion of
mitochondria)
4) Rapid changes in electrical potential across the membranes of neurons as basis of the
nervous system
5) Localization of receptors to facilitate response to physiological signals
6) Mediate cell-cell recognition and interaction

Membrane Characteristics:

1) Membranes are sheet like structures, two molecules thick, that form closed boundaries
between compartments
2) Membranes consist of mainly lipids and proteins, w/ carbohydrates linked to these
molecules
3) Membranes are built from amphipathic molecules
4) Membranes are largely impermeable to polar molecules
5) Specific membrane proteins mediate particular biological functions
6) Membranes are self assembling, non covalent structures
7) Membranes are fluid and dynamic structures
8) Membranes are highly specialized in their composition and distribution (asymmetric)

Lipid Bilayer is the Basic Structural Element of Membranes:

- Membrane formation is a consequence of the amphipathic nature of the membrane
lipids
- These molecules self assemble through the hydrophobic effect
- The structure formed depends on the ratio of cross-sectional areas of the polar head
group and the hydrophobic tail
- Fatty acids favor formation of micelles
- Lipids w/ two hydrocarbon tails (glycerophospholipids and sphingolipids)
tend to form bilayers
- The desire to have hydrophobic structures away from water and
hydrophilic heads towards it, drive this formation
Lipid Bilayers Form Membrane Vesicles:
- Exposure of hydrophobic tails at the edge of a bilayer to water is energetically
unfavourable
- Flat bilayer sheets are unstable and spontaneously form membrane vesicles w/ an
internal volume
- These vesicles are the basis of cells and organelles

Membranes are Impermeable to Polar Molecules:

- Lipid bilayer membranes have a very low permeability to ions and most polar
molecules
- Polar molecules like to associate w/ water (which lipids do not)
- For a polar molecule to pass through the membrane structure, it would have to
shed off its interactions w/ water molecules, and go into a very hydrophobic
water-less environment
- Need to be transported by something
- Permeability of a small molecule is correlated w/ their relative solubility in water
- Some small non-polar gases (like O2 and CO2) and small hydrophobic molecules
(like the fat soluble hormones) can pass directly through the membrane

Vesicles for Drug Delivery:

- The cell membrane can represent a critical barrier for polar drugs intended for
intracellular targets
- It needs to be taken into the cell by a transport system that already exists
- Encapsulation of a drug within a liposome can facilitate transport across the
membrane
- You can take your drug molecule w/ a lot of phospholipids present.
Sonification (putting sound waves through it to shake it) → lipids will
spontaneously form liposomes
- Within the internal volume of the liposomes will be the drug molecule
- As the liposome gets to the membrane, it fuses together and releases the drug
inside the cell
- Liposomes can also be used to target specific organelles within a cell
- You can do this if you incorporate proteins within the liposome w/ the drug
- You can also have a liposome within a liposome to get through multiple
membrane layers

Membrane Composition:
- Membrane are primarily composed of lipids and proteins
- More active membranes have higher ratio of protein to lipid
- Myelin sheath has little protein bc its inactive
- Active liver membranes are about 50% protein
- Composition of membrane components can be dynamic, in particular for prokaryotes
Fluid Mosaic Model of Membrane Structure:
- Membranes are dynamic structures due to the nature of the non covalent interactions
- Lipids and proteins freely diffuse in the plane of the membrane
- Lipid molecules have freedom to diffuse around in the plane of the membrane
(freedom of lateral diffusion)
- Proteins are not covalently bound, so they also have the opportunity to move
around
- Lateral movement of proteins and lipids within the membrane is very rapid
- Movement across the membrane is restricted

- Fluid mosaic model describes the freedom of lateral diffusion of lipids and proteins in
the face of the membrane
- The surface of the membrane looks still (calm), but when you look at the
water molecules, lipids, and proteins, they are moving around

Transbilayer Movement Requires Catalysis:

- Transbilayer movement requires a polar head group to pass through hydrophobic
environment
- There is a lot of energy barriers associated w/ a hydrophobic environment
- Uncatalyzed rate of lipid molecule crossing from one sheet to the other (flip flop
diffusion) is very slow
- Flip flow diffusion = when you have a membrane protein or lipid that goes
over to the other face of the membrane (moving from bottom layer to top
layer, or vice versa)
- Translocation of lipids from one side of bilayer to other is catalyze by enzymes called
flippases
- Flippases provide an environment that make it more likely for the polar head
group to be able to get through the hydrophobic core
- Provide a hydrophilic or polar environment for the polar heads to jump
over to the other side
- Lipid composition of the inner and outer sheets of the bilayer can be different,
allowing for specialization of the membrane faces
- Membranes are asymmetric, what is presented to each face is different. We
need a mechanism to get the asymmetry in place
- Once you position the lipid/proteins to the side you want them to be on, they
will stay there bc they can spontaneously come back

Lateral diffusion is simple and fast to do, but transverse diffusion is difficult to do (moving
from one side to the other)
Membrane Fluidity:
- Cells need to maintain an appropriate level of membrane fluidity
- Need things to have freedom of motion, but don’t want to compromise the
integrity of membrane
- Membranes undergo temperature dependent phase transitions
- Below the phase transition temp, membrane is too solid - to rigid
- Above the phase transition temp, membrane is too fluid
- At the phase transition temp, the hydrocarbon chains are partially ordered
but lateral diffusion is still possible (just right)
- Semi solid state that allows sufficient movement of the membrane
components
- Transition state between being a solid and a liquid
- Cells can adjust membrane composition to maintain liquid ordered state
- May not be able to control the temperature, but can control the molecules
making up the membranes and influence what the phase transition temperature
will be
- Bacteria can vary the length and saturation of the hydrocarbon tails of
membrane lipids
- Can change the length or degree of saturation of the hydrocarbon tails
(longer tails make it more likely to be a solid)
- Animals use cholesterol to mediate membrane fluidity

Temperature Dependent Changes to Membrane Composition:

- Growing e. Coli at different temperatures affect the hydrocarbon chains of the bacteria
- As the temperature increases, E. coli will produce more long chain saturated fats
(danger of it being too liquid, so bacteria makes its tails more solid
- As the temperature decreases, E. coli makes more unsaturated fats (danger of being
too solid, so makes tails more fluid)
- Will adjust the types of fatty acids present on the membrane lipids to suit the current
conditions

Specialization of Membrane Structure and Function:

- While the basic features of the bilayer are simple and consistent, there are
mechanisms to enable specialization:
- Composition of membrane components -
- can vary the amount of lipids/proteins present, and change the specific
identities of the molecules in order to perform different biological
functions
- Distribution of membrane components
- Static and dynamic - uneven distribution of these membrane
components, and have a static and dynamic component to it
- Can change where things are positioned in the membrane in response
to situations
 - Specialized membrane regions - Lipid rafts and fencing interactions
Specialized Compositions and distribution of Lipids in Membranes:
- The lipid composition of membranes varies across species and cell types (highly
specialized compositions and functions)
- There is specialization within the organelle, but also within each face of the
membrane
- This includes dynamic changes to composition and/or positioning to regulate
biological events
- There is static and dynamic component
- Static - you will establish an uneven distribution of membrane lipids
(specialization in terms of composition/position within the membrane)
- Dynamic - the movement of a molecule from one face of a membrane
to the other can signal important biological events
- For example, the movement of phosphatidylserine to outer leaf function in
initiating cell destruction (apoptosis)
- Normally, phosphatidylserine points to the inside of the cell
- If it gets in trouble, it can signal its own death (apoptosis)
- Phophatidylserine moves from the inside of the cell to the outside, to
indicate that it will die soon

Specialized Membrane Regions (Lipid Rafts):

- Lipid rafts arise from the spontaneous association of lipid molecules whose
hydrocarbon tails are of similar length
- Fluid mosaic model wants to gravitate to associate w/ other molecules w/
hydrocarbon tails of the same length
- They want to optimize packing interactions by the length of the tail
- Spontaneous grouping together of sphingolipids w/ longer tails - where they
come together, there will be bulges in the membrane (thicker portion) - this is
a lipid raft
- Sphingolipids (w/ longer tails) form clusters that exclude glycerophospholipids
- Lipid rafts = membrane components w/ longer hydrocarbon tails naturally
gravitating together
- These are non covalent structures and associate by non covalent forces
- The longer, saturated hydrocarbons of sphingolipids form stable associations making
the rafts thicker and more ordered than the rest of the membrane
- Rafts are docking points in lipid anchored proteins that contain long chain saturated
fatty acid anchors
- Lipid anchors (GPI anchors) are tails that want to bury themselves in
hydrophobic environments
- It will bury itself in the membrane, and in doing so, will tether a protein into
the membrane
- Bc these tails have hydrocarbon associated w/ it, they tend to move towards
lipid rafts
- The lipid linked proteins that associate w/ rafts often serve signalling functions
 - Tend to be involved in signal transduction - how the cell is communicating w/
the extracellular environment
- Prion proteins are GPI anchored proteins
- Brain can handle plaques and tangles of prion, they can’t handle the
signalling of prions

Fencing interactions -
- Some molecules are localized to one regions and cannot go beyond it

Membrane Proteins:
- Active roles of membranes often performed by proteins
- Receptors and transporters
- Three categories of membrane proteins are defined based on different mechanisms of
association w/ the membrane
- Peripheral (c and d) - associate w/ either the inner or outer face of the
membrane, and can associate w/ lipid molecules or protein molecules
- Lipid anchored (e) - have a hydrophobic tail associated w/ it, and the
hydrophobic tail wants to bury itself in the hydrophobic core and anchor a
protein (into a lipid raft)
- Integral membrane proteins (a and b) - membrane spanning proteins (have
an extracellular and intracellular component)
- Such as signalling cell - needs to receive the signal from the outside of
the cell, and then communicate it to the inside of the cell
- Transporters - need to bind a molecule on the inside/outside, and get it
to the other side

Peripheral Membrane Proteins:

- Associate w/ membrane through electrostatic or hydrogen bonding interactions
- Their interactions w/ the membrane (lipid or protein) are non covalent
- Usually electrostatic or hydrogen bond interactions - which are very weak and
easy to disrupt
- Lipid linked are more difficult to disrupt, bc they are embedded within the
hydrophobic core, and the last thing the tail wants to do is be exposed to an
aqueous environment
- The way these are removed are by cutting (by enzymes), where it cuts off the
group and leaves the lipid anchor embedded in the membrane
- Can dock to membrane lipids or integral membrane proteins
- Integral membrane proteins are even more difficult to remove
- The bulk of the peripheral membrane proteins is in the cytosol or extracellular space
- Changes in pH or ionic strength often releases these proteins from the membrane
Lipid Anchored Membrane Proteins:
- Lipid anchored molecules also called greasy fingers
- Covalently attached lipids can anchor proteins to the membrane
- When you attach a hydrocarbon tail onto protein, it will want to bury into the
hydrophobic core of a membrane
- You can then take another protein and add a lipid anchor and it will associate
w/ the other protein in the context of the membrane
- When it is associated w/ the membrane it represents its inactive form (storage
area) - these are post translational modifications
- These protein modifications are sometimes reversibly, allowing for regulation of
cellular location
- There are different types of fatty acids that can be linked to proteins
- Proteins correspond w/ 3 different types of fatty acids
- These fatty acids get attached to different points on the protein (such on
cysteine or serine residues, or on the N terminus, etc)
- This implies higher level organization and regulation
- GPI anchored proteins always at outer face
- GPI anchored are associated w/ extracellular proteins, and they hold the
protein to the outside of the cell, specifically to lipid rafts
- Proteins w/ single chain hydrocarbons always to inner face

Integral Membrane Proteins:

- Integral membrane proteins are immersed in, and usually span, the membrane
- Protein positioning within a membrane is specific and directional
- Integral membrane proteins tend to be of three varieties; single pass alpha-helical,
alpha helical bundles, and beta barrels
- Single pass alpha helix = polypeptide chain passes through the membrane
once
- Alpha helical bundles = have multiple passes (usually 7) → 7 alpha helicies
coming together
- Beta barrels = layering of beta sheets to form beta barrels

Distribution of Amino Acids in Integral Membrane Proteins:

- In integral membrane proteins, charged residues are located mostly within the intra
and extracellular portions of the protein
- In the transmembrane region (hydrophobic) there tend to be non polar
residues (no charge, no hydrogen bonding capability)
- Outside and inside the cell, there tends to be the polar groups
- Tryptophan and tyrosine w/ ring structures tend to form rings at the interface
of the membrane (where hydrophobic becomes hydrophilic)
- Residues w/ non polar side chains (white) dominate inside the hydrophobic slab of the
bilayer
- Tryptophan and tyrosine cluster at the interface between the hydrocarbon chain and
polar head group region
Predicting Membrane Spanning Regions of Integral Membrane Proteins:
- Membranes spanning regions can be predicted from the amino acid sequence (primary
structure)
- Stretches of about 24 hydrophobic residues in a row are likely to be membrane
spanning
- A hydropathy index looks at the hydrophobic characteristics of a protein to predict
transmembrane regions
- Hydropathy plots anticipate transmembrane regions
- Stretch of about 24 hydrophobic residues, and then banded by tyrosine or
tryptophan, is likely a transmembrane protein

Hydrogen Bonding within Membrane Spanning Regions:

- Side chains within the transmembrane region tend to be non polar, however the
carbonyl and amide groups of each peptide bond are polar
- Hydrogen bonds are non covalent interactions, and to some extent, the alpha
helices of the proteins are coming apart and rejoining
- For regular proteins, any temporary breaks in hydrogen bonding patterns can
easily satisfied and repaired by water molecules
- In a membrane there are no water molecules - so it is important to optimize
hydrogen bonds within the main chain bc there are no other options
- Alpha helix in a transmembrane region is much more permanent, and is
locked into the hydrogen bonding patterns (won’t have the same type of
deviations)
- Polar unpaired carbonyl and amide groups in the bilayer core are energetically
unfavourable
- Carbonyl and amide groups of the protein backbone within the bilayer have a higher
requirement for hydrogen bonding

Transport across Membranes:

Categories of Membrane Transport:
1) Simple diffusion - some molecules can pass through a membrane down a
concentration gradient
2) Facilitated diffusion - proteins enable movement of molecules down a concentration
gradient
a) Carriers
b) Channels
3) Active transport - movement of molecules against their concentration gradient using
energy
a) Primary - energy is ATP
b) secondary - energy is ion gradients
4) Ion transporters - propagation of nerve transmission
 Carrier Saturable Gradient Energy

Simple Diffusion No no down no

Facilitated Diffusion

Channels Yes No Down No

Carriers Yes Yes Down No

Active transport

Primary Yes Yes Up Yes (direct/ATP)

Secondary Yes Yes Up Indirect (Ion gradient)

Simple Diffusion:
- Non polar gases (O2 and CO2) and hydrophobic molecules can cross directly through
the membrane
- Limited to a small number of molecules // CO2 and O2 are soluble enough
that they can diffuse through
- Cholesterol and hormones derived from cholesterol can pass through
- Their direction and rate of movement determined by the relative concentrations on
either side of the membrane
- Diffusion = region of higher concentration to lower concentration
- Relative number of molecules moving in each direction depends on the
relative concentrations of each
- Diffusion can only result in the net movement down a concentration gradient

Facilitated Diffusion:
- Membrane transporters lower the activation energy barrier of crossing the bilayer
- Proteins can enable molecules that can’t get through the membrane, get
through it (doesn’t have to directly interact w/ the hydrophobic environment)
- Proteins are highly specific to the molecule they’re transporting
- Activation energy for removing the hydration shell from a polar solute and
transferring it into the non polar environment in the core of the bilayer is very high
- Membrane transporters lower the activation energy for crossing the membrane by
replacing the hydration shell w/ interactions w/ polar groups along the transfer path in
the protein interior
Facilitated Diffusion (Channels vs Carriers)
Channels:
- Look like an open corridor/passageway that molecule can pass through
- Membrane pores to transport molecules down concentration gradient
- High conductance rates bc they bind the substrate very weakly
- Very quick, tend to be much faster
- Do not saturate - no upper limit on how fast they can go
Carriers:
- Looks like a protein that is situated within the membrane, that binds to its substrate on
one side to create a transporter-substrate complex, it changes its conformation and
flips around, and spits the molecule on the other side. Repeat.
- Membrane proteins that undergo substrate induced conformational change, or
membrane repositioning to release substrate to the other side of the membrane
- Slower bc they bind the substrate quite strongly - tend to be slower than channels
- Can saturate - upper limit on how fast they can go

Facilitated Diffusion through a Carrier Glucose Permease of Erythrocytes:

- Facilitated diffusion of glucose at 50, 000x faster than simple diffusion
- Similar to an enzyme, protein binds, ligand-induced conformational change,
- Specific for D-glucose
- The rate of uptake follows a pattern resembling M-M kinetics
- Kt about ⅓ the concentration of blood glucose so the transporter is nearly saturated
and operates near Vmax
- Kt = rate constant of transport / / represents the concentration of extracellular
glucose in which the transporter is going ½ is maximum velocity
- In enzymes, Km values were very close to their substrate values → this is due
to their response rates
- In glucose, the concentration of substrate is 3x greater than the Kt
- They are working at 75% maximum capacity
- Nothing much goes on in the RBC, doesn’t need to have increased
response rates. This means its okay to run the system a bit faster
Coupled Transport:
- Transport of a single molecule is called uniport
- Some transporters couple the movement of two molecules
- Antiporters move molecules in opposite directions
- Symporters move molecules in the same direction
- In diffusion, co transport through antiport or symport depends on the charge of the
molecules in order to have a net neutral charge
- In diffusion, there is a priority to maintain the charge status of the cell.
- In these systems, the molecules being transported are always electric neutral
(so the net change in charge over the movement equals out to zero)
- E.g., if 2 molecules of negative charge, you would use antiport (pumping out
one negative, bringing in one negative, nothing changes)
- E.g. if 2 molecules were positive and negative, you would use symport
- In secondary active cotransport, system couples a molecule moving down its gradient
to one moving down its gradient

Active Transport:
- Input of energy allows movement of molecules against concentration gradients
- Primary Active Transport
- Driven by direct source of energy (ATP)
- High concentration of x outside the cell, and little x inside the cell, but we
want to move more x out of the cell. Therefore x is moving against its
concentration gradient, which requires energy input
- Includes P-type, V-type, and ABC Transporters
- Secondary Active Transport
- Couples the movement of one molecule down its concentration gradient to the
movement of a second molecule down its gradient
- Source of energy is a gradient of molecules, typically ions
- X’s are outside of the cells, there’s a concentration difference, and x’s want to
get back in. We want to take molecule S inside the cell against its
concentration gradient, which takes energy input
- What happens is a cotransport system where we couple x down its
concentration gradient w/ the movement of S up its concentration
gradient.
- Secondary active transport is dependent on primary active transport
Primary Active Transport P-Type ATPase:
- Cells maintain high gradients of Na+ outside the cell and K+ inside the cell
- This gradient controls cell volume, electrical excitability and enables uptake of
nutrients through secondary active transport systems
- Maintaining activity the Na+ - K+ pump requires about a third of your energy
- Na+ - K+ ATPase uses the energy of ATP hydrolysis to pump three Na+ out of the
cell and two K+ into the cell
- This is antiport
- This is the exception of the rule (must maintain a neutral charge) as it creates
the concentration gradient the whole system uses
- Called a P-type transporter as it undergoes a phosphorylated intermediate
- When using ATP hydrolysis, we phosphorylate a residue within that protein
- Unique to this system, it is aspartate that gets phosphorylated (not serine,
threonine, or tyrosine)
- Aspartate breaks down quickly and is unstable. This allows it to
dephosphorylate itself as part of its catalytic mechanism → has a auto
phosphatase mechanism associated with it
- Establishes and maintains membrane potential 2

Active Transport: (V-type ATPases and ABC Transporters)

V-type ATPases:
- V for vacuole
- Use the energy of ATP to move proteins against a concentration gradient
- Acidification of organelles - lysosomes are packed w/ destructive enzymes, and they
have a low pH within them.
- They become acidic by these V-type ATP-ases
- It uses the energy of ATP hydrolysis to pump protons against its gradient. We
pump protons into the lysosome, as the concentration of the protons goes up,
the pH goes down
- V-type ATPase use the energy of ATP hydrolysis to pump protons into an
organelle
- In chloroplasts and mitochondria, F-type ATP synthases reverse this reaction to use
proton gradients to generate ATP
- Use ATP as energy, producing ADP (dead batteries), ADP goes to
mitochondria, we can use the proton gradient created by oxidative metabolism,
run the system in reverse and recharge ADP
- F type ATPase opposite direction to produce ATP
ABC Transporters:
- Contain ATP-binding domains (ATP Binding Cassette)
- Transport of a variety of biomolecules out of the cell against a concentration gradient
- Multi drug resistance protein pumps drugs (chemotherapeutic) out of the cell
- rendering the drugs ineffective
- Purpose of this system is to detoxify the cell
- Invest the energy of ATP to pump out damaging molecules out of the cell
- Chemotherapeutic drugs are toxic, and ABC Transporter starts to pump it out
of the cell

Secondary Active Transport (Glucose Uptake into Intestinal Epithelial Cells):

- In intestinal epithelial cells, glucose uptake from the gut is driven through the symport
w/ Na+
- The food we eat passes through the digestive system, and the body wants to
take up as many nutrients as possible
- Sodium and glucose are taken up together (through symport)
- The movement of glucose up its concentration gradient is enabled by the movement
of Na+ ions down their concentration
- Active transport of glucose from the gut depends on the action of the Na+ - K+
ATPase to establish the gradient of Na+ ions
- Depends on the Na+-K+ ATPase
- Pump three Na+ out, big gradient of sodium ions outside of the cell, and low
concentrations of glucose outside the cell relative to inside the cell
- Sodium ions are moving down the concentration gradient acting as the source
of energy, and the source of energy is used to pull glucose against its
concentration gradient

Ion Channel:
- Ion channels enable rapid movement of ions across the membrane
- Actions of ion channels can cause changes in membrane potential (action potentials)
in neurons
- They need to regulated or gated → can’t have action potentials going off
randomly (open and close in response to a signal)
- Signal can be voltage gated (on going propagation of a signal) or ligand gated
- They also need to be extremely rapid (in terms of transport of molecules
across the cell
- Can’t be saturation limits → as soon as switch is flipped, there will be a
massive flood of ions to provide the opportunity to depolarize cells
- Ion channels are tightly regulated; voltage gated channels and ligand gated channels
- Ion channels differ from ion transporters (like the Na+ - K+ ATPase) in three ways:
- 1) Faster
- 2) No saturation limits
 - 3) Gated/Regulated (open and close in response to stimuli)
- Ion channels are highly selective for the molecule to be transported
Specificity of Ion Channels (K+ Channel):
- K+ channel allows rapid movement of K+ ions out of cell
- Although Na is smaller the channel is 100-fold more permeable to K
- Selectivity filter discriminates K and Na based on their ability to shed water
molecules to form electrostatic interactions within backbone carbonyls
- Na and K are similar in that they both carry a positive charge
- K is bigger than Na, so anything K can fit through Na can fit through
- So how did they allow K to fit through and not Na? → comes down to the
closeness of the fit
- Each molecule has a charge associated w/ them, they are going to have a
hydration layer associated w/ them
- What happens is the passageway through is narrow enough that in order to fit
through, they have to shed off all of their water molecules and replace them w/
interactions w/ the transporter
- Potassium ions fit through nicely, the energy they get from the dipole carbonyl
entering the channel will compensate for the molecules it had to shed off
- In contrast, sodium is smaller and can’t interact w/ carbonyls on both sides at
the same time. So the energy it takes to shed off water molecules will not be
compensated by the carbonyl

Speed of Ion Channels (K+ Channel):

- Selectivity filter has four equivalent binding sites for K+
- As K+ ions enter the filter, the electrostatic repulsion from other incoming K+ ions
helps to flow of ions from inside to outside the cell