Principles of Biochemistry

~
CHE 315

Chapter 11.
Biological Membranes
& Transport
 No membrane = no cell

Biological membranes define

cells by their very existence

Electron micrograph of the erythrocyte plasma

membrane showing the trilaminar appearance
 Electron micrographs of several different membrane structures

Plasma membrane of Menoidium,

a protozoan.

Gram-negative envelope Golgi apparatus Many membrane

of Aquaspirillum serpens structures are evident in
pancreatic acinar cells
 Membranes are…

…not just simple barriers – they must be:

• Flexible – movement and growth

• Self-sealing – fusion / fission

• Selectively permeable – retain and exclude

• Organizers – for organelles, proteins, nucleic

acids, etc.
Percentage of lipid and protein in various cellular membranes
 Membranes
Composition

Biological membranes are composed of:

1. Lipids
- Mostly phospholipids…unless you are a plant
- Sterols
- Sphingolipids
2. Proteins

• Membrane lipids - the primary structural component

(but can also be involved in signaling)

• Membrane proteins – the primary functional

component (signals, transporters, enzymes, anchors,
channels, etc.)
 Membranes
Composition

Protein composition
is the most variable component
in biological membranes
Why?
amtb_in_membrane
 amtb_in_membrane

Membranes
Composition

Protein composition
is the most variable component
in biological membranes
Why?

Proteins impart functional specialization

Lipid composition of cellular membranes isolated
from rat liver.

Amount of major lipid components Phospholipid composition as a

as percentage of total lipid. percentage of total phospholipid.

"Other" includes mono-, di-, and triacylglycerol, fatty acids, and cholesterol esters.
 Distribution of phospholipids between inner and outer layers
of the human erythrocyte membrane.

Values are percentage of each phospholipid in the membrane. Other include phosphatidylinositol,
phosphatidylinositol 4-phosphate, phosphatidylinositol 4,5-phosphate, and phosphatidic acid.
Lipid Bilayer
Membrane lipids are structurally diverse.
Lamellar gel Lamellar liquid crystalline
 An electron micrograph of
a multilamellar phospholipid
vesicle in which each layer is
a lipid bilayer.

Multilamellar Golgi
structure (x94,000)

An electron micrograph of
a liposome. Its wall consists
of a bilayer.
The phase-transition, or melting, temperature (Tm)
for a phospholipid membrane.

Lamellar gel Lamellar liquid crystalline

 The melting temperature of phosphatidyl choline
containing different pairs of identical fatty acid chains.
 Membranes
Composition – the carbohydrate connection

Rich in sulfated Carbohydrates (in the form of

sugars glycoconjugates) (Chapter 7.3)
are extremely important lipid
and protein derivative
molecules.
Glycoconjugates are:
Structural
Lipids:
- Glycolipids
- Lipopolysaccharides
Informational
Proteins:
- Glycoproteins
A proteoglycan integral
- Proteoglycans
membrane protein
 Membranes & Carbohydrate
Derivative Molecules
See Chapter 7

THE GREATER “PORTION” OF THE

MOLECULE IS USUALLY “LISTED”
LAST IN THESE CONJUGATES
 Membranes
Structure

Membranes are selectively permeable

• Impermeable to most polar compounds
• Permeable to non-polar compounds
• 50-80 Å thick
• Appear trilaminar

Studies have revealed motion of protein and lipid

molecules within the membrane leading to:
 Membranes
The Fluid Mosaic Model
Lipids:
•Nonpolar tails inside Proteins:
•Polar heads outside •Protrude asymmetrically
•Clustering reduces to give the membrane a
hydrophobic surface sidedness

The structure is thermodynamically stable – driven by hydrophobic interactions

 Membranes
The Fluid Mosaic Model

The fluid mosaic model is a dynamic model

• Both proteins and lipids are able to move laterally within
and on the bilayer
• Both proteins and lipids are also able to move between
faces in the bilayer though this is quite restricted.

WHY?

• Proteins protrude from selective sides of bilayer

Hydrophobic interactions hold it all together

and provide the thermodynamic driving force
for membrane formation
 Membranes
Amphipathic Lipid Aggregates
Reduction of hydrophobic exposure
•Glycerophospyholipids Virtually insoluble &
•Sphingolipids spontaneously
•Sterols form aggregates

/ Vesicle
Formation is favored when Two-lipid monolayers. Pre-cursors to cells? Form
the x-sectional area of the X-sectional area of head group from bilayers due to unstable
head group is greater than and tails are similar end regions of layers
that of the acyl side chains
Distribution of membrane lipids across an artificial membrane.
Lipid composition of the plasma and organelle membranes of rat hepatocyte.
Doxorubicin
 A computed tomography (CT) image of the upper abdomen of a dog
following administration of liposome-encapsulated iodine, a contrast
agent that improves the light/dark contrast of objects in the image.
 Phospholipids
Distribution in Plasma Membranes

• Asymmetrical distribution
between inner and outer
monolayers
• Distribution can change for
functional reasons
• Distribution can be determined
by treating cell with
phospholipase C:
- Can’t get to lipids inside
- Removes heads of lipids
outside
- Estimate lipid content by
evaluating head groups
Three Types of Membrane Proteins

These differ in how they are

associated with the membrane

◼ Integral Membrane Proteins

◼ Peripheral Membrane Proteins
◼ Amphitrophic Proteins
 peripheral_membrane_protein

Membrane Proteins
General Categories

Categorized based on (1) where they are & (2) how they are solubilized

1. Integral Proteins
- Firmly associated with the membrane
- Have hydrophobic regions that interact with hydrophobic lipid tails
- Commonly α-helices or multi-stranded β-barrels
- Can be predicted by secondary structure (# of hydrophobic AAs)
- Solubilized only by agents that interfere with hydrophobic interactions:
Detergents
Organic solvents
Needed to completely remove
Denaturants protein from entire membrane
Phospholipase C
 peripheral_membrane_protein

Membrane Proteins
General Categories
2. Peripheral Proteins
- Loosely associated with membrane
- Use H-bonds & ionic interactions (electrostatics)
- Occasionally attached via a lipid “anchor”
- Interact with polar head groups
- Solubilized by mild agents and conditions
Changing pH
Interfering with electrostatics
(ionic strength)
Breaking hydrogen bonds Needed to remove
protein from membrane
Urea surface (inner or outer)
Carbonate
Proteases
Denaturants
 Membrane Proteins
General Categories

3. Amphitrophic Proteins
• Found both in cytosol and in association
with membranes
• Association with membranes is:
1. Reversible – dependent on molecular
interactions in a pathway or mechanism
2. Regulated – e.g. phosphorylation
 Require “gentler”
Membrane Proteins
Conditions for removal Two Groups
Peripheral and Integral

Some protein types can be

distinguished by the
conditions required for
their removal

For example: peripheral

proteins require much
less drastic measures
than integral proteins for
their removal
Entire membrane
Disruption required
For removal
Monotopic proteins penetrate only one leaflet.
Lipid annuli associated with an integral protein.
Integral Proteins are Held
in the Membrane by
Hydrophobic Interactions
with Lipids
 Six Categories of Integral Proteins
Attachment is the direct result of interactions between
the membrane lipids and hydrophobic domains of the proteins

Type I and II – have only one

transmembrane helix (NH3+ out or
NH3+ in)

Type III – multiple helices in a single

polypeptide

Type IV – several polypeptides

assembled to form a channel

Type V – covalently linked by a lipid

anchor

Type VI – has both a transmembrane

helix and a lipid anchor
Integral proteins have been very hard to crystallize
 Membrane Proteins
Transbilayer disposition of Glycophorin
An integral protein
We said before that integral
proteins can often be predicted
by secondary structure (# of
hydrophobic AAs):
• Carbohydrates on outside
(tetrasaccharides with bound
Neu5Ac)
Hydrophobic
Transmembrane • Many polar residues both
segment outside and inside
• Bilayer spanning residues (18)
are largely hydrophobic
• Each red hexagon on outside
surface contains two Neu5Ac1
(Sialic acid) each of which has
one (-) charge

Glycophorin - a glycoprotein 1N-acetylneuraminic acid (Fig. 10-12)

 Bacteriorhodopsin
A well characterized membrane spanning protein
A light driven proton pump
• Single transmembrane
protein
• Seven segments of 20
hydrophobic residues =
α-helices that can span
the membrane (50-80 Å)
• 7 internal helices are a
popular motif in signal
reception
• These helices are colored
to coordinate with the
Provide transmembrane hydropathy plot (Fig. 11-
passage for protons 11)
 Bacteriorhodopsin & the retinal,
the prostetic group of
bacteriorhodopsin, forms a Schiff base
with Lys216 of the protein.
Transmembrane portions of an integral protein
can often be predicted from 1° structure
• 3-D structure hard to
determine
• Hard to xtallize

RULE OF THUMB
The presence of 20 or more
hydrophobic AA in a row
indicates the segment passes
through the lipid bilayer as an
anchor or a channel

Why?
α-helices of 20-25 AA residues
can span 30 Å of bilayer
because each turn is 1.5 Å!!
Photosynthetic reaction center
of a purple bacterium
 Hydropathy Plots
Use hydropathy index information (see chapter 3)
 Hydropathy Plots
Predicting transmembrane sequences in proteins

• Hydropathy index (from table

3-1) is plotted vs AA residue
number
• Windows of these are plotted
to give indications of the
hydrophobicity or hydrophilicity
of defined lengths of the
protein
• More than 20 residues of a
high hydropathy index is
presumed to be a
transmembrane helix
• Good correspondance here
between theory and known
sequences
 Membrane Proteins
β-barrels are another common structural motif
(Chapter 4.3)

Not all transmembrane proteins are

α-helices

β-barrels: 20 or more transmembrane

segments form β-sheets that
line a cylinder

Need only 7 or more AA to span

in a β-sheet
 Amino Acid Residue distribution at
water/lipid interface

• Charged residues (Lys, Arg, His, Glu, Asp) found almost

exclusively in aqueous phase
• Positively charged residues are found more frequently in
the cytosol – known as the positive-inside rule
• Trp and Tyr are commonly found at interface – can
function as anchors and can have polar interactions
Proteins may be bound to the membrane
via covalent lipid anchors
The lipid anchor is
Thioester linkage
hydrophobic
Association is weaker than
for integral proteins but
carbonate does not
release some of these
(ex. GPI-linked proteins1)
This makes it an integral
protein by strict definition.
Association may even be
reversible
Notice the “sidedness” of
the membrane proteins

1Glycosyl phosphatidylinositol
The ribosomal synthesis, membrane insertion, & initial glycosylation
of an integral membrane protein according to the signal hypothesis.
 The N-terminal sequences of some eukaryotic secretory proteins.
The hydrophobic cores (brown) of most signal peptides are preceded
by basic residues (blue).
The posttranslational processing of
integral membrane proteins.
Change in lipid composition of secretory vesicles
with their passage through the Golgi apparatus.
 Membrane Dynamics

Membranes are in constant motion

Membranes must be fluid to support life

 Membrane Dynamics

Membranes must be able to alter their integrity to

survive – they are boundaries for open systems
• Exocytosis
• Endocytosis
• Cell movement
• Exchange of metabolic precursors and products
• Fusion
• Fission
Basis of the dynamics?
• Noncovalent interactions – allow for fluidity
 Fluidity of Plasma Membrane
default.jpg
 Bilayers of lipids have two states

(a) Paracrystalline State:

ORDERED
• Occurs at low temps
• Semisolid state
• Polar head groups are
uniformly arranged
• Acyl side chains are
ordered/packed nearly
motionless

The states are temperature dependent!

 Bilayers of lipids have two states
(b) Fluid (or Liquid) State:

(MORE) DISORDERED

• Occurs at elevated temps

• There is much thermal motion
(almost constant motion)
• Acyl side chains have no
regular organization
• Can be
1. liquid-disordered (as seen
at lower left)

2. liquid-ordered which is in
b/t (a) and (b)

The states are temperature dependent!

 Bilayer Composition is Variable

Composition can change under different

conditions.

• Bacteria at ↓ temps will synthesize more

unsaturated fatty acids than saturated ones

What would this accomplish?

Ensures membrane fluidity!!!

TRANSBIAYER MOVEMENT
OF LIPIDS REQURES
CATALYSIS
Motion of single phospholipids in a bilayer.
Lipid Motion Within Bilayers
Single phospholipid motion
Simple diffusion is very
slow:
• Requires the polar head
group to “dive” into the
hydrophobic layer
• ΔGDiffusion =
large and (+)

One could also consider this

ΔG as a ΔG‡ of sorts
Uncatalyzed (since we are talking about
enzymes here)
Lipid Motion Within Bilayers
Single phospholipid motion

In contrast:
Lateral movement can
occur very rapidly and
requires no enzyme
catalysis

Lateral movement occurs

when the lipid
“changes places” with
the nearest
neighboring molecule
Uncatalyzed
Lipid Motion Within Bilayers
Single phospholipid motion

Translayer motion must

be “catalyzed” in order
to to occur at useful
rates

Flippases, Floppases, &

Scramblases
• Provide an
energetically favorable
transmembrane path
• Are (“enzyme-like”)
proteins
Catalyzed
• Facilitate diffusion
 FRAP – Fluorescence Recovery After Photobleaching:
An intense laser light pulse bleaches the fluorescent markers (green) from
a small region of an immobilized cell that has a fluorescence-labeled membrane
component. The fluorescence of the bleached area, as monitored by
fluorescence microscopy, recovers as the bleached molecules laterally diffuse
out of it & intact fluorescence-labeled molecules diffuse into it. The fluorescence
recovery rate depends on the diffusion rate of the labeled molecule.
Fusion of mouse and human cells.
Restricted motion of the erythrocyte chloride-bicarbonate exchanger
and glycophorin.
 Membrane Rafts
(form Microdomains)
Allow for specialization of membrane dynamics
• Proteins are functionally
associated with rafts
• Proximal association
increases rates of reactions
(rate enhancement by local
entropy reduction)
• Proteins may move
between rafts but perform
tasks mainly within rafts
However…
Certain rafts usually contain
certain subsets of proteins
Again, this is a functional
Palmitoyl (lipid) protein anchors form strong distinction
associations with membrane rafts
Atomic force microscopic view of
membrane rafts
Caveolin forces inward curvature of a membrane.
The human erythrocyte membrane skeleton.
Internal membranes of eukaryotes.
Movement of a protein into the nucleus.
Electron micrographs
of coated vesicles.
An electron micrograph of triskelions.
Dynamic Membrane Fusion
EXAMPLES

• Budding - Golgi complex

handles products from
the ER
• Exocytosis and
endocytosis - export and
import of metabolites,
signals, etc.
• Fusion of imports with
lysosomes for processing
• Viral infection requires
direct entry into cell
Three models of protein-induced
curvature of membranes.
Membrane fusion during neurotransmitter release at a synapse.
Vesicle fusion at the synapse.
Neurotransmitter release.
 Fusion of two membranes
requires that:
1. Two membranes simply start by recognizing each
other
2. Their surfaces become closely associated – this
entails the removal of H2O
3. Bilayers become locally disrupted (outer leaflets)
4. Bilayers fuse to form continuous bilayer
5. This all happens at the appropriate time (signaling)

Also requires that the cells don’t just explode!!!

Fusion proteins are mediators that assist in these events

 HIV Viral Fusion
C:\Users\ArtemAlfredo\Desktop\HIV_infection_ccr5.jpg
Solute Transport Across Membranes

The hydrophobic core of the lipid bilayer restricts

the movement of polar or charged molecules –
these require transport
EXCEPT FOR…
WATER
Water can diffuse slowly across the membrane

When water is needed in large quantities it is

transported by aquaporins
Solute Transport Across Membranes
Transport can be categorized based on:

• The need for energy

• Kinetic behavior
• The direction of movement

There are very few exceptions to mediated

transport because cells are constantly bathed in
a “soup” and “leakage” is not compatible with the
selectivity necessary for process control.
 2 Modes of Transmembrane
Transport
Passive transport
• Moves solutes across membranes and down
their respective concentration gradients
• Requires no direct input of energy

Active transport
• Moves solutes across membranes against their
electrochemical gradients
• DO require an input of some form of energy
Types of Membrane Transport
A membrane may:

• Simply facilitate diffusion

(down concentration or
charge gradient)
• Actively transport solutes up
gradients of charge,
concentration, or both (this
is “pumping” and requires
energy)
• Transport may be passage
through channels

Transport (almost always)

requires proteins in some
capacity
Kinetics of movement of a solute molecule through a membrane.

Initial rate of diffusion is directly proportional to the concentration of solute.

In mediated transport, rate will reach a Vmax when carrier is saturated.
PASSIVE TRANSPORT IS
FACILITATED BY
MEMBRANE PROTEINS
Movement of solutes across a permeable membrane.
 The Electrochemical Gradient
is also called
The Electrochemical Potential
=
The Chemical Gradient
(difference in solute concentration)
+
The Electrical Gradient
(Vm)

Minimization of this potential is in accordance with the

2nd Law of Thermodynamics
Energetics of Passing Through
a Lipid Bilayer
In order to pass:
• The polar or charged solute
must 1st give up its
interactions with H2O
• Then it must move through
an unfriendly environment

It takes energy to strip away the

hydration shell (endergonic,
ΔG = (+)
This energy is re-gained on the
other side (but the middle is
tough)

Transporter proteins reduce ΔG‡transport

Differences between channels and transporters.
 The ion transport modes of ionophores.
Carrier ionophores transport Channel-forming ionophores span
ions by diffusing through the the membrane with a channel
lipid bilayer. through which ions can diffuse.
 Carrier ionophores bind their selected ion, diffuse through the
membrane and release it. Their ionic complexes must be soluble
in nonpolar solvents.
Valinomycin is a K+ carrier – ring-shaped;
transports 104 K+ ions/sec.
For a carrier, cooling the membrane below the
transition temperature eliminates its activity.
Valinomycin is a cyclic depsipeptide (has both
ester and amide bonds) and contains D- and
L-amino acids.

X-ray structure puts K+ coordinated octahedrally by the carbonyl groups of Val residues. Methyl and
isopropyl sidechains stick out from backbone – hydrophobic exterior makes it soluble in lipid bilayer.
While K+ fits into it (dehydrated ionic radius 1.33 Å), Na+ (r = 0.95 Å) does not and valinomycin binds
K+ 10,000-fold better than Na+.
 Channel forming ionophores make transmembrane channels or
pores through which selected ions diffuse. Channel formers are
relatively insensitive to decreased temperature.

Gramicidin A allows 107 K+ ions/sec to cross. Gramicidin A

is a 15-amino acid polypeptide of alternating hydrophobic
L- and D-amino acids. Dimerizes to form channel – cannot
be α-helical (α-helix has no central channel and cannot
accommodate alternating D- and L-amino acids). Forms helix
with several residues per turn; side chains cover periphery
with polar backbone lining central channel (4 Å = d). Trp
residues in C-terminal half have polar N-H groups directed
towards bilayer surface that orient the helix perpendicular to
the bilayer surface.

The limited permeability of the lipid bilayer means that almost any molecule that needs to cross it requires help
from a membrane protein – nutrients needed to support metabolic processes and waste products produced by
the cell must be dealt with. All of the transport proteins identified to date pass through the membrane multiple
times. The proteins that do this use the same tricks the ionophores do.
 Carriers and Channels – Two Main Classes of
Membrane Transport Proteins
Carrier proteins (also called permeases or transporters)
bind the molecule to be transported and undergo a series
of conformational changes that allow them to release the
substance on the other side of the membrane.
Transport by carriers can be active or passive.

Channel proteins form pores that extend all the way

across the lipid bilayer. When the pore is open, the
substance (usually an inorganic ion) can go through.
Transport through channels is much faster than transport
by carriers.
Transport through channels is always passive and
channel proteins tend to interact with the solute to be
transported less tightly than carriers.
 Carrier Proteins – Mediators of Both
Passive and Active Membrane Transport
Carrier proteins exist in two states:
While the transported solute is not modified
by the carrier protein, it is moved across a
membrane. Each carrier must bind its
substrate on one site of the membrane; a
subsequent conformational change
exposes the bound substrate to the other
side of the membrane, where it can
dissociate.

ACTIVE vs. PASSIVE TRANSPORT

Passive transport – solutes cross

the membrane going down their
electrochemical gradient using a
channel protein or a carrier protein.

Active transport – solutes are

pumped uphill against their
electrochemical gradient. Mediated
by carriers (also called pumps) –
pumping activity is directional.
PASSIVE TRANSPORT: What makes passive transport different from simple diffusion
(non-mediated transport)? Carriers or channels provide polar compounds and ions that
cannot dissolve in the lipid bilayer with an alternative route across the bilayer. Carriers
bind their substrates stereospecifically, reducing the energy barrier for passage across
the membrane. Examples: the glucose transporter (GLUT), a carrier, and the aquaporins
(AQP), which forms channels.

Glucose Transporters (GLUT): The flux of

Glucose (Glc) (JGlucose) into human erythrocytes
varies as a function of the [Glc] in the medium;
in the example shown, the temp. was 5 oC,
and it is easy to see that the flux saturates –
the concentration at which the flux is
half-maximal defines the KM or Kt (Ktransport)
of the transport process. Kt reflects the affinity
of the transporter for its substrate.

Simple diffusion of Glc into erythrocytes increases

linearly with [Glc], but is so small (about 50,000-fold slower than facilitated diffusion) that the
line can’t be distinguished from the x-axis.
While D-Glc and D-mannitol have similar permeabilities for lipid bilayers, D-Glc uptake into
erythrocytes is almost 105-times as efficient as D-mannitol uptake.
The erythrocyte transporter has a Kt of 1.5 mM for D-Glc, 20-30 mM for D-Gal (D-galactose),
and 3,000 mM for L-Glc. Passive transport is both saturable, specific, and has high rates
of diffusion down a concentration gradient (three hallmarks of passive transport).
Kinetics of glucose transport in erythrocytes.
There are many types of
GLUT transporters
SDS-gel electrophoresis of erythrocyte membrane proteins (top)
and a densitometer tracing of the same gel (bottom).

Band 3 is the anion-

transporting protein

Band 4.5 is the

glucose transporter

The sarcoplasmic
reticulum
The photoreceptor membrane of
membranes of muscle cells.
retinal rod cells.
 Glucose Transporter (GLUT)
Example of Facilitated Diffusion / Passive Transport
Membrane topology of the glucose transporter GLUT1: multiple TM (transmembrane)
helices in a single protein (Type III TM Protein).

GLUT1
Hydropathy
Plot
 Model of glucose transport into erythrocytes by GLUT1
Glucose transport occurs in 4 steps
This is an example of a uniport system (one substrate at a time in one direction)

Kinetic “substrate” model

Transport of glucose into a myocyte by GLUT4 is
regulated by insulin.
ACTIVE TRANSPORT: To pump a solute uphill against its electrochemical gradient, the
conformational changes in the carrier protein are simply linked to a source of energy. Active
transport is typically driven in three different ways:

1. ATP-driven pumps – uphill

transport is coupled to ATP hydrolysis.
There are at least 4 types: P-, V-,
F-type ATPases & multi-drug
resistance (ABC cassette) transporters.

2. Light-driven pumps – uphill

transport is coupled to energy input from light – e.g., rhodopsin, bacteriorhodopsin.

3. Coupled carriers – uphill transport of one solute occurs along with downhill transport of
another solute.

Coupled carriers or secondary transport:

Coupled transport involves the simultaneous
movement of a second solute in the same
direction (symport) or in the opposite direction
(antiport) to the one of interest. The energy
stored in the electrochemical gradient of one
solute (usually an ion) is used to transport the
other solute. The E. coli genome encodes some
160 coupled transporters (out of about 4,000
total proteins).
Two types of active transport.
ATP-DRIVEN PUMPS: P-type ATPases
Plasma Membrane Na+/K+ Pump:
Intracellular Na+ levels are typically 5-15 mM
while extracellular levels are 145 mM.
Intracellular K+ levels are typically 140 mM
while extracellular levels are 5 mM. These
differences are maintained by the Na+/K+ pump.

The Na+/K+ ATPase is an antiporter, pumping Na+ out against its electrochemical gradient
and pumping K+ in. The resultant Na+ gradient is essential for transport of most nutrients
and for regulating cytosolic pH. The stoichiometry of the pump has 3 Na+ going out and 2 K+
coming in for every ATP that is hydrolyzed; this stoichiometry means that the pump is
electrogenic. Fully 1/3 rd of the energy expended by your average cell goes to the Na+/K+
pump. In addition to establishing the ion gradients that are so essential to other transport
processes, extrusion of Na+ by this pump so essential for control of water content.
 Na+/Glucose Symporter

In the plasma membrane, Na+ is usually the co-transported ion. The Na+ that
enters the cell in this way is then pumped out of the cell by the Na+/K+ ATPase,
an ATP-driven pump.
Glucose transport is driven by the Na+ gradient: Na+ and glucose bind
cooperatively – the high concentration of Na+ outside encourages glucose
binding.
With an external Na+ concentration of 143 mM, an internal Na+ concentration of
14 mM and a membrane potential of ‒50 mV, a 66-fold gradient of an uncharged
molecule like glucose can be generated.
 Chloride Bicarbonate Exchanger (Antiporter)
(Anion Exchange (AE) Protein) of the erythrocyte membrane.

This is a facilitated diffusion

system in erythrocytes.

This exchanger is essential for

CO2 transport to lungs.

Waste CO2 from tissues is

converted to bicarbonate
(HCO3-) by carbonic
anhydrase.

Note that the exchange of anions

is electroneutral.

This is an antiport system

 4 Types of Membrane ATPase
Transporters
1. P-type – Na+/K+ ATPase and Ca2+ ATPase
(SERCA). These pump cations.
DRIVEN BY ATP HYDROLYSIS AND PHOSHORYLATION!
2. V-type – for vacuolar pump. These pump
protons.
3. F-type – use proton motive force from electron
transport to make ATP and when operating in
reverse they hydrolyze ATP to generate proton
motive force
4. ABC transporters – pump various molecules
out of cells (protein, AAs, metals, drugs, etc)
 Postulated Mechanism for
the Na+/K+ ATPase

Sodium / Potassium ATPase

Na+/K+ ATPases (P-Type ATPase)

This is a primary active

antiporter for Na+ and K+

Maintains an ion
IMBALANCE

Phosphorylation forces a
conformational change
that drives the cation
across the membrane
 The leaves of the purple foxglove plant
(Digitalis purpurea) are the source of
the heart muscle stimulant digitalis.

Digitoxin (digitalin), the major component of digitalis; & ouabain, a cardiac

glycoside isolated from the East African Ouabio tree, are among the most
commonly prescribed cardiac drugs, used to increase sodium/calcium exchange.
 Cardiac Glycosides: Potent Drugs from Ancient Times

Oleander plant (Nerium oleander) Monarch butterfly (Danaus plexippus)

Common milkweed Lily of the Valley Viceroy butterfly(Limenitis Archippus)

(Asclepias syriaca) (Convallaria majalis)
 Vanadate is a transition state analog &
an inhibitor of the phosphorylation step.
The general structure of
the P-type ATPases.
 SERCA Pump
Ca2+ pump of the sarcoplasmic reticulum
Postulated mechanism of the SERCA pump.
Two proton pumps with similar structures.
 F-Type ATPases are
Reversible & ATP-Driven
This is a reversible ATP-
driven proton pump

FO integral membrane
protein is inhibited by
oligomycin

F1 peripheral membrane
protein was the 1st factor
isolated
Mitochondrial
F-type ATPase Reversibility
These can appropriately
also be called ATP-
synthases

Proton gradients supply the

energy to drive the
reverse rxn.

The forward rxn is an

ATPase

The reverse rxn is an ATP

synthase
 ATP-DRIVEN PUMPS: ABC Transporters.
The largest family of membrane transport proteins
is named for their ATP Binding Cassette (they are
also known as P-glycoproteins). These proteins
have two groups of 6 or more transmembrane
domains, each with an ATP-binding cassette. The
two halves can be part of a single polypeptide chain
or can be separate polypeptides that assemble.

ATP binding leads to dimerization of the

two ATP binding domains; subsequent
ATP hydrolysis leads to dissociation.
The structural changes that occur upon
ATP binding to the cytosolic domains is
thought to be transmitted to the
transmembrane domains, triggering the
conformational changes needed to
expose substrate binding sites on one
and then the other side of the
membrane. The ABC transporters use
ATP binding and hydrolysis to transport
a wide variety of molecules across
various membranes.
Substrates for these transporters include amino acids, sugars, inorganic ions,
polysaccharides, lipids, and peptides.
In eukaryotes, ABC transporters generally function in export.
Bacteria use them for both import and export functions.
ABC transporters (floppases) catalyze the transport of lipids (e.g., phosphatidylcholine &
cholesterol) from the cytosolic face of the bilayer to the outer face.
Yeast a-factor, a 12 amino acid peptide used as a mating factor, is produced in the cytosol
and its export requires an ABC transporter.
Secretory epithelia use these transporters to excrete metabolites (e.g., bile salts) against
steep gradients.
TAP (transporter associated with antigen processing), an ABC transporter in the ER,
transports peptides produced by protein degradation from the cytosol into the ER, the site of
MHC class I assembly; this step is key to surveillance by the immune system.

ABC transporters are important in removing drugs (xenotoxins) and drug conjugates and
the first family member identified was the multidrug resistance protein (MDR).
ABC Transporters
A defective ion channel in cystic fibrosis.
A defective ion channel in cystic fibrosis.
 Ion Gradients

The ion gradients formed by

primary transport of Na+ or K+
can then
– in turn –
provide the driving force for co-
transport of other molecules
Lactose Permease
A lactose symporter

Two components:
1. The primary
transport of H+
out of the cell
2. The secondary
active transport
of lactose into
the cell
 Na+/K+ Transport of Glucose
(ion gradient)
The energy for this
comes from two
sources
1. Chemical
potential (higher
[Na+] outside
2. Electrical
potential
(negative inside
and draws Na+
inward)
This is a Na+- glucose symporter
Glucose transport in the intestinal epithelium.
 Aquaporins (AQP)
When water has to move rapidly across membranes, a member of the aquaporin family is
used. Presence of the channels specific for water allows independent regulation of solute
and water. Aquaporins (AQPs) (11 mammalian have been identified) have 5 transmembrane
domains; some transport small molecules in addition to water (e.g., glycerol, urea). AQP1
forms tetramers; ach monomer forms a hydrophilic channel 2.8 Å in diameter. AQP1 allows
single file passage of water at a rate of 5 x 108 per sec – the water must move in a
continuous stream. (For comparison, catalase, the fastest enzyme known, has a turnover
rate of 0.4 x 108 per sec.
Loops B and E fold into the membrane to form the pore. Specificity for water is established
by the diameter of the channel and the orientation of a pair of dipoles formed by the
signature NPA motifs
(Asn-Pro-Ala sequences that
occur twice in each monomer) –
the dipoles interact with individual
water molecules, preventing
them from H-bonding to adjacent
water molecules.
When antidiuretic hormone levels
rise, AQP2 molecules stored in
intracellular vesicles in the
epithelial cells of the collecting
duct are placed on the plasma
membrane – water permeability
increases and water is
reabsorbed from the collecting duct.
Ribbon representation of
the AQP1 fold depicting
the six membrane-
spanning helices,
the two pore helices, &
the connecting loops
in different colors.
Schematic representation of AQP1 topology & hydropathy.
Ribbon and space-filled diagrams, representing
the structure of the aqueous pre in AQP1.
Schematic representations explaining the mechanism
for blocking proton permeation of AQP1.
 Ion Channels
Channel proteins form hydrophilic pores across the membrane. Over 100 types of ion
channels have been described. A given cell will only express a subset and many of those
expressed are localized to specific regions of the cell.
Ion channels are characterized by several key properties:
1) Their selectivity for different ions;
2) The existence of open and closed states – gating;
3) Regulated transitions between open and close states by ligand, voltage, or lateral pressure.
4) Spontaneous inactivation.
One of the keys to understanding ion channel function has been the ability to monitor the flow
of ions through a single channel using patch clamping. A high resistance seal between a glass
pipette and the membrane allows measurement in the cell-attached mode.
By pulling a small fragment
of the membrane,
the experimenter has
access to the part of
the channel that used
to be inside the cell.
 Gating: control of opening and closing.
Since permeant ions flow passively through channels, channel opening and closing must
be controlled carefully. Channels typically fluctuate between closed and open states.
The open state (when ions are passing rapidly through the channel) corresponds for an
enzyme to the enzyme-substrate complex; the closed state (no ions moving) corresponds
to an enzyme that just released its product.
Ion channels open in response to a variety of stimuli including changes in voltage
(voltage-gated channels), ligands that bind to the part of the channel exposed to
extracellular space (neurotransmitters, e.g., acetylcholine), ligands that bind to the
cytosolic part of the channel
Mechanically- or tension-gated
(e.g., cyclic nucleotides or ions
such as calcium) (chemically-
gated channels), and
mechanical stimuli in
response to changing lateral
pressure of the membrane
(mechanically-gated).
In addition, channel function
can be regulated by
phosphorylation and
dephosphorylation of the
channel protein and by the
subcellular localization of the channel.
 Ion Channels are distinct from
ion transporters
1. The rate of flux through channels can be
several orders of magnitude greater than
the turnover number for a transporter
2. Ion channels are NOT saturable: rates
do not approach a maximum at high
substrate concentration
3. They are “gated” ---opened or closed in
response to some cellular event
 Examples of Ion Channels
• Ligand-gated channels – binding of a
small molecule forces and allosteric
transition in the protein which opens or
closes the channel
• Voltage-gated channels – a change in
transmembrane electrical potential (Vm)
causes the channel to open or close (can
be measured electrically)….

We will see these in action in the next

chapter
Time course of an action potential.
 The K+ channel of Streptomyces lividans.

This is a ligand-gated uniporter

and is very specific
 A Voltage Gated Ion Channel
Work by sensing electrical gradients
across membranes
• Respond by opening or closing
• Within ms of opening, a channel closes
and remains inactive for many ms (till next
charge buildup)
• Activation followed by inactivation of Na+
channels is the basis for signaling by
neurons
Voltage-gated Na+ Channels of
Neurons
 Tetrodotoxin, an organic compound isolated
from the puffer fish, binds to sodium channels
with great affinity (Ki ≈ 1 nM) & blocks the channel.
Bactrachotoxin (BTX) is a steroidal alkaloid from the skin of Phyllobates
terribilis, a poisonous Columbian frog (source of the poison used on
blowgun darts). In the presence of BTX, sodium channels in an excised
patch stay persistently open when the membrane is depolarized.
 Aminobutyric acid (GABA) opens channels that are specific for
chloride ions. The GABAA receptor channel is pharmacologically important
because it is the target of Valium, which is used to diminish anxiety.

Librium
Valium Xanax

Clonazepan Alprozalam
Pharmacology of Valium (Diazepam)
➢ Diazepam is a benzodiazepine that binds to a specific subunit on the GABAA receptor at a site
that is distinct from the binding site of the endogenous GABA molecule. The GABAA receptor is an
inhibitory channel which, when activated, decreases neuronal activity. Benzodiazepines do
not supplement for the neurotransmitter GABA, rather benzodiazepines such as diazepam bind to
a different location on the GABAA receptor with the result that the effects of GABA are enhanced.
Benzodiazepines cause an increased opening of the chloride ion channel when GABA binds
to its site on the GABAA receptor leading to more chloride ions entering the neuron which
in turn leads to enhanced central nervous system depressant effects. Diazepam binds
non-selectively to α1, α2, α3 and α5 subunit containing GABAA receptors.
➢ Because of the role of diazepam as a positive allosteric modulator of GABA, when it binds to
benzodiazepine receptors it causes inhibitory effects. This arises from the hyperpolarization of
the post-synaptic membrane, owing to the control exerted over negative chloride ions by GABA A
receptors.
➢ Diazepam appears to act on areas of the limbic system, thalamus, and hypothalamus, inducing
anxiolytic effects. Its actions are due to the enhancement of GABA activity. Benzodiazepine drugs
including diazepam increase the inhibitory processes in the cerebral cortex.
➢ The anticonvulsant properties of diazepam and other benzodiazepines may be in part or entirely
due to binding to voltage-dependent sodium channels rather than benzodiazepine receptors.
Sustained repetitive firing seems to be limited by benzodiazepines' effect of slowing recovery of
sodium channels from inactivation. The muscle relaxant properties of Diazepam are produced via
inhibition of polysynaptic pathways in the spinal cord.
 Cholera
Cholera - an infection of the small intestine, caused by the bacterium Vibrio cholerae.
Main symptoms: profuse watery diarrhea & vomiting. Transmission: primarily by drinking
or eating water or food that has been contaminated by the diarrhea of an infected person
or the feces of an infected but asymptomatic person. The severity of the diarrhea &
vomiting can lead to rapid dehydration & electrolyte imbalance & death in some cases.
Primary treatment: oral rehydration solution (ORS) to replace water and electrolytes, &
if this is not tolerated or doesn't provides quick enough treatment, intravenous fluids can
also be used. Antibiotics are beneficial in those with severe disease to shorten
the duration and severity. Worldwide it affects 3–5 million people and causes 100,000–
130,000 deaths a year as of 2010. Cholera was one of the earliest infections to be
studied by epidemiological methods.
 Acetylcholine Receptors (AChR)
Bind acetylcholine (ACh) & transmit its signal

Muscarinic AChRs Nicotinic AChRs

GPCRs that mediate Ligand-gated ion
a slow metabolic channels that mediate
response via second a fast synaptic
messenger cascades. transmission of
the neurotransmitter.
 Ligand-gated Ion Channels
Ex: Nicotinic acetylcholine (N-Ac) receptor
(muscle contraction)
.

• Ac is released by motor neurons

• Ac diffuses and binds to membrane receptors
• Binding produces a conformational change
• Conformational changes open an ion channel
• There is an inward (+) charges
• which depolarize the plasma membrane
• Contraction results
 Acetylcholine Receptor I
(subunits and respective x-membrane helices)
Acetylcholine Receptor II
(cross-section of the M2 helices)
Acetylcholine receptors.
 The torpedo (Torpedo marmorata, also known as the electric ray)
has an electric organ, rich in acetylcholine receptors, that can deliver a shock
of as much as 200 V for approximately 1 sec.
Gap Junctions
 Classification of some transport proteins
based on mechanism and energetics.
I. Channels (Passive)
(A) Voltage-gated channels (example: Na+ channel)
(B) Chemically regulated channels (example: nicotinic acetylcholine receptor)
(C) Other (unregulated, pressure-sensitive, etc.)

II. Transporters
(A) Passive uniporters (example: erythrocyte glucose transporter)
(B) Active transporters
(i) Primary active transporters
(a) Redox coupled (example: cytochrome c oxidase)
(b) Light coupled (example: bacteriorhodopsin)
(c) ATPases (example: Na+/K+-ATPase)
(ii) Secondary active transporters
(a) Symporters (example: lactose permease; sodium/glucose symporter)
(b) Antiporters (example: Band 3; bicarbonate/chloride exchanger)
Some superfamilies of structurally related channels and transporters.
(1) Voltage-regulated ion channels:
Na+ channel
K+ channel
Ca2+ channel (dihydropyridine-sensitive)
(2) Neurotransmitter-regulated channels:
Nicotinic acetycholine receptor
γ-Aminobutyric acid (GABA) receptor
Glycine receptor
(3) Mitochondrial transporters:
ADP/ATP transporter
H+-Phosphate transporter
Uncoupling protein (H+/OH- transporter)
(4) Sugar transporters:
Mammalian glucose transporter
E. coli H+-arabinose transporter
E. coli H+-xylose transporter
(5) E1E2-type ion-motive ATPases:
H+/K+-ATPase (mammalian gastric mucosa)
Na+/K+-ATPase (plasma membrane)
Ca2+-ATPase (sarcoplasmic reticulum)
H+-ATPase (plasma membrane)
K+-ATPase (S. faecalis)