biological membranes and membrane transport · membrane transport previous knowledge (high school...
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Biological membranes and
membrane transport
Biological membranes: Textbook, pages 88-94.
Simple diffusion: Textbook, pages 276-278.
Electrodiffusion, diffusion of ions: Textbook, page 279.
Facilitated diffusion: Textbook, pages 279-282.
Ionophores: Textbook, page 280.
Active transport: Textbook, pages 282-283.
György Vámosi
Biological membranes and
membrane transport
Previous knowledge (high school biology, medical chemistry class):
• Chemical structure and properties of lipids and proteins
Why are these topics important?
• The plasma membrane is a barrier between the cell and its environment and a gateway forcommunication and transport. Membranes make it possible to form specialized compartments ineukaryotic cells. Transport processes involve exchange of ions, nutrients, metabolites and signalingmolecules with the environment.
What do we learn today?
• Structure and constituents of the plasma membrane: lipids, proteins and carbohydrates• Physical properties of the cell membrane: fluidity, phase transition• Transmembrane transport: simple and facilitated diffusion (passive), primary and secondary
active transport • Transport proteins
Aim:
• Understand the principles governing passive and active transmembrane transport processes
Biological membranes
Functions of the plasma membrane:
• separation of spaces
(whole cell, organelles)• electric insulation
• diffusion barrier
• transport• controlled movement of materials
• signal transduction• detection and transmission of electric and
chemical signals
!
What is the structure of the membrane like?
• lipids (40-60%)
• proteins (30-50%)
• carbohydrates (10%)
!
lipid bilayer, hydrophilic and hydrophobic parts
protein: integral, peripheral
carbohydrates – extracellular side
What is the structure of the membrane like?
integral proteins
peripheral protein
transmembrane protein
!
• sphingolipids are phospholipids with an amino-alcohol instead of the glycerol backbone, an
important example is sphingomyelin (below)
• the apolar part is formed by two side-chains of fatty acids with 14-22 carbon atoms,
connected by ester bonds to the glycerol
• polar head could be either serine, ethanolamine, choline, inositol, the corresponding lipids
are phosphatidyl-serine, phosphatidyl-ethanolamine, phosphatidyl-choline, phosph.-inositol
Membrane structure: phospholipids
serine ethanolamine choline inositol
sphingosine
oleic acid (fatty acid residue)
phosphocoline
R:
!
non-polar tail(hydrophobic)
polar head(hydrophilic)
double
bond
aggregation of phospholipids in aqueous medium
cis double bonds result in a higher average separation distance
cis trans
Phospholipids are amphipatic molecules !
Functions of membrane proteins:
Membrane structure: membrane proteins!
the carbohydrate components of the membrane
functions:
- surface protection
- cell communication,
recognition
- cell adhesion,
extracellular matrix
Membrane structure: carbohydrates
glycolipids (rare) and glycoproteins (common)
5*
T<Tm, gel phase,
tighter packing of the
lipids, limited molecular
motions and diffusion
T>Tm, liquid phase
looser packing of the lipids,
more intense molecular motions
and faster diffusion
Phases of the membrane
Tm: phase transition (or melting) temperature
!
length of fatty acyl chains: shorter chains – weaker
interaction - Tm
amount of unsaturated fatty acids: double bond –
bend in the chain – weaker interaction - Tm
What factors influence membrane fluidity?
amount of cholesterol (a steroid lipid, essential part
of membranes)
dual effect: increases fluidity below Tm, decreases
fluidity above Tm, stabilizes the membrane
cis trans
Cis double
bond
!
The dynamics of the membrane
the cell fusion experiment of Frye and Edidin
The fluid-mosaic model of Singer and Nicolson:
membrane proteins freely swim around in the lipid sea
Partially true, but in reality, membrane structure is more complex than that:
!
What are “lipid rafts” (DIG-microdomains)?
What is the function of lipid rafts?
Membrane domains of special composition (high sphingolipid,
glycolipid and cholesterol content) that also include proteins
(Detergent Insoluble Glycolipid microdomain)
Lateral organization of membrane proteins, keeping essential
elements of certain signaling events in each other’s vicinity to
enable their interaction
p56lck
CD
4/C
D8
TCR/
CD3
hDlg
Kv1.3
ZIP-1/2
1 inte
grin
Kv2
PKC
5*
What molecular motions take place in the membrane and how can these be examined?
proteins:
FRAP
Single Particle Tracking
Fluorescence Correlation Spectroscopy
lipids:
DPH fluorescence polarization or
anisotropy (rotational freedom)
fluidity
lateral diffusion
whipping motion
(flexibility)
rotation
flip-flop
(exchange)
5*
!
5*
Membrane transport
What does the rate of diffusion of
molecules across membranes depend on?
decreasing
permeability
Permeability
decreases with
increasing size and
increasing
polarity/electric
charge of the particle
!
Classification of transport mechanismspassive
toward lower concentration
no energy required
simple diffusion
facilitated diffusion
activetoward higher concentration
requires energy
primary active
secondary activedirect use of ATP
transport protein aiding the
passage of the target molecule
indirect use of ATP: transport of
the target molecule using
energy from the gradient of
another molecule
!
transport protein
Passive transport
simple diffusion: only small and lipid-
soluble molecules can cross the membrane
e.g. steroid hormones, O2 and CO2
facilitated diffusion: ion channel or carrier
molecule helps transport toward the lower
concentration
selective, saturable, can be selectively
inhibited
e.g. glucose transporter, ion channels, water
channels
!
partition coefficient
units:m sm
m
KDp
d
• permeability constant
m2 m1m m m
c ccJ D D
x d
• cm1 and cm2: concentration of the solute molecule in the lipidmembrane at the two membrane/water interfaces
• cw1 and cw2: concentrations of the solute in the aqueousphases at the two interfaces
• Dm: diffusion coefficient of solute in the membrane
m1 m2
1 2w w
c cK
c c
Simple diffusion through the membrane
(measure of hydrophobicity: solubility in the lipid membrane vs. water)
pm depends on• K (hydrophobic/hydrophilic character of the molecule)• Dm (size and shape of the molecule)• d (thickness of the membrane)
mJ
d
cw1
cm1
cm2
cw2
con
cen
tra
tio
n
H2O H2Olipid membrane
If ic, & ec. conc. are unequal → matter flow density through the membrane (Fick I):
K>1 (hydrophobic)
K<1 (hydrophilic)
1 22 1
1 2
w wm m mm m w w
Kc Kcc c D KD D c c
d d d
1 2m w wp c c
!
• the rate of diffusion is substantially larger than thatexpected from Fick’s law
• strongly selective (just one type, or narrow range ofstructurally related substances)
• the rate of transport can be saturated
• can theoretically work in both directions, its direction isdetermined by the (electro)chemical potential gradient ofthe substance being transported
• can be selectively inhibited by inhibitors
• examples: glucose transporters (GLUT), water transporter(aquaporin) in kidney and bladder cells, ion channels(though they are not saturable)
Facilitated diffusion
vv max
M
c
K c
KM: Michaelis constant(concentration at which the rate oftransport is half of the maximal)
c (M)
Rate of transport (Michaelis-Menten equation):
!
Problem 21.2.2
GLUT-1 GLUT-3 are high glucose-affinity uniporter proteins. GLUT-1 is present in most
tissue types, and is responsible for the basal glucose uptake of cells, whereas GLUT-3 is
present in neurons and a few other cell types. Their KM value is ~1 mM. Calculate the value
of v/vmax
a) at 4 mM blood glucose concentration on an empty stomach,
b) and shortly after meal, at a blood glucose concentration of 10 mM.
Solution:
a) We apply the Michaelis-Menten equation:
max
40.8
1 4M
v c mM
v K c mM mM
At a concentration on empty stomach, the rate of the transport is 80% of the maximum rate (at a given
GLUT expression level).
b) at a blood glcusose cocncentration of 10 mM :max
100.909
1 10M
v c mM
v K c mM mM
At a blood glucose concentration of 10 mM the rate of transport is 90.9% of the maximal rate. (Note: It can be seen that, in the physiological range, the blood glucose level has only a slight effect on the rate of
GLUT-1 and GLUT-3 transport, since these concentrations are well above the value of KM, and the transporters
function nearly at maximal rate.)
Problem 21.2.4
The GLUT-2 glucose transporter is expressed e.g. in hepatocytes and in the membrane of
beta-cells of the pancreas. It has a low glucose affinity, its KM value is ~17 mM. Calculate
the v/vmax value at blood glucose concentrations on empty stomach, and after meal (4 mM,
10 mM). How many times will the rate increase by the glucose concentration increase?
Solution:
At 4 mM blood glucose concentration on empty stomach:
max
40.19 19%
17 4M
v c mM
v K c mM mM
The rate of the transport is 19% of the maximum rate.
At a blood glucose concentration of 10 mM, after meal:
max
100.37
17 10M
v c mM
v K c mM mM
The increase of transport rate: 0.37/0.19=1.95 fold.(Note: The low affinity GLUT-2 functions as a sensor, at a higher blood glucose concentration it is
more active.)
Two major classes:
Channel forming: introduce a hydrophilic pore into the membrane, allowing ions
to pass through
Gramicidin (monovalent cations), Nystatin (monovelant ions)
Mobile ion carriers: molecules with a ring like structure, hydrophobic outside,
binding the ion inside, shuttle between the ec. and ic. side
Valinomycin, Nonactin, Monactin, Nigericin, A23187, Ionomycin, CCCP
Ionophores
Ionophores are small, hydrophobic molecules that can transport
ions across membranes down their electrochemical gradient, by
shielding their charge.
They are usually synthesized by microorganisms to kill other,
competing microorganisms. They have an antibiotic effect.
Valinomycin
!
5*
5*
5*
Active transport
primary active: the protein pumps ions
across the membrane against the gradient
using energy from ATP hydrolysis
e.g. Na+/K+, Ca2+, H+ pumps
secondary active: moves ions/molecules across
the membrane against the gradient using
energy from the energy stored in the gradient
of another ion, created by a primary active
mechanism
e.g. Na+-glucose or Na+/Ca2+ cotransport
driven by the Na+ gradient
direct ATP usage
indirect ATP usage: energy
from the gradient of another
ion
!
facilitated diffusion
channel or carrier protein
Classification of transport proteins based on
the number of transported species and the
direction of transport
secondary active
simultaneous transport of two/more
molecules/ions using the energy stored in the
gradient produced by other pumps utilizing ATP
symport: Na+-glucose
antiport: Na+/Ca2+ exchanger (3:1)
!
How does the Na+/K+ pump work
and what is its function?
Role of the maintenance of Na+ and K+ gradients:
• membrane potential (electrogenic: 3 Na+(out)/2 K+(in), source of diffusion
potential)
• decrease of osmotic pressure (decreased solute concentration)
• provide energy for secondary active transport
!
Glucose uptake in the small intestine
ec. space lumen of the
small intestine
high Na+
low K+
low glucose
low Na+
high K+
high glucose
high Na+
glucose (from
food)
Na+-glucose
symport
glucose uniport
glucoseglucoseglucose
Na+/K+ ATPase
epithelial
cell
The Na+-glucose symport uses the free energy of the Na+ gradient to transport
glucose against its own concentration gradient
basolateral
membrane
apical
membrane
!
Conclusion
Self-control questions:
• What are the main functions of the cell membrane?
• What is the current model of the cell membrane?
• What are the constituents of the cell membrane?
• What determines the permeability of the membrane to different particles?
• How can we classify transport processes?
• What is the difference between facilitated diffusion and active transport?
Take-home message:
• The cell membrane is the barrier between the cell and its environment, where selective and
regulated transport and signal transduction processes take place
• The cell membrane is very dynamic (Singer Nicolson fluid mosaic model), but it is not
homogenous (membrane domains), and there are constraints hindering the diffusion of its
constituents (membrane skeleton, membrane domains, aggregation)
• Small, neutral and hydrophobic molecules can cross the membrane by simple diffusion, large
polar or charged molecules require transport proteins for passing across the membrane
• Simple and facilitated diffusion occur “downhill” the electrochemical potential gradient, active
transport goes “uphill” and requires ATP directly (primary) or indirectly (secondary)
Supplementary material
Facilitated diffusion: Lineweaver-Burk plot
vv max
M
c
K c
1 1 1M
max max
K
v v v c
Intracellular
Extracellulularcw2
cw1
Electrodiffusion: transport of charged
particles (eg. ions)
2 1diff
cJ D p( )
xw wc c
diff,k E,k
c z FJ J J D c
x RT x
k kk k k k kL X
c z FI J Fz z FD c
x RT x
k kk k k k k k
for ion “k”:
E E
zF zFJ u c , u =D J Dc
x RT RT xel el
Δ Δx
material current
density
Electric current
density