Biological membranes and membrane transport · membrane transport Previous knowledge (high school biology, medical chemistry class): • Chemical structure and properties of lipids

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