BIOL 266 CELL BIOLOGY

Lecture 7:
Membrane transport
 The relative permeability of a synthetic lipid
bilayer to different classes of molecules

O2, CO2, N2,

Small hydrophobic molecules benzene

18 46 H2O, glycerol,
daltons Small uncharged polar molecules
ethanol

92 180 amino acids,

Large uncharged polar molecules glucose,
daltons nucleotides

H+, Na+, K+, Ca2+,

Charged molecules (IONS) Mg2+, Cl-, HCO3-
Protein-free artificial (synthetic) lipid bilayers are
impermeable to large uncharged polar molecules
(amino acids, glucose and nucleotides) and charged
molecules (ions)
Protein-free artificial lipid bilayer

= charged and polar molecules

Cell membranes are permeable to large uncharged polar
molecules (amino acids, glucose and nucleotides) and
charged molecules (ions)
Transfer of these water-soluble molecules depends on
membrane transport proteins

Cell membrane

membrane
transport proteins

= charged and polar molecules

Each type of transport protein in a cell membrane transfers a
particular type of molecules, causing a selective set of water-soluble
molecules to end up inside the membrane-bounded compartment

Cell membrane
transport protein specific for

transport protein specific for

transport protein specific for

The set of transport proteins in the plasma membrane, or in the
membrane of an intracellular organelle, determines exactly what
water-soluble molecules can pass into and out of that cell or organelle
Therefore, each type of membrane has its own characteristic set of
transport proteins
plasma membrane

cytosol

organelle A organelle B
 The concentrations of positively charged ions (cations)
inside and outside of a typical mammalian cell
OUTSIDE
[K+]out = 5 mM

INSIDE
[Na+]in = 12 mM [K+]in = 140 mM

[Na+]out = 145 mM

[K+]in is ~ 30 times higher than [K+]out

[Na+]in is ~ 12 times lower than [Na+]out
[K+]in + [Na+]in = [K+]out + [Na+]out = 150 mM
 The concentrations of negatively charged ions (anions)
inside and outside of a typical mammalian cell
OUTSIDE
[Cl-]out = 120 mM

[Proteins-]in = 140 mM INSIDE [Cl-]in = 5 mM

[HCO3-]in = 15 mM

[Proteins-]out = 10 mM
[HCO3-]out = 30 mM

[Cl-]in + [Proteins-]in + [HCO3-]in = [Cl-]out + [Proteins-]out + [HCO3-]out

= 160 mM
 If a cell is not to be torn apart by electrical forces:
The quantity of positive charge inside the cell must be balanced
by an almost exactly equal quantity of negative charge

Cations inside: Anions inside:

[K+]in = 140 mM
[Proteins-]in = 140 mM
[Na+]in = 12 mM
[HCO3-]in = 15 mM
[Cl-]in = 5 mM

[K+]in + [Na+]in = [Proteins-]in + [HCO3-]in + [Cl-]in = 150 mM

[Cations+]in = [Anions-]in
If a cell is not to be torn apart by electrical forces:
The quantity of positive charge outside the cell must be balanced
by an almost exactly equal quantity of negative charge

Cations outside: Anions outside:

[K+]out = 5 mM
[Cl-]out = 120 mM
[Na+]out = 145 mM
[HCO3-]out = 30 mM
[Proteins-]out = 10 mM

[K+]out + [Na+]out = [Cl-]out + [HCO3-]out + [Proteins-]out = 150 mM

[Cations+]out = [Anions-]out
1st mechanism by which small molecules cross lipid bilayers
passive diffusion
Gases (O2, CO2), hydrophobic molecules (benzene) and small
polar uncharged molecules (H2O, ethanol) are able to dissolve in
the lipid bilayer
During passive diffusion, a molecule simply dissolves in the lipid
bilayer, diffuses across it, and then dissolves in the aqueous
solution at the other side of the membrane
hydrophobic
molecules

gases small polar

molecules
 1st mechanism by which small molecules cross lipid bilayers
passive diffusion
No membrane proteins are involved
No external source of energy is required
The net flow of molecules is always down their concentration
gradient: the direction of transport is determined only by the
relative concentrations of the molecule inside and outside of the cell

Concentration gradient of Concentration gradient of

Direction of transport of Direction of transport of

Transport No transport Transport No transport

(Passive diffusion) (Equilibrium) (Passive diffusion) (Equilibrium)
2nd mechanism by which small molecules cross lipid bilayers
facilitated diffusion (passive transport)
Large polar uncharged molecules (amino acids, nucleotides and
sugars) and charged molecules (ions) are unable to dissolve in
the lipid bilayer
During facilitated diffusion (passive transport), the passage of
polar and charged molecules is mediated by proteins that enable
the transported molecules to cross the membrane without
directly interacting with its hydrophobic interior
Membrane transport proteins

charged large polar

molecules molecules
 2nd mechanism by which small molecules cross lipid bilayers
facilitated diffusion (passive transport)
Membrane transport proteins are involved
No external source of energy is required
The net flow of molecules is always down their concentration
gradient: the direction of transport is determined only by the
relative concentrations of the molecule inside and outside of the cell

Concentration gradient of Concentration gradient of

Direction of transport of Direction of transport of

Passive transport No transport Passive transport No transport

(Facilitated diffusion) (Equilibrium) (Facilitated diffusion) (Equilibrium)
 3rd mechanism by which small molecules cross lipid bilayers
active transport
Membrane transport proteins are involved
External source of energy is required
The net flow of molecules is against their concentration gradient:
energy provided by another coupled reaction (the hydrolysis of
ATP) is used to drive the uphill transport of molecules

Concentration gradient of Concentration gradient of

Direction of transport of Direction of transport of
Energy-providing coupled Energy-providing coupled
reaction reaction

Active transport No transport Active transport No transport

 1st class of transport proteins ATP-powered pumps
ATPases that use the energy of ATP hydrolysis to move water-soluble
molecules (large polar molecules and ions) across a membrane
against their concentration gradient
Transport of molecules against a concentration gradient across a
membrane, which requires energy, is coupled to the hydrolysis of
ATP (adenosine triphosphate) to ADP and Pi, which releases energy
Transport of molecules by the ATP-powered pumps is driven by the
hydrolysis of ATP
Concentration
External gradient of
medium
Cell Energy-requiring
reaction
membrane
Cytosol Energy-producing
ADP + Pi reaction
ATP
Rate of transport = 100 103 molecules/s
 2nd class of transport proteins channel proteins (ion channels)
Channel proteins transport specific types of ions down their
concentration gradient
A channel protein forms a hydrophilic pore across the lipid bilayer
through which specific inorganic ions can diffuse
Ion channels can exist in either an open or a closed conformation and
transport only in the open conformation
Multiple ions move simultaneously through the ion channel

External conformational
change
medium
Cell
membrane
Cytosol
closed open
Concentration conformation conformation
gradient of
Rate of transport = 107 108 molecules/s
 3rd class of transport proteins carrier proteins (transporters)
Carrier proteins bind a water-soluble molecule on one side of the
membrane and deliver it to the other side through a change in the
conformation of the carrier protein
In contrast to channel proteins, carrier proteins (transporters) bind
only one (or a few) substrate molecules at a time

conformational
change
External
medium
Cell
membrane
Cytosol

Concentration
gradient of Rate of transport = 102 104 molecules/s
 The 1st group of transporters (carrier proteins) - uniporters
Transport a single type of molecule down its concentration
gradient
This type of transporter moves glucose or amino acids across
the plasma membrane into mammalian cells

conformational
change
External
medium
Cell
membrane
Cytosol

Concentration
gradient of
 The 2nd group of transporters (carrier proteins) - symporters
Couple the movement of one type ion or polar molecule against its
concentration gradient to the movement of a different ion or polar
molecule down its concentration gradient
Symporter moves both ions or polar molecules in the same direction
across the membrane
Like ATP-powered pumps, symporters mediate coupled reactions in
which an energetically unfavorable reaction (transport of ) is
coupled to and is driven by, an energetically favorable reaction
(transport of )
conformational
External change
medium
Cell
membrane
Cytosol
Concentration
gradient of
Concentration
gradient of
 The 3rd group of transporters (carrier proteins) - antiporters
Couple the movement of one type ion or polar molecule against its
concentration gradient to the movement of a different ion or polar
molecule down its concentration gradient
Antiporter moves both ions or polar molecules in opposite directions
across the membrane
Like ATP-powered pumps, antiporters mediate coupled reactions in
which an energetically unfavorable reaction (transport of ) is
coupled to and is driven by, an energetically favorable reaction
(transport of )
conformational
External change
medium
Cell
membrane
Cytosol
Concentration
gradient of
Concentration
gradient of
 Symporters and antiporters are called coupled transporters
or cotransporters
conformational
External change
medium

Symporter
Cytosol

conformational
change

Antiporter

Concentration
gradient of
Concentration
gradient of
 Na+/K+ ATPase maintains the intracellular Na+ and K+
concentrations in mammalian cells
Na+/K+ ATPase is responsible for the coupled movement of K+ and
Na+ into and out of the cell, respectively
Hydrolysis of one molecule of ATP to ADP and Pi is coupled to
export of three Na+ ions (blue circles) and import of two K+ ions
(red triangles) against their concentration gradients across the
plasma membrane

3 Na+
External Na+
medium
Plasma
membrane
Cytosol
K+ ADP
2 K+
+ Pi
ATP
 Mechanism of action of the Na+/K+ ATPase in the plasma membrane
1st step: binding of 3 Na+ ions
In its E1 conformation, the Na+/K+ ATPase has three high-affinity
Na+-binding sites and two low-affinity K+-binding sites on the
cytosolic-facing surface of the protein
Due to the high affinity of E1 conformation to Na+, 3 Na+ ions bind to
the Na+-binding sites despite the low intracellular Na+ concentration
Despite the high intracellular K+ concentration, K+ ions are unable to
bind to the low-affinity K+-binding sites
External Na+
medium
Plasma E1
membrane
Cytosol
ATP site K+

High-affinity Na+-binding sites Low-affinity K+-binding sites

 Mechanism of action of the Na+/K+ ATPase in the plasma membrane
2nd step: binding of ATP, phosphorylation of aspartate
ATP binds to its site on the cytosolic surface
The bound ATP is hydrolyzed to ADP and the liberated phosphate is
transferred to a specific aspartate residue in the Na+/K+ ATPase,
forming a high-energy acyl phosphate bond, denoted by E1~P

External Na+
medium
Plasma E1~P
membrane
Cytosol
P
K+
ADP
ATP
 Mechanism of action of the Na+/K+ ATPase in the plasma membrane
3rd step: E1~P E2~P conformational change, outward
transport of 3 Na+ ions
The protein changes its conformation to E2~P
During the E1~P E2~P transition, the 3 bound Na+ ions move
outward through the protein
Transition to the E2~P conformation also generates two high-affinity
K+ sites and three low-affinity Na+ sites on the extracellular face

Low-affinity Na+-binding sites High-affinity K+-binding sites

External Na+
medium
Plasma
membrane E2~P
Cytosol
P
K+
 Mechanism of action of the Na+/K+ ATPase in the plasma membrane
4th step: dissociation of 3 Na+ ions, binding of 2 K+ ions
The 3 Na+ ions, transported outward through the protein, dissociate
into the extracellular medium from the low-affinity Na+ sites despite
the high extracellular Na+ concentration
Due to the high affinity of E2~P conformation to K+, 2 K+ ions bind to
the K+-binding sites despite the low extracellular K+ concentration

External Na+
medium
Plasma
membrane E2~P
Cytosol
P
K+
 Mechanism of action of the Na+/K+ ATPase in the plasma membrane
5th step: hydrolysis of aspartyl phosphate, E2~P E1
conformational change
The aspartyl-phosphate bond in E2~P is hydrolyzed, causing E2~P to
revert to E1
During the E2~P E1 transition, the two bound K+ ions move inward
through the protein

External Na+
medium
Plasma
membrane E1
Cytosol
K+
Pi
 Mechanism of action of the Na+/K+ ATPase in the plasma membrane
6th step: dissociation of 2 K+ ions
The two K+ ions, transported inward through the protein, dissociate
into the cytosol from the low-affinity K+ sites on the cytosolic surface
despite the high intracellular K+ concentration

External Na+
medium
Plasma
membrane E1
Cytosol
K+
 1st property of ion channels ion selectivity
Ion channels are highly selective because narrow pores in the channel
restrict passage to ions of the appropriate size and charge

K+-H2O K+-H2O

Na+-H2O

Ion selectivity of Na+ channels:

A narrow pore permits the passage of Na+ bound to a single water
molecule but interferes with the passage of K+ or larger ions
 2nd property of ion channels gating
Ion channels are not permanently open
Ion channels are gated: they can switch between an open
and a closed state by a change in conformation
This transition is regulated by gates that transiently open
in response to specific stimuli
conformational change in
response to specific stimuli

External
medium
Cell
membrane
Cytosol
closed open
Concentration conformation conformation
gradient of
GATE
 Three types of gated ion channels:
(1) voltage-gated open in response to changes in electric
potential across the plasma membrane
(2) ligand-gated open in response to the binding of a chemical
ligand to the channel, outside or inside the cell
(3) stress-activated open in response to a mechanical force
applied to the channel conformational change in
response to specific stimuli

External
medium
Cell
membrane
Cytosol
closed open
conformation conformation

GATE
 Medically important ligand-gated ion channel:
Cl- channel in epithelial cells,
a.k.a. CFTR (cystic fibrosis transmembrane conductance regulator)

The CFTR Cl- channel is unusual in that it requires both

ATP hydrolysis and phosphorylation in order to open

ATP ATP
Cl- open
closed ADP + Pi ADP
conformation conformation
External P
medium
Plasma membrane
of an epithelial cell
Cytosol

Cl-
Recessive mutations in the CFTR gene cause Cystic Fibrosis:

The most common lethal inherited disease of Caucasians,

which is rare in other races
Incidence of the disease: 1 in 2500 newborns
The characteristic dysfunction: the production of thick sticky
mucus by several types of epithelial cells, including the cells
lining the respiratory and gastrointestinal tracts
The primary clinical manifestation: respiratory disease
Diagnostics: the presence of excessive salt in sweat
Cystic fibrosis is fatal, with lung disease being responsible
for 95% of mortality