Transport Mechanisms
Alvin Shrier
alvin.shrier@mcgill.ca
School of Biomedical Sciences
Anatomy & Cell
Biology
Biochemistry
Biomedical
Engineering
Goodman
Cancer Institute
Human Genetics
Dahdaleh Institute
for Genomics
Medicine
Microbiology &
Immunology
Pharmacology &
Therapeutics
Physiology
McGill Biomedical Research
Accelerator (MBRA)
•
•
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$7,500 Summer Studentship
15 students selected
15-week internship
Labs in BIOC, PHAR, BME,
MIMM, GCI, Lady Davies
Institute, CRBS, MRM
• Weekly seminars
• Final Sumposium,
Milieu intérieur or Internal Environment was used by
Claude Bernard in 1854 to describe the role of the body in
maintaining the uniformity of the conditions of life. So that
the external variations are at each instant compensated
for and equilibrated.
Homeostasis, from the Greek words for "same" and "steady,"
Refers to any process that living things use to actively maintain
fairly stable conditions necessary for survival. The term was
coined in 1930 by the physician Walter Cannon.
In order to preserve the constancy of the Milieu Intérieur
and the homeostasis we need to exchange nutrients, salts,
gases, and waste in and out of the body.
Exchange between Compartments
lungs
Interstitial Fluid
GI tract
plasma
ISF
skin
kidneys
capillary wall
cell membrane
(plasma membrane)
Intracellular Fluid
ICF
H2O-60% body wt
ICF 28L, 11L ISF, plasma 3L
Permeability Characteristics of Cell Membrane
Highly Permeable to:
H2O
Lipid-soluble substances
Dissolved Gases (O2, CO2)
Small uncharged molecules
Less Permeable to:
Larger molecules
Charged particles
Impermeable to :
Very Large molecules
Cell Membrane
Bimolecular Phospholipid Layer
( Phosholipid Bilayer)
polar
Cell (plasma) membrane
6-10nM thick
nonpolar
Amphipathic
Polar and Nonpolar ends
Phospholipids comprise 40-50%
of the plasma membrane by weight.
Cholesterol, inserted into
the phospholipid bilayer
functions as a buffer
preventing lower
temperatures from
inhibiting fluidity and
higher temperatures
increasing fluidity too
much. Cholesterol is also
involved in the formation
of vesicles that pinch off
the plasma membrane
and in lipid rafts. Slightly
amphipatic.
Integral
Proteins: Most diverse
macromolecules, 25-75%
membrane by weight
Integral – closely associated
with phospholipids, mostly
cross the membrane (Transmembrane)
Peripheral – more loosely
associated, mostly on the
cytoplasmic side
Peripheral
Glycocalyx is a fuzzy
coating that sourrounds
the cell membrane formed
of glycans, glycoproteins
and glycolipids. The
glycocalyx contributes to
cell-cell recognition,
communication, adhesion
and protection. It helps to
control vascular
pemeability.
glycoproteins, and glycolipids.
Fluid Mosaic Model of Cell Membrane
Amino acid transport
Na-K pump
G-protein receptor
Insulin receptor
ACh receptor
Channels and
Transporters
Actin
Microtubules
Septins
CD4 T Lymphocytes
CD45 Leucocytes
CD68 Monocytes
CAMs
Cadherins
Integrins
Transmembrane Pathways
a. via phospholipid bilayer
Channel
b. via interaction with a
transmembrane protein
Carrier
Exchanger
Transporter
Cell Membrane Transport Mechanisms
PASSIVE
ACTIVE
(energy independent)
(energy dependent)
1. Diffusion
2. Facilitated Diffusion
3. Osmosis
1. Active Transport
a) Primary
b) Secondary
2. Pino/Phagocytosis
Diffusion
Simple diffusion is the movement molecules from one
location to another as a result of random thermal motion.
Lump of
sugar
Sugar
molecules
Flux is the amount of particles crossing a surface per unit time
Net flux is from high concentration to lower concentration.
At equilibrium, diffusion fluxes are equal and net flux is zero.
Diffusion occurs even in the presence of a
mechanical partition (membrane) as long as it is
permeable to diffusing particles
At equilibrium, net movement = 0
Equilibrium
Gradient
Fig. 04.02
Fig. 04.03
Concentration C
Co = extracellular concentration
Ci = Co
Ci = intracellular concentration
0
0
Time
Consider the case of a single cell perfused extracellularly with a physiological
solution. At time 0 the physiological solution is changed by adding a molecule
at a concentration C0 ,which remains constant. The intracellular concentration Ci
is zero at time 0 and increases over time until Ci = C0.
FICK’s LAW of DIFFUSION
J = PA (C0 – Ci)
J
Flux: moles of solute crossing a unit
area per unit time - rate of diffusion
P
Permeability (or diffusion) coefficient:
a constant based on the ease with which
a molecule moves through a membrane
A
Surface area of the membrane
Co – Ci
Concentration gradient of the diffusing
molecule across the membrane
Diffusion time increases in proportion to the
square of the distance travelled by the solute
molecules. Diffusion is an effective transport
process only over short distances. (Einstein's
approximation equation)
Time for Glucose
Diffusion
1 um =
1 msec
10 um =
100 msec
100 um = 10,000 msec
Cell Membrane Diffusion
1. Mass of the molecule
2. Concentration gradient
3. Lipid Solubility
4. Electrical charge
5. Ion channels
6. Membrane carriers
Diffusion
a) Diiffusion of non-polar molecules and gases
across the lipid bilayer
b) Diffusion of ions through channels
Diffusion is related to the concentration gradient
Ion Channels
Ion channels are transmembrane proteins that
show ion selectivity
The movement of ions is also affected by the
presence of an electrical gradient. The
simultaneous existence of an electrical and a
concentration gradient for a particular ion is
known as an electrochemical gradient.
K+
Intracellular
milieu
K+
K+
K+
K+ Concentration
Gradient
K+
K
+
K+ K+ K+ +
+
+
K+ K K K
-
K+
K+
Extracellular
milieu
K+
- 90 mV
Electrical Gradient
0 mV
The simultaneous existence of an electrical and a concentration
gradient for a particular ion is known as the electrochemical gradient.
Ion channels can exist in
open or closed state as
they undergo
conformational changes.
This is known as gating.
Channels may be gated
in 3 ways:
a. Ligand-gated
Cooper
b. Voltage-gated
Hanrahan,
Lukacs Shrier,
c. Mechanically-gated Sharif
Voltage Gated Ion Channels
Na+ channels
K+ channels
Ca+ channels
Cl- channels
Current flow through single ion channels depends upon:
1. Channel conductance
2. Channel open time
3. Frequency of channel opening
Patch Clamp Experiment
Voltage step
Evoked single
K+ channels
Averaged Evoked
Single K+ Channels
Whole cell K+
Current
Mediated Transport
Mediated transport is the movement of ions and other molecules
(glucose, amino acids) by integral membrane proteins called
transporters or carriers.
Ion movement across membranes via transporters is much
slower than through ion channels.
1. Facilitated Diffusion (Passive)
2. Active Transport
1. Primary Active Transport
2. Secondary Active Transport
Characteristics of Mediated Transport
a. Specificity – system usually transports one particular
type of molecule only
b. Saturation – rate of transport
reaches a maximum when all
binding sites on all transporters
are occupied. Thus, a limit – the
transport maximum (Tm) – exists
for a given substance across a
given membrane
c. Competition – occurs
when structurally similar
substances compete for the
same binding site on a
membrane carrier
Tm
Factors that Determine Mediated Transport
1. Solute concentration
2. Affinity of transporter for the solute
3. Numbers of transporters
4. Rate of transporter conformational change
Mediated Transport Systems
1. Facilitated Diffusion
2. Active Transport
a. Primary Active Transport
b. Secondary Active Transport
Faciltated Diffusion
FACILITATED DIFFUSION involves the presence
of a “transporter” or “carrier” molecule, which
enables a solute to penetrate more readily than it
would be expected to by simple diffusion.
a) Solute binds transporter
b) Transporter changes
configuration
c) Solute is delivered to
other side of membrane
d) Transporter resumes
original configuration
Facilitated Diffusion
- transporter (carrier) mediated
- passive (no-energy)
- net flux from high to low concentration
Hormones may increase the number and/or
affinity of transporters in some membranes
Glut-4 transports glucose in muscle that is
increased by insulin
Active Transport
1. Transporter-mediated
2. Requires supply of chemical energy (usually
derived form enzymatic hydrolysis of ATP)
3. Susceptible to metabolic inhibitors
4. Can transport solute against its concentration
gradient (i.e., “uphill transport”)
Primary Active Transport
Active transport involves the hydrolysis of ATP by a
transporter (carrier).
Phosphorylation of the transporter changes the
conformation of the transporter and its solute
binding affinity.
Na+/K+-ATPase
Na+/K+-ATPase
phosphorylation
dephosphorylation
Changes in the binding site affinity for a transported solute
are produced by phosphorylation and dephosphorylation of
the Na+/K+-ATPase.
Other active transporters
Ca2+-ATPase
Maintain low intracellular Ca2+ levels
H+-ATPase
Maintain low lysosomal pH
H+/ K+- ATPase
Acidification of the stomach
Secondary Active Transport
In secondary active transport the movement of Na+
down its concentration gradient is coupled to the
transport of another solute molecule (ion, glucose,
amino acid) uphill against its concentration
gradient. Secondary active transport uses the
energy stored of the electrochemical gradient to
move both the Na+ and the transported solute. The
creation and maintenance of the electrochemical
gradient depends on primary active transport.
1. Na+ binds to a transporter outside the cell (where the
Na+ concentration is high) allowing glucose or amino acid
to bind to the same carrier.
2. Through a change in configuration, the transporter
“delivers” both Na+ and glucose or amino acid into the cell.
3. The transporter then reverts to its original
configuration, and the Na+ is extruded from the cell by the
Na+/K+ -ATPase.
Secondary Active Transport Mechanisms
Symport or Cotransport is
when solute X is transported
in the same direction as Na+.
Antiport, Countertransport or
Exchange is when solute X is
transported in the opposite
direction to Na+.
Symport
Antiport
Na+/HCO3− cotransporter
(NBC)
Na+/H+ exchanger
(NHE)
Na+/amino acid cotransporter
(NAcT)
Na+/Ca2+ exchanger
(NCX)
Na+/glucose cotransporter
(SGLT)
Summary of Transport Mechanisms
Fig. 04.15
Endocytosis and Exocytosis
Active transport mechanisms (energy-dependent)
involving participation of the cell membrane itself.
Endocytosis – the cell membrane invaginates and
pinches off to form a vesicle.
Exocytosis – an intracellular vesicle fuses with the cell
membrane, and its contents are released into the ECF.
Endocytosis
Exocytosis
Exocytosis
Excocytosis is the process of moving material from the inside
to the outside of the cell.
Types:
1) Constituitive Exocytosis
Non-regulated. Functions to replace plasma membrane, deliver
membrane proteins to the cell membrane and to get rid of substances
from the cell.
2) Regulated Exocytosis
Tends to triggered by extracellular signals and the increase of
cytosolic Ca2+. Responsible for the secretion of hormones, digestive
enzymes, and neurotransmitters.
Endocytosis
1. Pinocytosis also known as fluid endocytosis, involves an
endocytotic vesicle that engulfs the extracellular fluid including
whatever solutes are present. It is nonspecific and constituitive.
The vesicles travel into the cytoplasm and fuse with other vesicles
such as endosomes or lysosomes.
2.Phagocytosis is the process by which cells bind and internalize
particulate matter (>0.75 µm) such as small-sized dust particles,
cell debris and microorganiisms. It is specific and triggered.
Extensions of the cell membrane called pseudopodia fold around
the particle and fully engulf it. The pseudopodia fuse to form large
vesicles, called phagosomes, that pinch off the membrane.
Phagosomes migrate to and fuse with lysosomes where the
contents of the phagosome are degraded. Defend against
infection and scavenge senescent and dead cells.
Nonspecific
uptake of
solutes
and H2O
Nucleus
Ligand
Receptor
Nucleus
Vesicle
Solutes
Golgi
apparatus
Clathrin proteins
forming a
Unbound
clathrinligand
coated pit
Plasma
membrane
Vesicle
Lysosome
Extracellular fluid
Clathrin proteins
being released
from vesicle
(a) Fluid endocytosis
Bacterium
Lysosome
Nucleus
Receptor
Endosome
Cytosol
Receptor recycled
to membrane
Vesicle
formation
Pseudopodia
(c) Receptor-mediated endocytosis
Phagosome
Extracellular fluid
(b) Phagocytosis
Involves macrophages, neutrophils and dendrtitic cells
Receptor-mediated Endocytosis
In receptor-mediated endocyosis molecules in the extrtacellular
fluid (ligands) bind with high affinity to specific protein receptors
on the plasma membrane.
a. Clathrin-dependent receptor-mediated endocytosis.
When the ligand binds the receptor undergoes conformational
change and clathrin is recruited to the plasma membrane.
Adaptor proteins link the ligand-receptor to the clathrin. The
complex forms a cagelike structure that leads to the
aggregation of ligand bound receptors. A clathrin coated pit is
formed which then invaginates and forms a “clathrin-coated
vesicle”. Once the vesicle pinches off it sheds the clathrin coat
and vesicles can fuse with the membrane of cellular organelles
such as endosomes and lysosomes. Or, they can sometimes
fuse with the membrane on another side of the cell
(transcytosis) . Receptors and clathrin protein are recycled
back to the cell membrane. An example is the LDL receptor.
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Nonspecific
uptake of
solutes
and H2O
Nucleus
Cholesterol is transported in the
Solutes as lipid-proteinVesicle
blood
particles
known as low-density lipoproteins
Plasma
membrane
(LDL). The lipoprotein
is recognized
by PM LDL receptors and
Extracellular fluid
endocytosis follows.
(a) Fluid endocytosis
Bacterium
Lysosome
Nucleus
Ligand
Golgi
apparatus
Receptor
Nucleus
Clathrin proteins
forming a
Unbound
clathrinligand
coated pit
Vesicle
Lysosome
Clathrin proteins
being released
from vesicle
Receptor
Endosome
Cytosol
Receptor recycled
to membrane
Vesicle
formation
Pseudopodia
(c) Receptor-mediated endocytosis
Phagosome
Extracellular fluid
(b) Phagocytosis
b. Potocytosis is the process by which molecules are
sequestered and transported by tiny vesicles called caveolae.
These vesicles are clathrin-independent. Caveolae can deliver
their contents directly into the cell cytoplasm as well as to the
endoplasmic reticulum (ER) or other organelles and to the
plasma membrane on the opposite side of the cell
(transcytosis). Potocytosis has been implicated in uptake of
low molecular wieght molecules such as vitamins.
DIFFUSION of WATER
Water diffuses freely across most cell membranes
This is facilitated by groups of proteins (aquaporins)
that form water permeable channels (Peter Agre)
Osmosis
Osmosis – the net diffusion of H2O across a
semipermeable membrane (i.e., permeable to
solvent, but not to all solute)
1L
2M
Glucose
53.5 M
H2O
1L
55.5 M
H2O
M.W. H2O = 18g/mol
1 L H2O = 1000 g
1000/18 = 55.5 M
Osmosis
53.5 M
H2O
55.5 M
H2O
Osmotic Pressure
The pressure required to prevent the movement of water
across a semi-permeable membrane is referred to as the
osmotic pressure. This pressure is equal to the difference
in the hydrostatic pressures of the two solutions.
In an ideal solution, the osmotic pressure is related to
temperature in the same way as the pressure of a gas
P = nRT/V
Van’t Hoff Equation for osmotic pressure
n= # of particles, R = gas constant, T = abs., temp, V =
volume
Thus the osmotic pressure is proportional to the number
of particles in solution/unit volume and not to their size,
configuration or charge.
Osmolarity
Osmolarity (Osm) is the total solute concentration of a
solution.
1 osmol = 1 mol of solute particles
1 mol glucose = 1 osmol of solute
1 mol NaCl
= 1 mol Na+ + 1 mol Cl- = 2 osmol
1 mol MgCl2 = 1 mol Mg2+ + 2 mol Cl- = 3 osmol
Osm = osmol/liter
1 mol glucose/L = 1 osmol/L = 1 Osm
1 mol NaCl/L
= 2 osmol/L = 2 Osm
1 mol MgCl2/L = 3 osmol/L = 3 Osm
Osmotic pressure is proportional to osmolarity (Osm)
Osmotic Pressure of Physiological Saline
1.Determine Molarity
0.9% saline = 0.9 NaCl /100g H2O
= 9.0 g NaCl/L H2O (L=1000g)
Moles = 9g NaCl x 1 mole /58.5 g NaCl = 0.15 moles NaCl
Molarity = moles/L = 0.15 moles NaCl/L = 0.15 M NaCl solution
2. Determine Osmolarity
0.15 M NaCl solution
= 0.15 moles Na+ + 0.15 moles Cl= 0.15 osmol + 0.15 osmol = 0.30 osmol
= 0.30 osmol/L H2O = 0.30 Osm = 300 mOsm
3. Calculate Osmotic Pressure
Osmotic pressure = 0.30 Osm x 22.4 atm/Osm = 6.7 atm
= 6.7 atm x 760 mmHg/atm = 5092 mm Hg
When ISF is 3 mOsm> ICF osmotic pressure gradient is 50.92 mm Hg
Osmolarity
Solutions which have the same osmolarity (concentration
of osmotically active particles) as normal extracellular (or
intracellular) solution (300 mOsm) are called ISOSMOTIC
Solutions which have an osmolarity lower than 300 mOsm
are called HYPOOSMOTIC
Solutions which have an osmolarity greater than 300
mOsm are called HYPEROSMOTIC
To be effective in exerting a sustained osmotic
pressure, particles must not be able to cross the
membrane and are referred to as nonpenetrating.
Extracellular Na+ behaves as a nonpenetrating solute
because the Na+ that moves into the cell is pumped out
by the Na-K ATPase.
Tonicity
a. If the solution has a concentration of 300 mOsm of
nonpenetrating solute particles, there will be no net shift
of water. ISOTONIC solution
b. If the solution has a concentration of nonpenetrating
solute particles less than 300 mOsm, water will enter the
cell and the cell will swell. HYPOTONIC solution
c. If the solution has a concentration of nonpenetrating
solute particles greater than 300 mOsm, water will leave
the cell and the cell will shrink. HYPERTONIC solution
Intracellular
Fluid (ICF)
Extracellular
Fluid (ECF)
Normal cell
20 mM urea
140 mM NaCl
Initial
penetrating
nonpenetrating
20 mOsm urea
280 mOsm Na+ + Cl300 mOsm
300 mOsm
Isoosmotic
penetrating
20 mOsm urea
Equilibrium/
300 mOsm
Final
280 mOsm
Cell Swelling
Total
300 mOsm
20 mOsm urea
280 mOsm Na+ + ClH 2O
Hypotonic
300 mOsm
CONDITION
ECF
Osm.
ECF Vol. ICF Osm. ICF Vol
Excessive
H2O intake
iv infusion of
0.9% NaCl
=
=
=
Hemorrhage
=
=
=
Drinking sea
water
Severe
sweating
Capillaries
• An adult has ~40 km of capillaries
• Capillaries contain ~5% of total circulating blood
• Each capillary is ~1mm long, inner diameter ~8 µm
CAPILLARY WALL
A single layer of flattened endothelial cells and a supporting
basement membrane.
CAPILLARY STRUCTURE and PERMEABILITY
4. Bulk flow
2.
1.
3.
TRANSPORT ACROSS CAPILLARY WALL
1, 2- DIFFUSION through across cell membrane is the most
important means of transport. Diffusion also occurs through
water filled channels (intercellular clefts and fused vesical
channels).
3. Transcytosis– Endocytosis on the luminal side followed
migration of the vesicle across the cell and then exocytosis
on the interstitial side.
4. BULK FLOW distributes the extracellular fluid volume
between the plamsa and ISF. Magnitude of bulk flow is
proportional to the hydrostatic pressure difference between
the plasma and the ISF. Caplillary wall acts as a filter that
permits protein free plasma to move from caplillaries to the
ISF.
Department of Physiology
Good luck!