Module 0220502
Membrane Biogenesis and Transport
Lecture 16
Water Transport,
and Transport across Epithelia
Dale Sanders
16 March 2009
Aims:
By the end of the lecture you should
understand…
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
•

•
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•
How water moves through bilayers by solubility in
lipid and through aquaporins;
How nutrients move across epithelia;
How absorptive epithelia move water across
themselves;
How secretory epithelia move water across
themselves.
Reading
Lodish et al. (2008) Molecular Cell Biology 6th ed pp. 444-446
& 470-473
A good introduction to aquaporins and epithelial transport
Fujiyoshi et al. (2002) Structure and function of water channels.
Curr. Opin. Struct. Biol. 12: 509-515
Murata et al (2000) Structural determinants of water permeation
through aquaporin-1. Nature 407: 599-605
Water Transport
Osmosis is rapid, yet membranes have hydrophobic interior.
How do we reconcile these observations??
H-bonding props of water allow significant solubility in hydrophobic
bilayer:
Passive permeabilities of solutes through lipid bilayers can be
measured in an artificial system. E.g. for water
Teflon chamber
1H O
2
3H O
2
Lipid bilayer
Measure radioactivity appearing on trans side
Results
codeine
slope = 1
3
butyric acid
log (diffusional
permeability
coefficient)
2
H2O
H2O “corrected” for high diffusion
coefficient (small molecule)
1,2 propanediol
1
formamide
1,4 butanediol
acetamide
1,2 ethanediol
-1
-1
inorganic
ions
3
4
log (oil:water partition coefficient)
urea
glycerol
For water, diffusive, passive permeability measured with 3H2O
(Pw) 2.10-5 cm.s –1 – not very different from value expected on
basis of oil:water partition coefficient
For many biological membranes, Pw has the value observed in
bilayers:
Conclude: solvation then diffusion of water through the lipid phase is
the primary mechanism of transport
HOWEVER in many other membranes,
water ALSO crosses through specific
WATER CHANNELS
Evidence
1. Physical/chemical
A 2nd way to measure water permeability:
impose an osmotic gradient
H2O
= solute molecule
If water diffusion through lipid, “osmotic permeability” for H2O (Lp)
should = Pw
in some cells, it doesn’t e.g.
Cell type
+
Hg2+
Lp
Pw
10-5 cm s-1
Red blood cell
+
Kidney prox. tubule
+
20
1.8
40
3.2
• >10x discrepancy between Lp and Pw
• Discrepancy largely abolished by Hg2+
2.0
1.8
2.0
1.0
Hypothesis to explain discrepancy
A single file, multiple-occupancy water channel:
H2O
Lp is NET flow: 1 molecule entering on left knocks out 1 molecule
on right.
Pw is unidirectional flow: 3H2O entering on left could diffuse back
and has many others to pass in pore – slows down the passage of
radiotracer
2. Molecular/Biochemical evidence
AQUAPORINS: members of the Major Intrinsic
Protein family
MIP: lens epithelium
TIP: plant tonoplast
CHIP: Channel-forming intrinsic protein (red blood
cells)
Mr = 28,000 – A homotetramer with each monomer
forming a channel
Evidence that CHIP and TIP are H2O
channels:
Inject mRNA into Xenopus oocytes and look at
swelling rate in response to hypo-osmotic solutions
• Swelling response also blocked by Hg2+
CHIP-expressing
1.6
Relativevolume
Control
1.4
30 s
1 min
TIP
CHIP
1.2
2 min
3 min
Water control
1.0
0 1 2 3 4 5
t (min) in hypotonic solution
Chrispeels & Agre (1994) Trend. Biochem. Sci. 19: 421-425
The Hourglass Model
repeat 1
repeat 2
NPA
1 2
NPA
3
4
5
6
C
N
Connecting loop B
Connecting loop E
Connecting loops B & E dip into membrane from
opposite sides to form aqueous pathway
Aquaporin monomer
End-on view from extracellular side
• 2-fold symmetry
around H1-3/H4-6
• Insertion of
connecting loops
B & E into
structure
Murata et al (2000)
Nature 407: 599605
Aquaporin monomer
Side view
Connecting
loops B & E
entering
from
opposite
sides
Murata et al (2000)
Nature 407: 599605
Limits of
membrane
Space-filling models of the
pore region of the
aquaporin monomer
•Large number of hydrophobic
residues (yellow) line the
pore
•Asn76 and Asn192 (red) are in
the NPA motifs: contribute
to the tight constriction and
H-bond with H2O
•Cys189 (green) is site of Hg
binding
Murata et al (2000)
Nature 407: 599605
Transport Across Epithelia
So far, we have considered cells as spatially uniform for transport:
S
S
S
S
A good model for most cells.
But an important exception:
Epithelial cells: asymmetry
with respect to transport
because they must transport
across the tissue.
http://cellbio.utmb.edu/microanatomy/epithelia/00004493.jpg
Generalized representation of epithelial cells and
some definitions
Mucosal
(lumenal)
side
Blood
(serosal)
side
Intercellular
space
Junction
(tight or leaky)
Brush border
(apical) membrane
Basolateral
membrane
Epithelia can be broadly classified as either
1. Absorptive: Take up nutrients, salts, water
from mucosal side and deposit them on
serosal side
2. Secretory: Transport water to mucosal
side
Transepithelial transport involves coordinated activity of pumps, carriers and
channels.
1. Absorptive Epithelia
e.g. Small intestine, kidney tubules
(a) Solute transport, e.g: absorption of glucose in small intestine
M (BB)
S (BL)
–
Na+
Na+
ATP
glucose
K+
+
ouabain
glucose
phlorizin
cytochalasin
Relative
electrochemical
Activities:
Na+
glucose
K+
BB membrane
M
Intracellular
BL membrane
S
A similar picture for other solutes eg amino acids
Evidence for this model:
1.
Net transport of Na+ and glucose from M S implies
charge translocation:
A trans-epithelial potential difference of about -10 mV,
mucosa negative, is measured…
Sensitive to inhibition of (Na+ + K+)-ATPase by ouabain
2.
BB and BL membranes can be fractionated:
Na+ - coupled solute transport only at BB membrane,
ATPase activity only BL membrane
3.
Selective use of inhibitors: they inhibit glucose transport
in a side-specific manner.
(b) Absorption of water by epithelia:
Small intestine, large intestine, kidney (proximal and distal tubules
and collecting duct)
eg kidney, collecting tubule
peritubular space
lumen
–
BB
Cl–
H2O
Na+
Na+, K+, 2Cl–
K+
+
H2O
ATP
H2O Na+
K+
Na+
K+
BL
Cl–
Cl–
collecting
tubule
proximal
tubule
distal
tubule
loop
of
Henle
1.
(Na+ - K+)- ATPase generates electrochemical potential
difference for Na+ across BL membrane
2.
Na+ moves passively across BB membrane, and also drives
uptake of K+ and Cl- from lumen
3.
K+ channels at both membranes allow recirculation of K+ and
control Δ.
4.
Trans-epithelial Na+ transport sets up trans-epithelial Δ,
which drives paracellular Cl- transport into cleft.
5.
Cl- channels in BL membrane release Cl- on serosal side.
6.
H2O absorbed from lumen in response to osmotic gradient
generated by NaCl, especially in cleft.
Note that the whole mechanism of H2O transport driven ultimately
by the (Na+/K+) – ATPase: sets up the ionic gradients.
2. Secretory Epithelia
eg salivary glands, sweat glands, lacrimal glands, exocrine
pancreas, gastric mucosa, tracheal epithelium
lumen
–
serosa
BL
Na+, K+, 2Cl-
Na+
H2O
Secretagogues
H2O
Cl
–
CFTR
BB
–
Cl
+
cAMP
Ca2+
+
Ca2+
Na+
eg ACh,
catecholamines
ATP
K+
Ca2+-activated K+
channel maintains
intracellular Δ
negative,
sustaining the
driving force for Cl
release
1.
Secretagogues generate  [Ca2+]i through opening of nonspecific cation channels.
2.
Ca2+ activates K+ channels at BL membrane causing K+
release.
3.
K+ reabsorbed by Na+,K+,2Cl carrier at BL membrane, and
by (Na+ + K+)-ATPase, which also removes incoming Na+.
4.
Cl release occurs through cAMP activated Cl channels at
BB membrane, resulting in formation of transepithelial Δ
(lumen-negative)
5.
Na+ moves paracellularly into lumen, driven by Δ.
6.
H2O moves into lumen in response to osmotic gradient, set
up by net movement of NaCI.
-
-
-
Summary
1.
2.
3.
4.
5.
6.
Water moves through bilayer membranes by H bonding in
hydrophobic environment.
In some membranes, aquaporins greatly increase rate of
osmotically driven H20 flow.
Aquaporins are small (28 kDa) polypeptides with
6 t/m spans and NPA motifs on connecting loops
Solutes are moved across absorptive epithelia by Na+coupling in BB membrane and non-coupled transporters
(carriers) in BL membrane.
(Na++K+)-ATPase is exclusively in the BL membrane.
Flow of water across epithelia is driven by NaCI movement,
with the membrane location of the Na+, K+, 2Cl carrier
determining direction of flow.