MEMBRANE TRANSPORT
Bob Mercer
rmercer@wustl.edu
September 19, 2013
POLYCYSTIC RENAL DISEASE
1 in 500 autopsies
1 in 3000 hospital
admissions
Accounts for ≈10% of
end-stage renal failure
Autosomal dominant
inheritance
WILSON’S DISEASE
1 in 100 individuals
carry mutation in
ATP7B gene (CuATPase)
1-4 per 100,000
people
Autosomal recessive
inheritance
Neurological or
psychiatric symptoms
Liver disease
Kaysar-Fleischer (KF) ring
CYSTIC FIBROSIS
1/2000 births in white
Americans
Median age for survival
late 30s
Autosomal recessive
inheritance
THE RELATIVE PERMEABILITY OF A SYNTHETIC LIPID
BILAYER TO DIFFERENT MOLECULES
HYDROPHOBIC
MOLECULES
O2
CO2
N2
benzene
SMALL
UNCHARGED
POLAR
MOLECULES
H2O
urea
glycerol
LARGE
UNCHARGED
POLAR
MOLECULES
glucose
sucrose
IONS
H+, Na +
HCO¯3 , K+
Ca2+, Cl¯
Mg 2+
synthetic
lipid
bilayer
COMPARISON OF ION CONCENTRATIONS INSIDE AND
OUTSIDE A TYPICAL MAMMALIAN CELL
Component
Intracellular
Concentration
(mM)
Extracellular
Concentration
(mM)
5-15
140
0.5
10-4
8 x 10-5 (pH 7.1)
145
5
1-2
1-2
4 x 10-5 (pH 7.4)
Cations
Na
K
Mg
Ca
H
Anions
Cl
5-15
110
Because the cell is electrically neutral the large deficit in intracellular anions reflects the
fact that most cellular constituents are negatively charged. The concentrations for Mg and
Ca are given for free ions.
Tuesday, July 1, 1980
A Cross between Human Beings and Plants . . .
SCIENTISTS ON VERGE OF CREATING
PLANT PEOPLE . . .
Bizarre Creatures Could do Anything You Want
Brain water (g/100 g dry wt)
500
Normal
Died
Asymptomatic
139 baseline
119 in 2 h
122 in 3.5 d
Lethargic
450
400
350
300
99 in 16 d
Plasma Na concentration (mEq/l)
Woman drinks so much water she dies
January 13, 2007
SACRAM ENTO, California (AP) -- A woman who competed in a radio station's
contest to see how much water she could drink without going to the bathroom
died of water intoxication, the coroner's office said Saturday.
Jennifer Strange, 28, w as f ound dead Friday in her suburban Rancho Cordova
home hours after taking part in the "Ho ld Your Wee for a Wii" contest in which
KDN D 107.9 promise d a Nintendo Wii video game system for the winner.
"She said to one of our supervisors that she was on her way home and her
head w as hurting her real bad," said Laura Rios, one of Strange's co-workers
at Rad iological Associates of Sacramento. "She w as crying, and that was the
last that anyone had heard f rom her."
Copyright 2007 The Associated Press. All rights reserved.This material may not
be pub lished, b roadcast, rewritten, or redistributed.
Simple Diffusion
Flux
• Flux is proportional to
external concentration
• Flux never saturates
[S]o
PROTEIN MEDIATED MEMBRANE
TRANSPORT
• PRIMARY ACTIVE
• SECONDARY ACTIVE TRANSPORT
• FACILITATED DIFFUSION
• ENDOCYTOSIS/TRANSCYTOSIS
Membrane Flux (moles of solute/sec)
•
•
•
Simple Diffusion
Carrier Mediated Transport
• Facilitated Diffusion
• Primary Active Transport
• Secondary Active Transport
Ion Channels
TRANSPORT OF MOLECULES THROUGH MEMBRANES
transported molecule
lipid
bilayer
electrochemical
gradient
channelmediated
dif f usion
carriermediated
dif f usion
PA SSIVE TRA NSPORT
(FA CILITATED DIFFUSION)
GY
ER
EN
simple
dif f usion
ACTIV E TRANSPORT
CARRIER MEDIATED TRANSPORT
lipid
bilayer
UNIPORT
SYMPORT OR
COTRANSPORT
ANTIPORT OR
COUNTERTRANSPORT
COUPLED TRANSPORT
Membrane Potential Review
•
•
•
•
•
The lipid bilayer is impermeable to ions and acts like an
electrical capacitor.
Cells express ion channels, as well as pumps and exchangers,
to equalize internal and external osmolarity.
Cells are permeable to K and Cl but nearly impermeable to Na.
Ions that are permeable will flow toward electrochemical
equilibrium as given by the Nernst Equation.
Eion = (60 mV / z) * log ([ion]out / [ion]in) @ 30°C
The Goldman-Hodgkin-Katz equation is used to calculate the
steady-state resting potential in cells with significant relative
permeability to sodium.
 PK  [K]out  PNa  [Na]out  PCl  [Cl]in 

Vm  60mV log
 PK  [K]in  PNa  [Na]in  PCl  [Cl]out 
Structure of a Potassium Channel
Doyle et al., 1998
Carrier-Mediated Transport
• Higher flux than predicted
by solute permeability
• Flux saturates
• Binding is selective
(D- versus L-forms)
• Competition
• Kinetics:
Flux
Mmax
0.5
Km
[S]o
[S]o << Km M a [S]
[S]o = Km M = Mmax / 2
[S]o >> Km M = Mmax
MEMBRANE ION TRANSPORT PROTEINS
Na,K-ATPas e
Ca-ATPase
out
2 major subunits
a Mr = 112 kD
ß Mr - 60 kD
in
out
in
Mr = 110 kD
2K
ADP + Pi
ATP
3Na
inhibited by ouabain,
digoxin, digitalis
ADP + Pi
SR Ca-ATPase inhibited
by thapsigargin,
cyclopiazonic acid
Other P-Type Ion Transport A TPases:
H,K-ATPas e
2 subunits
a Mr = 114 kD
ß Mr - 60-85 kD
out
in
K
ADP + Pi
ATP
H
inhibited by Schering
28080,omeprazole,
lansoprazole
Cu-ATPase
H-ATPase
Cd-ATPase
K-ATPase
FoF1-Type A TPases:
H-ATPase
K-ATPase
ATP
2Ca
EXCHANGERS/COUNTERTRANPORTERS:
Na/H Exchange r
out
Mr = 90 kD
Anion Exchanger
Cl/HCO3 Exchanger
in
out
Na
in
Cl
Mr - 102 kD
inhibited by amiloride
H
inhibited by DIDS, SITS
phenyl isothiocyanate
HCO3
Na/Ca Exchanger
out
Mr = 108 kD
in
3Na
inhibited by dichlorobenzamil, exchanger
inhibitory peptide (XIP)
Ca
COTRANSPORTERS:
Na,K,2Cl Cotrans porte r
out
Mr = 120 kD
in
Na,glucos e
Cotransporter
Na,K
out
2Na
Mr = 73 kD
inhibited by bumetanide
furosemide
2Cl
inhibited by
phlorizin
glucose
in
Transport Kinetics
So + Co
k+
k-
SCo
Si
S = Solute C = Carrier
dSCo/dt = k+ [S]o [C]o – k- [SC]o = 0 at equilibrium
 k+ [S]o [C]o = k- [SC]o
k- / k+ = ([S]o [C]o)/[SC]o = Km
 [SC]o = ([S]o [C]o)/Km
Fractional Rate = M / Mmax = [SC]o / ([C]o + [SC]o)
M = Mmax / (1 + [C]o/[SC]o) = Mmax / (1 + Km/[S]o)
Reversible Transport
Co
Ci
So
Si
SCo
SCi
Mnet = Min – Mout =
Mmax
(
1
1 + Km / [S]o
1
1 + Km / [S]i
)
Facilitated Diffusion
• Uses bidirectional, symmetric carrier proteins
• Flux is always in the directions you expect for simple diffusion
• Binding is equivalent on each side of the membrane
Examples include: Glucose Transporters (GLUT); Anion Exchanger;
Organic Anion Transporters; Urea Transporters; Monocarboxylate
(lactate) Transporters (MCTs); Amino Acid Transporters; Zn Transporters
(ZIP)
Facilitated Diffusion
• Uses bidirectional, symmetric carrier proteins
• Flux is always in the directions you expect for simple
diffusion
• Binding is equivalent on each side of the membrane
Facilitated Diffusion: Band 3/AE1
Facilitated Diffusion: Band 3/AE1
Cytoskeletal/AE1 Interactions
Primary Active Transport: Driven by ATP
• Class P – all have a phosphorylated intermediate
•
•
•
•
•
•
Na,K-ATPase
H,K-ATPase
Ca-ATPase
Cu-ATPase
H-ATPase
bacterial K-ATPase
Phospholipid Flippase
• Class V
•
H+ transport for intracellular organelles
• Class F
•
Synthesize ATP in mitochondria
Primary Active Transport: Na,K-ATPase
3 Na
ATP
ADP + Pi
2K
•
•
•
•
•
•
•
3 Na outward / 2 K inward / 1 ATP
Km values: Nain = 20 mM Kout = 2 mM
Inhibited by digitalis and ouabain
Palytoxin “opens” ion channel
2 subunits, beta and alpha (the pump)
Two major conformations E1 & E2
Turnover = 300 Na+ / sec / pump site @ 37 °C
Na,K-ATPase Reaction Scheme
Extracellular
3Na
2K
E2P.K2
E2P
E1P
E1P.Na3
Pi
E2.(K2)
ATP
E1P.(Na3)
ADP
E2.(K2)ATP
E2.ATP.K2
E2.ATP
2K
Intracellular
3Na
E1.ATP.Na3
E2P.ADP.Na3
Membrane Transport and Cellular Functions that Depend on
the Na,K-ATPase
Amino Acid Homology Among the Na,K-ATPase Subunit Isoforms
K
L T L D T E
I
R
E C K A Y G E N
Y L Q P L L
A V Q F T N
I
L
Y Y
F F G G M G F Y
P Y Y Q L P G
F G
P
Q
L K G Y Y
I
T W D D R W I
R E
N D V E D
S
a
M1
T
A
S
R
G I
L A Y
L C F
I
I G A
L L W 100
M
G F S
L F G
NH 2
G
R
D
K
Y
E
P
A
A
V
S
E
H
G
D
K
K
S
K
K
A
K
K
E
R
D
M
D
E
L
K
K
E
V
S
M
D
D
H
K
E E E P
L S L D
Q
R
C
F
K
V
W
E
P
T
T
P
P
P
T
L
R
Q
P
G
D
R
A
L
I
E
A
P
R
A
P
T
L
G
R
S
L
D
T
G
Y 50
K
R
H
L
E
F
P
N
D
D
L
Y L G
S
V V L
V
M2 A V T G
I I
C F
S
Y
Y
Q
E
A
K
S
S 150
K
I
M
E
S
F
K
N
M
V
P
Q
Q
A
L
V
I
R
N
G
E
K
M
S
I
N
A
E
D
V
V
V
G
D
L
V
E
V K
P
Y T W L
E
I L
L S L
I
300 S F F
G V
M3 L A V F
V
I T G
L
F I H
H
E
I
E
E
A
I
P
T
Q
G
G
E
L
G
S
A
L
T
A
I
R
G
M
V
T
R
D
G
T
Y
V
V
I
G
R 250
A
G E S
E
T
P
L
Q
S
T
S
R
N
S
D
P
V
D
K
F
C
T
G
N
N
E
A
N
S
P
I
L
I 200
E
R
T
L
R
D
T
N
A
G
I
P
E
A
I
V
F
R
C
F
D
N
S T
G
G
E
A V I F
L I G I
I V A N
V P E G
M4
L L A T
V T V C
L T L T A
K
R
M
A
R
K
N 350
C
L
V
K
N
L
E
A
V
E
T
L
G
S
T
S
T
I
C
S
D
K
T
G
T
L
T
Q
N
R
M
T
A
R
P
P
D
P
K H
I
S F R P N D P K S Y
I
I K V
I M V T G D H P
I
T A K A
I
A K G V G
L G V F C L N D V P F N V E D T D F Q F G E P F Q E D P L
500
L L V M K G A P E R
I
P
L K
E
S
L T I
L M V
A N
P N K H
I
Q I
I F I
G T
G A
L C
G
Y V
I F
Y
F L
L I
52
M1

I
D E
K
F
W
S
G
G
T
R
G
L
T
E
N
G
I S E
550
L L H C F G L V R E
NONE
F
E
K
K
E
S
N
W
I
F
K
K
W
S
G
E
E
K
A
K
G
R
A
M
V
3/3 IDENTICAL
2/3 IDENTICAL
NH 2
G
L
G
I
L D R C S S
I
L L H G K E Q P L D E E L
S L Q Y K N T S N F P
I
E V
I
K D A F Q N A Y L E
K T Y K E R M E M V S G C C V E
400
A D T T E N Q S G V S F D K T S A T W F A L S R
I
V L N I
V R D Q Y T
L F G N
E
600
A V P D A V G K C R S A G
E A Y
100
Q T L G P P A
P
E
S
H E
Q
I Q K T E
P L
I M S
V
A
H
M
W
F
D
N
Q
E I
I
Y
E
I K R Q
A
V
R M
P
G
L
Y
L
E
T V 800
I
P L K
F T C
G A A
N I
F V
A
L
M
Y
G
I
I
F
P
T
A
P T W
I
T
C
F
H T
L
F
C I D
W F C
L I F
L G G
F V S
F L S Y
D
A
A
T
Q
A
F
G
V
L
A F P
I
V
I V
M5 P
M6 M7
M8
M10
A M9
T
G M
M V P
E E T
P E I
Q I
Q W A
Y S L
F
S N I
A I S
A Y G 850 D L V
L I F
F G L
M
L
T
A
S
L
Y
I L I 950
V Y D
Y T
I
L I
A
E
K
R
C
E
I
Q
E
K
N
V
S
A
T
K
R 1000
N
K
R
M
K
E
V
K
L
R
G
L
S
L
K
N
Q
D
I
S
N
Q
I
D
I
V F
D
M
T
R
F
K
K
R
I
R
P
R
L
Q
N
P
P R
R
G
G
G
750
E
E V G T V I S A F N D D
W
L
V
L
E
M I
K
V G S D V S K Q A A D
I
E
G
T
M
Y
A V G I D A K K L A P S D N V G D G T V A V I
A
Y COOH
G
700
Q
H T E I V F A R T S P Q Q K L I I V E G C Q R
Y
R
L
650
I
D D L E E S T M D K L D S G H V V C A K A D R P N V Q N V P I N L R A A
L
I
Cytoplasmic
G
Q
Q 900
W
T
L
L
H
COOH
K P C I I I K L
E G K
G Y K
Y
S
E D N L G S C N G L W D L K F R C V K R E G R E H N F E G R E K P E S P M S G C D E F
150
Y
G
E V K S
N G V K D K D E D R K G T C Q V P L V N P
N
Y
N R V L G F K P K P P K N E S L E T Y P L T M K
200
250
Extracellular
I G Y S E K D R F Q G R F D V K I
300
I
L G
450
C K L L A S E S
A G L C N R A V F Q A N Q E N L P
I
L
K R A V
A
D
G
A
4/4 IDENTICAL
3/4 IDENTICAL
2/4 IDENTICAL
NONE
I
I M D D
K
Q
A
R F L E K Y K D S
QuickTime™ and a
Sorenson Video 3 decompressor
are needed to see this picture.
The Na,K-ATPase As a Receptor For Signal Transduction
SR Ca-ATPase
FoF1 ATPase
QuickTime™ and a
H.264 decompressor
are needed to see this picture.
Nat Commun. 2012 February
21; 3: 687
Experimental Evidence for Rotation
Secondary Active Transport
• Energy stored in the Na+ (or H +) gradient is used to
power the transport of a variety of solutes
glucose, amino acids, ions and other molecules are pumped
in (cotransport)
Ca2+ or H+ are pumped out 2 or 3 Na+ / 1 Ca2+ ; 1 Na+ / 1 H+
(countertransport)
• These transport proteins do not hydrolyze ATP directly;
but they work at the expense of the ion gradient which
must be maintained by an ATPase
Secondary Active Transport
• In humans over 40 families of Na coupled transporters
Examples include: Na+/H+ exchanger; Na+/Ca2+
exchanger; Na+/aspartate cotransporter; Na+/amino
acid cotransporter; Na+/glucose cotransporter; Na+/urea
cotransporter; Na+/PO4; cotransporter; (H+/Na+)/Zn2+
exchanger (ZnT)
Secondary Active Transport
• Energy stored in the Na+ gradient is used to power the
transport of a variety of solutes
glucose, amino acids and other molecules are pumped in
(cotransport)
Ca2+ or H+ are pumped out 2 or 3 Na+ / 1 Ca2+ ; 1 Na+ / 1 H+
(countertransport)
• These transport proteins do not hydrolyze ATP directly;
but they work at the expense of the Na+ gradient which
must be maintained by the Na,K-ATPase
Energy available from ATP
H2O
ATP
ADP + Pi
DG = Gproducts – G reactants
Chemical Energy (G) = RT ln [C]
DG = DG° + 2.3 RT (log ([ADP] [Pi]) – log [ATP])
2.3 RT = 5.6 kiloJoules / mole @ 20° C
DG° = -30 kiloJoules /mole @ 20°C, pH 7.0 and 1M
[reactants] and [products] “Standard Conditions”
Energy Depends on Substrate
Concentrations
DG = -30 – 5.6 log
[ATP]
[ADP] [Pi]
kJ / mole
The energy available per molecule of ATP depends on:
[ATP] @ 4mM, [ADP] @ 400 µM, [Pi] @ 2 mM
per mole of ATP hydrolyzed:
DG = -30 kJ – 5.6 kJ * log
= -30 kJ - 21 kJ
=
4 x 10-3
2 x 10-3 * 4 x 10-4
-51 kiloJoules per mole of ATP
Converting to approximately -530 meV/molecule of ATP
Energy in the Sodium Gradient
Consider Na+ movement from outside to inside:
DG = Gproducts – Greactants = Ginside – Goutside
DGtotal = DGelectrical + DGchemical
Conditions for our sample calculation:
Vm = -60 mV
[Na+]out = 140 mM
and 2.3 RT = 60 meV / molecule
[Na+]in = 14 mM
Energy in the Na Gradient: Electrical Term
DGelectrical = e * mVin – e * mVout
= +1e * -60 mV – (+1e) * 0 mV
= -60 meV
• negative sign means energy is released moving from
outside to inside
• 60 meV is the energy required to move a charged ion
(z=1) up a voltage gradient of 60 mV (assuming zero
concentration gradient)
Energy in the Na Gradient: Chemical Term
DGchemical = 2.3 RT (log [Na+]in – log [Na+]out)
= 60 meV * (-1)
= -60 meV
• negative sign means energy is released moving from outside to
inside
• 60 meV is the energy required to move a molecule up a 10 fold
concentration gradient (true for an uncharged molecule or for a
charged molecule when there is no voltage gradient)
Energy in the Na Gradient: Total
DGtotal = DGelectrical + DGchemical = -120 meV
• 120 milli-electron-Volts of energy would be required to pump a
single Na+ ion out of the cell up a 10 fold concentration
gradient and a 60 mV voltage gradient.
• Hydrolysis of a single ATP molecule can provide at least 500
meV of energy – enough to pump 4 Na+ ions.
• A single Na+ ion moving from outside to inside would be able to
provide 120 meV of energy, which could be used to pump
some other molecule, such as glucose, an amino acid, Ca2+ or
H+ up a concentration gradient
Example: Na+/Ca2+ exchange
Compare the internal [Ca2+] for exchange ratios of
2 Na+ : 1 Ca2+
vs.
3 Na+ : 1 Ca2+
Vm = -60 mV, [Ca2+]out = 1.5 mM [Ca2+]in = ?
Ca2+ moves from inside to outside
DG = Gproducts – Greactants = Goutside – Ginside
DGelectrical = (+2e) * (0 mV) – (+2e) * (-60 mV)
= +120 meV
DGchemical = 60 meV (log 1.5 – log ?)
Na+/Ca2+ exchange
DGtotal = DGE + DGC = 120 meV + 60 meV log (1.5 / ?)
Internal [Ca2+]
can be reduced
100 fold lower
for 3 Na : 1 Ca
vs 2 Na : 1 Ca
2 Na+
240
120 / 60
102
?

=
=
=
=
240 meV
120 + 60 log (1.5 / ?)
log (1.5 / ?)
1.5 / ?
15 µM
3 Na+
360
240 / 60
104
?

=
=
=
=
360 meV
120 + 60 log (1.5 / ?)
log (1.5 / ?)
1.5 / ?
0.15 µM
Structure of the Na/Ca Exchanger
Summary: Energetics
Transport Energetics
• A molecule of ATP donates about 500 meV
• It takes 60 meV to transport up a 60 mV
electrical gradient
• It takes 60 meV to transport up a 10 fold
concentration gradient
• A single sodium ion donates approximately
120 meV
Summary: Membrane Flux (moles of solute/sec)
Simple Diffusion
• Flux is directly proportional to external concentration
• Flux never saturates
Carrier-Mediated Transport
• Higher flux than predicted by solute permeability
• Flux saturates
• Binding is selective D- versus L-forms
• Competition
• Kinetics
Facilitated Diffusion
• Uses bidirectional, symmetric carrier proteins
• Flux is in the direction expected for simple diffusion
• Binding is equivalent on each side of the membrane
Primary Active Transport – driven by ATP hydrolysis
Secondary Active Transport – driven by ion gradients
Ion Channels
Transporters Regulated by Signaling Cascades
Na/H Exchangers
Na/Phosphate Cotransporter
Na/K/2Cl Cotransporter
Na/Cl Cotransporter
K/Cl Cotransporter
Na/Ca Exchanger
Na Channels
K Channels
Na,K-ATPase
H,K-ATPase
Unidirectional Transport Assays
1. Cells washed in isotonic buffered solution
2. Required transport inhibitor(s) added
3. Flux medium containing radioactive isotope added
Cells growing
in multi-well plates
4. At required times flux medium rapidly removed and
cells washed (3-4 x) in ice-cold isotonic saline
5. Final wash removed, cells lysed and radioactivity and
protein content of samples determined
Unidirectional Transport Assays
Calculations:
Specific Activity of medium:
Measure radioactivity in known volume of flux medium.
For example:
For unidirectional uptake of Na into cells in medium containing:
50 mM Na
100 mM choline Cl
25 mM K-Hepes, pH 7.4
22Na (≈ 1 µCu/ml)
Measure radioactivity in 5 µl flux medium
cpm (22Na)
5x
10-6
L
X
1L
0.050 moles Na
X
1 mole
109
nmoles
=
cpm ( 22Na)
nmoles Na
Measure radioactivity and protein content in sample.
Determine Na influx using specific activity of medium
Determine transport rate/protein content (Na uptake nmoles/µg protein/min)
THICK ASCENDING LIMB CELL
Na +
K+
Na +
Na + K+
Cl
2Cl
K+
Na +
K+
K+
Na +
K+
Lumen
Blood
GASTRIC PARIETAL CELL
K+
BLOOD
Na +
K+
"alkaline
tide"
H+
H+ HCO3
K+
Cl
CA
H2O
+
CO2
HCO3
HCO3
Cl
Na +
H+
CO2
Lumen
SMALL INTESTINAL CELL
Na +
K+
H2O
Na +
Na + K+
2Cl
Cl
cAMP
Lumen
K+