Transcript protein mediated membrane transport

POLYCYSTIC RENAL DISEASE
1 in 500 autopsies
1 in 3000 hospital
admissions
Accounts for ≈10% of
end-stage renal failure
Autosomal dominant
inheritance
CYSTIC FIBROSIS
1/2000 births in white
Americans
Median age for survival
late 30s
Autosomal recessive
inheritance
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)
Woman drinks so much water she dies
January 13, 2007
SACRAMENTO, 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, was found dead Friday in her suburban Rancho Cordova
home hours after taking part in the "Hold Your Wee for a Wii" contest in which
KDND 107.9 promised 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 was hurting her real bad," said Laura Rios, one of Strange's co-workers
at Radiological Associates of Sacramento. "She was crying, and that was the
last that anyone had heard from her."
Copyright 2007 The Associated Press. All rights reserved.This material may not
be published, broadcast, rewritten, or redistributed .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
CARRIER MEDIATED 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 
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
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
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
• Ca-ATPase
• H,K-ATPase
• Cu-ATPase
• 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
Membrane Transport and Cellular Functions that Depend on
the Na,K-ATPase
Amino Acid Homology Among the Na,K-ATPase Subunit Isoforms
The Na,K-ATPase As a Receptor For Signal Transduction
Association of Src With the Na,K-ATPase
SR Ca-ATPase
FoF1 ATPase
Experimental Evidence for Rotation
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
III. 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
GASTRIC PARIETAL CELL
SMALL INTESTINAL CELL