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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
Intracellular Extracellular Concentration ConcentrationComponent (mM) (mM)
Cations Na 5-15 145 K 140 5 Mg 0.5 1-2 Ca 10-4 1-2 H 8 x 10-5 (pH 7.1) 4 x 10-5 (pH 7.4)
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.
COMPARISON OF ION CONCENTRATIONS INSIDE AND OUTSIDE A TYPICAL MAMMALIAN CELL
A Cross between Human Beings and Plants . . .
SCIENTISTS ON VERGE OF CREATING PLANT PEOPLE . . .
Bizarre Creatures Could do Anything You Want
Tuesday, July 1, 1980
Bra
in w
ate
r (g
/10
0 g
dry
wt)
Simple DiffusionF
lux
[S]o
• Flux is proportional to external concentration
• Flux never saturates
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.
outClinNainK
inCloutNaoutKm [Cl]P[Na]P[K]P
[Cl]P[Na]P[K]Plog60mVV
• Higher flux than predicted by solute permeability
• Flux saturates• Binding is selective
(D- versus L-forms)• Competition• Kinetics:
[S]o << KmM [S]
[S]o = Km M = Mmax / 2
[S]o >> Km M = Mmax
Carrier-Mediated Transport
[S]oKm
Mmax
0.5Flu
x
MEMBRANE ION TRANSPORT PROTEINS
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)
Transport Kinetics
So + Co SCo Si S = Solute C = Carrierk +
k -
Reversible Transport
Co Ci
So Si
SCo SCi
Mnet = Min – Mout =
Mmax 1 11 + Km / [S]o 1 + Km / [S]i
-( )
• 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
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 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
3 Na
2 K
ATP
ADP + Pi
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 H2OATP ADP + Pi
G = Gproducts – G reactants
Chemical Energy (G) = RT ln [C]
G = G° + 2.3 RT (log ([ADP] [Pi]) – log [ATP])
2.3 RT = 5.6 kiloJoules / mole @ 20° C
G° = -30 kiloJoules /mole @ 20°C, pH 7.0 and 1M [reactants] and [products] “Standard Conditions”
Energy Depends on Substrate Concentrations
G = -30 – 5.6 log [ATP] kJ / mole [ADP] [Pi]
The energy available per molecule of ATP depends on: [ATP] 4mM, [ADP] 400 µM, [Pi] 2 mM
per mole of ATP hydrolyzed:
G = -30 kJ – 5.6 kJ * log 4 x 10-3 2 x 10-3 * 4 x 10-4
= -30 kJ - 21 kJ = -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:
G = Gproducts – Greactants = Ginside – Goutside
Gtotal = Gelectrical + Gchemical
Conditions for our sample calculation: Vm = -60 mV [Na+]out = 140 mM [Na+]in = 14 mM
and 2.3 RT = 60 meV / molecule
Energy in the Na Gradient: Electrical Term
Gelectrical = 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
Gchemical = 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
Gtotal = Gelectrical + Gchemical = -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
G = Gproducts – Greactants = Goutside – Ginside
Gelectrical = (+2e) * (0 mV) – (+2e) * (-60 mV)
= +120 meV
Gchemical = 60 meV (log 1.5 – log ?)
Na+/Ca2+ exchange
2 Na+ 240 meV 240 = 120 + 60 log (1.5 / ?)120 / 60 = log (1.5 / ?) 102 = 1.5 / ? ? = 15 µM
3 Na+ 360 meV 360 = 120 + 60 log (1.5 / ?)240 / 60 = log (1.5 / ?) 104 = 1.5 / ? ? = 0.15 µM
Gtotal = GE + GC = 120 meV + 60 meV log (1.5 / ?)
Internal [Ca2+]can be reduced 100 fold lowerfor 3 Na : 1 Cavs 2 Na : 1 Ca
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
Cells growingin multi-well plates
1. Cells washed in isotonic buffered solution
2. Required transport inhibitor(s) added
3. Flux medium containing radioactive isotope added
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
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 Na100 mM choline Cl25 mM K-Hepes, pH 7.422Na (≈ 1 µCu/ml)
Measure radioactivity in 5 µl flux medium
Measure radioactivity and protein content in sample.
Determine Na influx using specific activity of mediumDetermine transport rate/protein content (Na uptake nmoles/µg protein/min)
Unidirectional Transport Assays
cpm (22Na) 1 L 1 mole cpm (22Na)
5 x 10-6 L 0.050 moles Na 109 nmoles nmoles NaX X =
THICK ASCENDING LIMB CELL
GASTRIC PARIETAL CELL
SMALL INTESTINAL CELL