POLYCYSTIC RENAL DISEASE

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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
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CYSTIC FIBROSIS
1/2000 births in white Americans Median age for
survival late 30s Autosomal recessive
inheritance
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COMPARISON OF ION CONCENTRATIONS INSIDE AND
OUTSIDE A TYPICAL MAMMALIAN CELL
Intracellular Extracellular
Concentration Concentration Component
(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.
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CONCENTRATION OF THE MAJOR CATIONS IN THE ECF AND
ICF
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Brain water (g/100 g dry wt)
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Simple Diffusion

• Flux is proportional to external concentration
• Flux never saturates

Flux
So
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PROTEIN MEDIATED MEMBRANE TRANSPORT

• PRIMARY ACTIVE
• SECONDARY ACTIVE TRANSPORT
• FACILITATED DIFFUSION
• ENDOCYTOSIS/TRANSCYTOSIS

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Membrane Flux (moles of solute/sec)

• Simple Diffusion
• Carrier Mediated Transport
• Facilitated Diffusion
• Primary Active Transport
• Secondary Active Transport
• Ion Channels

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TRANSPORT OF MOLECULES THROUGH MEMBRANES
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CARRIER MEDIATED TRANSPORT
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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 (ionout / ionin)
  _at_ 30C
• The Goldman-Hodgkin-Katz equation is used to
  calculate the steady-state resting potential in
  cells with significant relative permeability to
  sodium.

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Carrier-Mediated Transport

• Higher flux than predicted by solute permeability
• Flux saturates
• Binding is selective (D- versus L-forms)
• Competition
• Kinetics
• So ltlt Km M a S
• So Km M Mmax / 2
• So gtgt Km M Mmax

Mmax
Flux
0.5
Km
So
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MEMBRANE ION TRANSPORT PROTEINS
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Transport Kinetics
dSCo/dt k So Co k- SCo 0 at
equilibrium Þ k So Co k- SCo k- / k
(So Co)/SCo Km Þ SCo (So
Co)/Km Fractional Rate M / Mmax SCo /
(Co SCo) M Mmax / (1 Co/SCo)
Mmax / (1 Km/So)
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Reversible Transport
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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

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Facilitated Diffusion Band 3/AE1
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Facilitated Diffusion Band 3/AE1
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Cytoskeletal/AE1 Interactions
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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

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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 _at_ 37 C

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Palytoxin
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Na,K-ATPase Reaction Scheme
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Membrane Transport and Cellular Functions that
Depend on the Na,K-ATPase
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Amino Acid Homology Among the Na,K-ATPase Subunit
Isoforms
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The Na,K-ATPase As a Receptor For Signal
Transduction
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Association of Src With the Na,K-ATPase
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SR Ca-ATPase
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FoF1 ATPase
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Experimental Evidence for Rotation
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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

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Energy available from ATP
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 _at_
20 C DG -30 kiloJoules /mole _at_ 20C, pH 7.0
and 1M reactants and
products Standard Conditions
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Energy Depends on Substrate Concentrations

• The energy available per molecule of ATP depends
  on
• ATP _at_ 4mM, ADP _at_ 400 µM, Pi _at_ 2 mM
• Þ per mole of ATP hydrolyzed
• DG -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

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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 Naout
140 mM Nain 14 mM and 2.3 RT 60
meV / molecule
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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 (z1) up a voltage gradient of 60 mV
  (assuming zero concentration gradient)

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Energy in the Na Gradient Chemical Term

• DGchemical 2.3 RT (log Nain log Naout)
• 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)

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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

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Example Na/Ca2 exchange
Compare the internal Ca2 for exchange ratios
of 2 Na 1 Ca2 vs. 3 Na 1
Ca2 Vm -60 mV, Ca2out 1.5 mM
Ca2in ? 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 ?)
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Na/Ca2 exchange
DGtotal DGE DGC 120 meV 60 meV log (1.5 /
?)
2 Na Þ 240 meV 240 120 60
log (1.5 / ?) 120 / 60 log (1.5 / ?) 102
1.5 / ? ? 15 µM 3 Na
Þ 360 meV 360 120 60 log
(1.5 / ?) 240 / 60 log (1.5 / ?) 104
1.5 / ? ? 0.15 µM
Internal Ca2can be reduced 100 fold
lowerfor 3 Na 1 Cavs 2 Na 1 Ca
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Structure of the Na/Ca Exchanger
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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

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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

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Transporters Regulated by Signaling Cascades
Na/H Exchangers Na/Phosphate Cotransporter Na/K/2C
l Cotransporter Na/Cl Cotransporter K/Cl
Cotransporter Na/Ca Exchanger Na,K-ATPase H,K-ATPa
se Na Channels K Channels
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THICK ASCENDING LIMB CELL
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GASTRIC PARIETAL CELL
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SMALL INTESTINAL CELL