Calcium Homeostasis

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Energy Transduction

• toni scarpa
• physiology biophysics
• c.w.r.u.
• east 541
• axs15_at_po.cwru.edu

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General Resources

• Nicholls, D. and Ferguson, S. Bioenergetics, 2nd
  Edition, Academic Press, 1992
• Scarpa, A. Transport across mitochondrial
  membranes, in Giebisch, Tosteson and Ussing,
  Membrane Transport in Biology, Springer Verlag
  vol 2, 1979 pggs 263-355.

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Oxidation of Biological Molecules

• Aside for mineral and water our body is
  constituted by carbon molecules, not in a stable
  form
• The energetically stable form of proteins,
  carbohydrates and fat is CO2 and H2O
• The process is not explosive and it is usually
  coupled to a reaction called oxidation
• Oxidation seldom requires movement of oxygen and
  it usually involves the removal of electrons (and
  vice versa in case of reduction)

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The Main Role of Mitochondria
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ATP Turnover
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Number, Size and Distribution of Mitochondria

• All mammalian cells
• (-erythrocytes)
• Size 2 x 1 ?m
• Number varies depending on cellular ATP needed
  (1 by vol in keratinocytes,
• 25 in myocytes)

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Mitochondrial Preparations
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Lipid Composition of Mitochondria
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Degrading Chemical Energy
Chemical energy (carbon containing molecules in
the form of fat, carbohydrate, protein) are
converted within the mitochondria into ATP, a
common and widely used energy currency
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Mitochondrial Respiratory Chain

• Malate Succinate
• Pyruvate Glycerol
  phosphate
• Glutamate
• NAD Flavoprotein Co Enz Q Cyt b
  Cyt c1-c Cyt a-a3 02
• Rotenone
  Antimycin
  Cyanide

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Respiratory Chain
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Energy Drop
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Oxidative Phosphorylation

• Chemical energy (pyruvate, fatty acids)
• Ion gradient energy
• Chemical Energy (ATP)

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Chemiosmotic Coupling
The coupling is not a chemical intermediate but
a state, the electrochemical H
gradient, generated by the oxidation
of substrates trough the resp chain
The electrochemical H gradient across the matrix
results in two distinct but interchangeable compon
ents, a ?pH (a conc.term) and a ?? (an
electrical term)
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Mitchell's Theory

• Peter Mitchell, Nobel Prize for Chemistry, 1978
• Proton motive force , ?µH
• Basic Tenets
• The electron transfer respiratory chain should
  translocate protons
• The ATP synthase should function as reversible
  proton translocating ATPase
• Mitochondria and other energy transducing
  membranes should have low proton conductance
• Mitochondria should have carriers for metabolites
  functioning in the presence of a large ?µH

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Respiratory Chain and Proton Pumping
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Permeability to Protons
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Proton Pumping
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ATP Synthase
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Reversibility of H Synthase
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?µH - F?? 2.3 RT ?pH
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?? and ?pH

• The respiratory chain pumping H across the
  membrane generate the protonmotive force, which
  consists of a ?? and ?pH
• The first is an electrical terms due to the
  charge translocation of H which generates
  transmembrane potential across the membrane
• The second is a chemical term referring to the
  concentration gradient of H across the membrane
• Those terms are interchangeable and, depending of
  the use of these components in driving other
  metabolites, the protonmotive force could in
  principle be only ?? or ?pH

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The Use of ?? and ?pH

• Both H trough the ATP Synthase
• ?? ADP/ATP exchange
• Ca Uptake
• Aspartate/Glutamate
• ?pH Pi/OH
• Anions

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Proton Transport

• H is largely impermeable, it is transported
  outside mitochondria by the respiratory chain and
  it is accumulated by the ATP synthase
• pH equilibration can occur though the operation
  of OH/substrate exchange or the movement of
  protonated species
• because the anion traffic the ?pH across
  mitochondria in situ is minimal
• the rate of H leak is more than 20 times slower
  than the maximal rate of translocation across the
  respiratory chain

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Cation Transport

• Na is mostly impermeable, H/Na and Ca/Na
  exchanges have been reported
• K is mostly impermeable and the concentrations of
  K in and out are similar
• Fe can be actively transported
• Mg can be extruded upon addition of ADP or in the
  presence of cAMP

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Operation of Ionophores
Valinomycin Gramicidin Nigericin
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Ionophores

• Channel Forming
• gramicidin
• dimer, monovalent cation, H
• Neutral Shuttles
• valinomycin
• the complex is charged
• KRbgtgtgtgtNa, H
• transport electrophoretic, movement according to
  potential, can collapse existing potential
• Charged Shuttles
• nigericin, A23187
• monocarboxylic acids, complex neutral
• exist as protonated or metal-complex
• electroneutrally exchange of H/Cation
• distribution according to ?pH, can collapse pH

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Mitochondrial Ca Transport

• Mitochondria accumulate Ca in the presence of
  coupled respiration
• Mitochondria have high capacity and low affinity
  for Ca
• At the resting cytosolic Ca concentrations there
  is controversy on whether or not mitochondrial Ca
  accumulation occurs
• The transport of Ca occurs though a non
  characterized uniport and the distribution of Ca
  is driven by the transmembrane potential

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• Energy Dependent Ca Transport

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Site of Inhibitors on Ca Transport
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Uncouplers

• Chemicals of different structure and size which
  uncouple oxidative phosphorylation
• The common feature is that are lipid soluble
  compounds which can be protonated/deprotonated
  and can transport H across mitochondria (proton
  ionophores)
• The net effect is a short circuit of protons
  because those ejected through the respiratory
  chain will enter through the uncoupler. This will
  collapse the ?? and will result in maximal
  respiration and no ATP synthesis

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Ionophores in Energized and not Energized
Mitochondria
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Oxygen Electrode
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Respiratory Control Ratio
State 4
State 3
State 4
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Electrical Equivalency
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Oxygen Consumption by Mitochondria
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Shuttles of Reducing Equivalents
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Anion Permeability and Volume Change
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Anion Translocation in Mitochondria
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Anions Antiporters
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Adenine Nucleotide Translocase

• The must abundant and probably the most important
  protein within the inner membrane
• It exchanges ATP produced within the matrix with
  cytosolic ADP
• The exchange is electrogenic and the ratio is 1/1
• The enzyme is a 32KD hydrophobic protein most
  likely working as a dimer
• It has two specific inhibitors (actractyloside
  and bonkgregic acid)

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Function of the ATP/ADP Translocase

• It maintains low cytosolic ADP and high ATP (and
  vice versa)
• Because of the stoichiometry, it separates but
  maintains two pools for ADP and ATP
• It makes the ATP synthase specific for ATP/ADP
• It creates two environments for ATP hydrolysis
  and ATP synthesis
• It is driven by, and consumes, ??
• It creates two environments where AMP and other
  nucleotides are handled differently