Membrane Transport

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Membrane Transport

• BL4010 11.28.05

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Outline

• Passive Diffusion
• Facilitated Diffusion
• Active Transport
• Transport Driven by ATP, light, etc.
• Specialized Membrane Pores
• Ionophore Antibiotics

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Trp (red) and Tyr (gold) at the interface
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The Na/K ATPase

• ATP hydrolysis drives 3Na out and 2K in

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Na/K Transport

• Hypertension involves apparent inhibition of
  sodium pump. Inhibition in cells lining blood
  vessels
• accumulation of Na, Ca2
• this inhibitor is the cardiac glycosyide, ouabain

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The Gastric H/K ATPaseThe enzyme that keeps
the stomach at pH 0.8

• The parietal cells of the gastric mucosa (lining
  of the stomach) have an internal pH of 7.4

• H,K-ATPase pumps protons from these cells into
  the stomach to maintain a pH difference across a
  single plasma membrane of 6.6!
• This is the largest concentration gradient across
  a membrane in eukaryotic organisms!
• H,K-ATPase is similar in many respects to
  Na,K-ATPase and Ca-ATPase (P-type)

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Osteoclast Proton Pumps

• How your body takes your bones apart!
• Bone material undergoes ongoing remodeling
• osteoclasts tear down bone tissue
• osteoblasts build it back up

• Osteoclasts function by secreting acid into the
  space between the osteoclast membrane and the
  bone surface - acid dissolves the Ca-phosphate
  matrix of the bone
• An ATP-driven proton pump in the membrane does
  this!

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The MDR ATPase

• aka the P-glycoprotein
• Animal cells have a transport system that is
  designed to recognize foreign organic molecules
  and transport them out of the cell usingthe
  hydrolytic energy of ATP
• MDR ATPase is a member of a "superfamily" of
  genes/proteins that appear to have arisen as a
  "tandem repeat"
• MDR ATPase interferes with drug treatments such
  as chemotherapy

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Secondary Active TransportTransport processes
driven by ion gradients

• Many amino acids and sugars are accumulated by
  cells in transport processes driven by ion
  gradients
• Symport - ion and the amino acid or sugar are
  transported in the same direction across the
  membrane
• Antiport - ion and transported species move in
  opposite directions

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Porins

• Found in Gram-negative bacteria and in
  mitochondrial outer membrane
• Porins are pore-forming proteins - 30-50 kD
• General or specific - exclusion limits 600-6000
• Most arrange in membrane as trimers
• High homology between various porins
• Porin from Rhodobacter capsulatus has 16-stranded
  beta barrel that traverses the membrane to form
  the pore

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Why Beta Sheets?

• Genetic economy?
• ?-helix requires 21-25 residues per transmembrane
  strand
• ?-strand requires only 9-11 residues per
  transmembrane strand

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The Pore-Forming Toxins

• Lethal molecules produced by many organisms
• They insert themselves into the host cell plasma
  membrane
• They kill by collapsing ion gradients,
  facilitating entry by toxic agents, or
  introducing a harmful catalytic activity

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Colicins

• Produced by E. coli
• Inhibit growth of other bacteria (even other
  strains of E. coli)
• Single colicin molecule can kill a host!
• Three domains translocation (T),
  receptor-binding (R), and channel-forming (C)

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Channel Formation

• C-domain 10-helix bundle, with H8 and H9 forming
  a hydrophobic hairpin
• Other helices amphipathic
• H8 and H9 insert, with others splayed on the
  membrane surface
• A transmembrane potential causes the amphipathic
  helices to insert!

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Other Pore-Forming Toxins

• Hemolysin from Staphylococcus aureus forms a
  symmetrical pore
• Aerolysin may form a heptameric pore - with each
  monomer providing 3 beta strands to a
  membrane-spanning barrel

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Amphipathic Helicesform Transmembrane Ion
Channels

• Many natural peptides form oligomeric
  transmembrane channels
• The peptides form amphiphilic ?-helices
• Aggregates of these helices form channels that
  have a hydrophobic surface and a polar center
• Melittin (bee venom), magainins (frogs) and
  cecropin (from cecropia moths) are examples

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Amphipathic Helices

• Melittin - bee venom toxin - 26 residues
• Cecropin A - cecropia moths - 37 residues
• Magainin 2 amide - frogs - 23 residues
• See Figure 10.35 to appreciate helical wheel
  presentation of the amphipathic helix

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The Magainin Peptides

• Incisions on Xenopus laevis (African clawed frog)
  healed without infection, even in bacteria-filled
  aquarium water

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The Cecropins

• Produced by Hyalophora cecropia when the moth is
  challenged by bacterial infections
• These peptides are thought to form ?-helical
  aggregates in membranes, creating an ion channel
  in the center of the aggregate

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Gap Junctions

• Animal cells
• Provide metabolic connections
• Provide a means of chemical transfer
• Provide a means of communication
• Permit large number of cells to act in synchrony

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Gap Junctions

• Hexameric arrays of a single 32 kD protein
• Subunits are tilted with respect to central axis
• Pore in center can be opened or closed by the
  tilting of the subunits, e.g. as response to
  stress

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Ionophore Antibiotics

• Mobile carrier or pore (channel)
• How to distinguish? Temperature!
• Pores will not be greatly affected by
  temperature, so transport rates are approximately
  constant over large temperature ranges
• Carriers depend on the fluidity of the membrane,
  so transport rates are highly sensitive to
  temperature, especially near the phase transition
  of the membrane lipids

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Valinomycin

• A classic mobile carrier
• A depsipeptide - a molecule with both peptide and
  ester bonds
• Valinomycin is a dodecadepsipeptide
• The structure places several carbonyl oxygens in
  the center of the ring structure
• Potassium and other ions coordinate the oxygens
• Valinomycin-potassium complex diffuses freely and
  rapid across membranes

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Selectivity of Valinomycin

• Why?
• K and Rb bind tightly, but affinities for Na
  and Li are about a thousand-fold lower
• Radius of the ions is one consideration
• Hydration is another - see page 324 for solvation
  energies
• It "costs more" energetically to desolvate Na
  and Li than K

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Gramicidin

• A classic channel ionophore
• Linear 15-residue peptide - alternating D L
• Structure in organic solvents is double helical
• Structure in water is end-to-end helical dimer
• Unusual helix - 6.3 residues per turn with a
  central hole - 0.4 nm or 4 A diameter
• Ions migrate through the central pore

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