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Membrane Transport
BL4010 11.28.05
Outline
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Passive Diffusion
Facilitated Diffusion
Active Transport
Transport Driven by ATP, light, etc.
Specialized Membrane Pores
• Ionophore Antibiotics
Trp (red) and Tyr (gold) at the interface
The Na+/K+ ATPase
ATP hydrolysis drives 3Na+ out and 2K+ in
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
The Gastric H+/K+ ATPase
The 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 CaATPase (P-type)
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!
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
Secondary Active Transport
Transport 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
Porins
Found in Gram-negative bacteria and in mitochondrial outer membrane
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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 16stranded beta barrel that traverses the membrane
to form the pore
Why Beta Sheets?
• Genetic economy?
• -helix requires 21-25 residues per
transmembrane strand
• -strand requires only 9-11 residues per
transmembrane strand
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
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 channelforming (C)
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!
Other PoreForming Toxins
• Hemolysin from
Staphylococcus aureus
forms a symmetrical
pore
• Aerolysin may form a
heptameric pore - with
each monomer
providing 3 beta strands
to a membranespanning barrel
Amphipathic Helices
form 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
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
The Magainin Peptides
• Incisions on Xenopus laevis (African clawed
frog) healed without infection, even in
bacteria-filled aquarium water
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
Gap Junctions
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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
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
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
Valinomycin
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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
Selectivity of Valinomycin
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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+
Gramicidin
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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