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Chapter 10
Membrane Transport
Outline
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10.1 Passive Diffusion
10.2 Facilitated Diffusion
10.3 Active Transport
10.4 - 10.6 Transport Driven by ATP, light, etc.
10.7 Group Translocation
10.8 Specialized Membrane Pores
10.9 Ionophore Antibiotics
Passive Diffusion
No special proteins needed
• Transported species simply moves down its concentration
gradient - from high [c] to low [c]
• Be able to use Eq. 10.1 and 10.4
• High permeability coefficients usually mean that passive diffusion
is not the whole story
Facilitated Diffusion
Delta G negative, but proteins assist
• Solutes only move in the thermodynamically favored direction
• But proteins may "facilitate" transport, increasing the rates of
transport
• Understand plots in Figure 10.3 Two important distinguising
features:
– solute flows only in the favored direction
– transport displays saturation kinetics
Active Transport Systems
Energy input drives transport
• Some transport must occur such that solutes flow against
thermodynamic potential
• Energy input drives transport
• Energy source and transport machinery are "coupled"
• Energy source may be ATP, light or a concentration gradient
The Sodium Pump
aka Na,K-ATPase
• Large protein - 120 kD and 35 kD subunits
• Maintains intracellular Na low and K high
• Crucial for all organs, but especially for neural tissue and the brain
• ATP hydrolysis drives Na out and K in
• Alpha subunit has ten transmembrane helices with large cytoplasmic
domain
Na,K Transport
• ATP hydrolysis occurs via an E-P intermediate
• Mechanism involves two enzyme conformations known as E1 and
E2
• Cardiac glycosides inhibit by binding to outside
Na,K Transport
• Hypertension involves apparent inhibition of sodium pump.
Inhibition in cells lining blood
• vessel walls results in Na,Ca accumulation
• Studies show this inhibitor to be ouabain!
Calcium Transport in Muscle
A process akin to Na,K transport
• Calcium levels in resting muscle cytoplasm are maintained low by
Ca-ATPase - a Ca pump
• Calcium is pumped into the sarcoplasmic reticulum (SR) by a 110 kD
protein that is very similar to the alpha subunit of Na,K-ATPase
• Aspartyl phosphate E-P intermediate is at Asp-351 and Ca-pump also fits
the E1-E2 model
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!!!
The Gastric H,K-ATPase
• 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
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
• This organic molecule pump recognizes a broad variety of
molecules and transports them out of the cell using the hydrolytic
energy of ATP
The MDR ATPase
• MDR ATPase is a member of a "superfamily" of genes/proteins
that appear to have arisen as a "tandem repeat"
• MDR ATPase defeats efforts of chemotherapy
Light-Driven H + Transport
The Bacteriorhodopsin story
• Halobacterium halobium, the salt-loving bacterium, carries out normal
respiration if O2 and substrates are plentiful
• But when substrates are lacking, it can survive by using
bacteriorhodopsin and halorhodopsin to capture light energy
• Purple patches of H. halobium are 75% bR and 25% lipid - a "2D crystal"
of bR - ideal for structural studies
Bacteriorhodopsin
Protein opsin and retinal chromophore
• Retinal is bound to opsin via a Schiff base link
• The Schiff base (at Lys-216) can be protonated, and this site is
one of the sites that participate in H+ transport
Bacteriorhodopsin
• Lys-216 is buried in the middle of the 7-TMS structure of bR and
retinal lies mostly parallel to the membrane and between the
helices
• Light absorption converts all-trans retinal to 13-cis configuration see Figure 10.22
Bacteriorhodopsin
The protons visit the aspartates....
• Asp-85 and Asp-96 lie on opposite sides of a membrane-spanning
helix
• These remarkable aspartates have pKa values around 11!
(WHY?)
• Protons are driven from Asp-96 to the Schiff base at Lys-216 to
Asp-85 and out of the cell
Bacteriorhodopsin
• Halorhodopsin transports Cl - instead of H +
• Halorhodopsin has Lys-242 Schiff base but no aspartates and no
deprotonation of Schiff base during the transport cycle
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
Secondary Active Transport
• Symport - ion and the aa or sugar are transported in the same
direction across the membrane
• Antiport - ion and transported species move in opposite directions
• Lactose permease in E. coli is a good example
• His-322 and Glu-325 are proton carriers
Group Translocation
The phosphotransferase system (PTS)
• Discovered by Saul Roseman in 1964
• Sugars are phosphorylated from PEP during transport into E. coli
cells
• Four proteins required: EI, HPr, EII, and EIII
Group Translocation
• EI and HPr are universal and work for all sugars
• EII and EIII are specific for each sugar
• Mechanism involves transfer of P from PEP to EI and then to HPr
and then to 2 sites on EIII and then finally phosphorylation of
sugar
Porins
Found both 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 (with eyelet!)
Why Beta Sheets?
for membrane proteins??
• Genetic economy
• Alpha helix requires 21-25 residues per transmembrane strand
• Beta-strand requires only 9-11 residues per transmembrane strand
• Thus, with beta strands , a given amount of genetic material can make
a larger number of trans-membrane segments
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
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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)
Clues to Channel Formation!
• C-domain: 10-helix bundle, with H8 and H9 forming a hydrophobic
hairpin
• Other helices amphipathic (Fig. 10.30)
• H8 and H9 insert, with others splayed on the membrane surface
• A transmembrane potential causes the amphipathic helices to
insert!
Other Pore-Forming Toxins
• Delta endotoxin also possesses a helix-bundle and may work the
same way
• There are other mechanisms at work in other toxins
• Hemolysin forms a symmetrical pore
• Aerolysin may form a heptameric pore - with each monomer
providing 3 beta strands to a membrane-spanning barrel
Gap Junctions
Vital connections for 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
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
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" 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
Amphipathic Helices
Alpha helices with a polar face and a hydrophobic face
• Aggregates of these helices arrange in membranes with their
polar faces to the center and nonpolar faces toward the lipid
bilayer
Amphipathic Helices
• Melittin - bee venom toxin - 26 residue peptide
• Cecropin A - cecropia moth peptide - 37 residues
• See Figure 10.35 to appreciate helical wheel presentation of the
amphipathic helix