Membrane Transport

1. Membrane Transport BL4010 11.28.05

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

4. Trp (red) and Tyr (gold) at the interface

5. The Na+/K+ ATPase • ATP hydrolysis drives 3Na+ out and 2K+ in

6. 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

9. 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) The Gastric H+/K+ ATPaseThe enzyme that keeps the stomach at pH 0.8

10. 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! Osteoclast Proton Pumps

11. 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

13. 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

15. 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

16. Why Beta Sheets? • Genetic economy? • -helix requires 21-25 residues per transmembrane strand • -strand requires only 9-11 residues per transmembrane strand

17. 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

18. 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)

19. 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!

20. 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

21. 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

22. 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

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

24. 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

25. 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

26. 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

28. 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

30. 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

32. 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+

33. 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