Membranes and Transport - Lehninger Chapter 11

1. Membranes and Transport - Lehninger Chapter 11 • 11.1 The Composition and Architecture of Membranes • 11.2 Membrane Transport • 11.3 Solute Transport Across Membranes

2. 11.1 The Composition and Architecture of Membranes • Membranes contain specialized lipids and proteins • Membranes are fluid mosaics • Lipid Bilayer Structure • Peripheral Membrane Proteins • Integral membrane proteins • Sequence predictions of Membrane spanning domains • Lipid anchors

3. Membranes contain specialized lipids and proteins • Proteins 30-70% • Phospholipids 7-40% • Sterols 0-25% • Specialized membranes More than 90% Rhodopsin in photoreceptor disc membrane Protein rich mitochondrial membranes Transport optimized Red Blood Cell membrane

4. Membranes are fluid mosaics • Proteins/specialized structures as the tiles • Lipids as the mortar • Components are constrained to a plane but can diffuse laterally • Individual components diffuse, associate, dissociate in 2D

5. Lipid Bilayer Structure • Lipids assemble to segregate polar end nonpolar substituents • Micelles are globular • Exterior polar head group, Interior hydrophobic tail • Favored by single acyl chains • Vesicles or liposomes have an internal aqueous compartments • Inner and Outer leaflets • Bilayers are locally planar structures • Inner and Outer leaflets are asymmetrical • Bio membranes are ~3 nm (30 Angstroms) thick

6. Membrane Proteins • Peripheral membrane proteins • Associate with the membrane surface (lipids or protein) • Can be dissociated by changes in solution conditions (salt, pH) • Integral membrane proteins • Interact with hydrophobic bilayer core • Membrane Spanning • Lipid Anchors

7. Integral membrane proteins • Specific structure and orientation • Topology and orientation can be probed in intact membranes Protease digestion Chemical reactivity (lysine, cysteine modifications) • Membrane spanning segments expose nonpolar surfaces to bilayer interior • Structures of a few transmembrane proteins reveal common helical membrane spanning elements

8. Sequence predictions of Membrane spanning domains • Hydropathy plots average hydrophobicity over N successive residues (N=7-20) alpha helix dimensions 3.6 residues/turn over 5.4 Angstroms Bilayer dimensions ~30 Angstroms / 5.4 = ~6 turns of helix or 21 AAs • Hydrophobic stretches of ~20 residues are often membrane spanning helices • 7-9 residues of (more extended) beta strands can span the membrane

9. Lipid anchors • Post translational modification of Amino acids with Fatty Acids -- Palmitate, Myristate Isoprenoids -- Farnesyl, geranyl (Inner leaflet) Sterols Glycosyl Phosphatidyl Inositol (GPI) - (Outer leaflet)

10. 11.1 Summary • Membrane compartments • The fluid mosaic model • Peripheral and integral membrane proteins • Membrane spanning proteins • Asymmetry

11. 11.2 Membrane Transport • Acyl Chains - order-disorder transition • Transbilayer transport by flippases • Lateral diffusion • Lipid Rafts • Caveolae • Cell adhesion proteins • Membrane fusion

12. Acyl Chains - order-disorder transition • Sterols and straight chains favor order • Double bonds and short chains favor disorder • Lipid composition is adjusted to maintain constant fluidity High temperature - more saturated FAs, sterols Low temperature - more unsaturated FAs, shorter chains

13. Transbilayer transport by flippases • Barriers to transbilayer lipid movement are high • Lipid biosynthesis on one side of a membrane is coupled to catalyzed transport

14. Lateral diffusion • Lipids and proteins can diffuse in 2D • Measured by Fluorescence Recovery After Photo-bleaching FRAP requires fluorescent tag on lipid or protein Photobleaching of a small area by intense light pulse makes a dark spot Recovery depends on diffusion of undamaged fluorophores to the bleached spot

15. Lateral diffusion may be limited by protein networks Cytoskeletal connections or membrane patches

16. Lipid Rafts • Glycosphingolipids cluster in the outer membrane • Cholesterol also enriched in Lipid Rafts • GPI, palmitoyl and myristoyl anchors on signaling proteins enriched

17. Caveolae • Caveolin is an integral membrane protein Binds to the inner membrane Dimer, Palmitoyl anchors Induces curvature • caveolae (as in cave) on the extracellular side • bulges on thy cytoplasmic side Caveolae seem to be the locus for signalling

18. Cell adhesion proteins - extracellular domains • Integrins - attach to the extracellular matrix Bind collagen and fibronectin • Cadherins - Homotypic association Side by side dimers on the same cell Interactions between adjacent cells • Selectins - bind to oligosaccharides Cell surface lectins

19. Membrane fusion • Budding and fusion are two sides of the same coin • Fusion 1. Recognition 2. Apposition 3. Disruption 4. Bilayer fusion

20. Neurotransmitter release due to vesicle fusion at gap junctions SNAP - NSF Attachment Protein SNARE - Soluble NSF Attachment protein REceptor

21. 11.2 Summary • Order and fluidity • Leaflets are isolated except for catalyzed exchange • Lateral diffusion allows for assembly of lipid rafts • Caveolae as signalling centers • Integrins, adhesins • Membrane fusion

22. 11.3 Solute Transport Across Membranes • Passive transport • Transport super-families • Erythrocyte glucose transporter • Chloride-Bicarbonate Exchange • Active Transport • P-type ATPases • F-type ATPases • ABC transporter • Ion Gradients • Aquaporins • Ion selective channels • Sodium Channels and Nerve function • Acetycholine receptor - a ligand gated ion channel • Ion channel defects and inhibitors

23. 11.3 Solute Transport Across Membranes Simple Diffusion Facilitated Diffusion Pores, Channels Ionophores Active Transport Direct Coupled

24. Energetics of Transport - Chemical Potential • Free energy defines the driving force • Transport of electro-neutral species is governed by chemical potential differences The chemical potential μ is made up of a standard state potential μ0 and a concentration dependent term μ = μ0 + RTlnC The free energy difference between two solutions that differ only in the concentration of one component is the difference between the chemical potentials ΔG = μ1 - μ2 = (μ0 + RTlnC1) - (μ0 + RTlnC2) = RTln(C1/C2)

25. For the free energy difference between the inside and outside • ΔG = RTln(Cin /Cout) • This is the driving force (or cost) of the transport reaction

26. Energetics of Transport - Electrical Potential • The energetic driving force (or cost) of transporting a charged object across an electric field ΔGel = Z F Δψ Δψ is the electrical potential in volts Z is the charge on the ion F is the Faraday constant 96,480 J/(V mole)

27. Energetics of Transport - Electro-chemical Potential • ΔG = RTln(Cinn/Cout) + ZF Δψ • If you forget the signs, remember: Free energy of a spontaneous reaction is negative Diffusion is spontaneous from high concentration to low Electrical transport is spontaneous for charges (Z) with signs opposite the potential difference (Δψ)

28. Passive transport - facilitated diffusion by proteins • Simple diffusion Rate determined by lipid/aqueous solubility Driving force is the sum of • Simple chemical potential and • electrochemical potential Not saturable (no Vmax) • Facilitated Diffusion - "passive transport” Transporters or Permeases are proteins Directionality determined by concentration and electrochemical gradients

29. Transport superfamilies • Carriers • Bind specific ligands • Catalyze transport across the membrane • In a sense they are enzymes • Transport is saturable • Channels • Less specific (often size specific) • Can be fluid filled • Transport may not be easily saturable

30. Erythrocyte glucose transporter - Uniporter • GlUT1 transports glucose into red blood cells • Specific for glucose, over other sugars

31. Michaelis Menten Kinetics • E + S <==> ES --> E + P • E + Sout <==> { ESo <==> ESi } --> E + Sin • Transition state is now conformational change of transporter • v0 = (Vmax [S]out) / (KM + [S]out) • Again v0 is the initial rate, - applies only when [S]out = constant, [S]in is negligible • If [S]in is not negligible, back reaction must be considered • E + Sout <==> { ESo <==> ESi } <==> E + Sin

32. Kinetics of glucose transport into erythrocytes

34. Chloride-Bicarbonate Exchange - Antiporter • Chloride and Bicarbonate • carry the same charge • move in opposite directions - ping pong mechanism • maintain electro-neutrality • Cl-in + E <=> E + Cl-out • HCO3-out + E <=> E + HCO3-in

35. Active Transport • Energy coupling can transport against a concentration gradient • Primary • Transport is coupled to a chemical process (ATP hydrolysis) • Secondary • Transport is coupled to a favorable transport process

36. P-type ATPases - Active Transport • Transport phosphate coupled to ATP hydrolysis • Inhibited by vanadate • Na+K+ ATPase • 2K+out + 3Na+in + ATP --> 2K+in + 3Na+out + ADP + Pi • Net charge (+1) transfer out results in a -50-70 mV membrane potential • Energetically costly but membrane potential essential for action potential and other processes

37. F-type ATPases - Proton Gradients <==> ATP • Can either use ATP to pump protons or proton gradients to make ATP

38. ABC transporters - homologous family • classified by sequence and structure - not by function • ATP dependent transport • Multidrug resistance transporter pumps out foreign compounds • The chloride channel CFTR responsible for cystic fibrosis • Flippases for transbilayer lipid transport

39. Ion Gradients - Na+ or H+ can drive secondary transport • lac permease - bacterial lactose proton symport • Active transport of Lactose depends on maintenance of proton gradient

40. Na+- Glucose Symport in human intestine • 2 Na+out + Glucoseout --> 2 Na+in + Glucosein • Combination of sodium chemical potential and membrane potential • provide driving force for ~9000 fold concentration [Glucose]in/[Glucose]out

41. Aquaporins • Allow passive transport of water • Respond to changes in osmotic pressure

42. Ion selective channels • Ligand gated • Acetylcholine Receptor • Neuromuscular junction • Voltage Gated • K+ Channel

43. Ion channel measurements • Patch Clamping can measure the characteristics of a single channel • Glass micropipette can be used to capture a "patch" of membrane with one or more channels • Patch detached from the cell - seals the pipet opening

44. Solute Transport Summary • Passive and Active Transport • Carriers • Electrochemical Driving Forces • Coupled Transport • Ion Channels