Membranes and Transport - Lehninger Chapter 11

Biochemistry 471/671 - M. Mossing

2007/11/16 5:20 PM

Membranes and Transport - Outline

• 11.1 The Composition and Architecture of Membranes
• 11.2 Membrane Transport
• 11.3 Solute Transport Across Membranes

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

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

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

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

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

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

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

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)

11.1 Summary

• Membrane compartments
• The fluid mosaic model
• Peripheral and integral membrane proteins
• Membrane spanning proteins
• Asymmetry

11.2 Membrane Transport

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

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

Transbilayer transport by flippases

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

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

Lipid Rafts

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

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

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

Membrane fusion

• Budding and fusion are two sides of the same coin
• Fusion
  1. Recognition
  2. Apposition
  3. Disruption
  4. Bilayer fusion
• Influenza Hemaglutinin
  • pH triggers conformational changes
  • Anchors in both cell membrane and viral envelope stimulate fusion
• Neurotransmitter release due to vesicle fusion at gap junctions
  • SNAP - NSF attachment protein
  • SNARE - soluble NSF attachment protein receptor

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

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

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

Energetics of Transport - Electrical Potential

• The energetic driving force (or cost) of transporting a charged object across an electric field
  • ΔG_el = 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)

Energetics of Transport - Electro-chemical Potential

• ΔG = RTln(C_inn/C_out) + 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 spontaeous for charges (Z) with signs opposite the potential difference (Δψ)

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

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

Erythrocyte glucose transporter - Uniporter

• GlUT1 transports glucose into red blood cells
• Specific for glucose, over other sugars
• Michaelis Menten Kinetics
  • E + S <==> ES --> E + P
  • E + S_out <==> { ESo <==> ESi } --> E + S_in
  • Transition state is now conformational change of transporter
  • v₀ = (Vmax [S]_out) / (K_M + [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 + S_out <==> { ESo <==> ESi } <==> E + S_in

Chloride-Bicarbonate Exchange - Antiporter

• Chloride and Bicarbonate
  • carry the same charge
  • move in opposite directions - ping pong mechanism
  • maintain electroneutrality
• Cl^-_in + E <==> E + Cl^-_out
• HCO₃^-_out + E <==> E + HCO₃^-_in

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

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

F-type ATPases - Proton Gradients <==> ATP

• Can either use ATP to pump protons or proton gradients to make ATP

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

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
• Na⁺- Glucose Symport in human intestine
  • 2 Na⁺_out + Glucose_out --> 2 Na⁺_in + Glucose_in
  • Combination of sodium chemical potential and membrane potential
  • provide driving force for ~9000 fold concentration [Glucose]_in/[Glucose]_out

Aquaporins

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

Ion selective channels

• 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
  • Voltages can be applied across the membrane ("clamped" at a constant V)
  • Ligands can be added
  • Currents can be measured on many samples to assess the properties of individual
• Ligand or Voltage gated channels

Sodium Channels (Voltage gated) and Nerve function

• At normal transmembrane potential channels are closed
• When membranes depolarize - channels open transiently
• Burst of Na⁺ influx leads to an overpotential

Acetycholine receptor - a ligand gated ion channel

• Acetylcholine is released into the synapse by exocytosis
• Acetylcholine binds to the outside of a channel on an adjacent cell as an allosteric activator
• Burst of Na⁺ influx leads to an overpotential...

Ion channel defects, inhibitors

• Cystic Fibrosis
• Many natural neurotoxins

11.3 Summary

• Transporters allow for transmembrane movement
• Carriers are saturable and substrate specific
• GLUT uniporters, transproters symporters
• Na K ATPase
• ABC transporters
• Ionophores
• Aquaporins - water
• Ion channels