Membrane Lipids and Transport Notes

Isoprene Based Lipids and Palmitic Acid

• Isoprene-based lipids: Fortisil and Jernil.
• Palmitic acid is linked to spectrin, forming long bundles.
• Submembrane cytoskeleton gives the biconcave shape to cells.
• The membrane is linked to the spectrin skeleton by proteins, including anchoring protein 4.2.
• Spectrin bundles restrict the diffusion of integral membrane proteins within the membrane.

Membrane Organization and Lipid Composition

• Membranes proceed from the ER to the Golgi and then to other locations.
• Proteins going to the mitochondria use a different pathway than the secondary pathway.
• Vesicles have different lipid compositions.
• Lipid composition varies between membrane leaflets.

Erythrocyte Membrane

• In the erythrocyte membrane:
  • The outer leaflet faces the outside world.
  • The inner leaflet faces the cytoplasm.
• Lipid composition differs between the inner and outer leaflets.
• Lipids are asymmetrically distributed.

Phosphatidylserine and Apoptosis

• Phosphatidylserine is found on the inner leaflet in all cells.
• It signals apoptosis (regulated cell death).
• Apoptosis prevents cells from falling apart and causing inflammation by being cleaned up by macrophages.
• Macrophages recognize cells undergoing apoptosis via phosphatidylserine, which flips to the outer leaflet to signal "eat me".

Asymmetric Distribution of Lipids

• Models for asymmetric distribution:
  • Asymmetric synthesis: Lipids made at one leaflet stay there.
  • Selective transfer: Lipids are made at one leaflet and then transferred to the other.
• Experiments on bacterial cells study lipid localization and synthesis.

Studying Lipid Localization

• Two questions to answer:
  • Where are specific lipids located (inner vs. outer leaflet)?
  • Where are lipids being synthesized?
• Marking lipids:
  • Use a reagent that covalently binds to lipids but cannot cross the membrane to mark lipids on the outer leaflet.
  • Use radioactive precursors to label newly synthesized lipids.

Experiment with Radioactive Phosphate and TMBS

• Radioactive phosphate labels newly synthesized lipids.
• TMBS (tri-nitro-benzene sulfonic acid) reacts with amino groups on lipid head groups and cannot pass through the membrane.

Experiment Results and Interpretation

• Simultaneous incubation with radioactive phosphate and TMBS:
  • Radioactive lipids are never labeled with TMBS and vice versa.
  • This indicates lipids are synthesized in the cytoplasmic leaflet.
• Incubation with radioactive phosphate followed by TMBS after a delay:
  • Some lipids are both radioactive and labeled with TMBS.
  • This indicates lipids can transfer to the outer leaflet over time.

Lipid Transfer and Enzymes

• Lipids are synthesized on one leaflet and can be transferred to the other.
• Enzymes mediate transfer of specific lipids, causing asymmetric distribution.
• Flipases (phospholipid translocases) facilitate the movement of lipids from one leaflet to another.
• These proteins contain a hydrophilic group that allows the hydrophilic head group of the lipid to traverse the hydrophobic area of the membrane.

Thermodynamics of Transport

• Three subchapters in membrane transport:
  • Thermodynamics of transport
  • Mediated transport
  • Active transport

Channels and Transport Proteins

• Channels are usually gated (closed) to maintain ion gradients.
• Transport proteins:
  • Uniports: transport a single molecule
  • Symports: transport two molecules in the same direction
  • Antiports: transport two molecules in opposite directions
• Examples:
  • Sodium-potassium ATPase
  • Lactose permease

Why Transport is Needed

• Membranes are hydrophobic, hindering hydrophilic molecules from crossing.
• Transport mechanisms are needed to:
  • Move fuel and building blocks.
  • Export waste.
  • Transport molecules between organelles.
  • Regulate osmotic pressure and ion concentrations.
  • Maintain ion gradients.

Classification of Transport

• Non-mediated transport: doesn't require help.
  • Involves nonpolar molecules like oxygen and carbon dioxide.
• Mediated transport: requires assistance.
  • Passive: no energy input, from high to low electrochemical potential.
  • Active: requires energy input, from low to high electrochemical potential.

Driving Force and Chemical Potential

• The driving force is the chemical potential (partial molar free energy).
• $\Delta G = G<em>{\text{destination}} - G</em>{\text{origin}}$
• If concentration at origin > destination, ln(concentration ratio) is negative.

Membrane Potential

• The membrane potential is the charge difference across the membrane.
• Electrical potential depends on:
  • Ionic charge (z)
  • Potential difference ($\Delta \Psi$)
• $\Delta G = RT \ln \frac{[A]<em>{\text{destination}}}{[A]</em>{\text{origin}}} + zF\Delta \Psi$

Energetics of Transport

• Example: Transporting $Na^+$ from outside to inside:
  • If $\Delta \Psi$ is -100 mV (inside negative), it favors movement of positive ions inside.
• If concentration inside is larger than outside, it requires energy (non-spontaneous).
• Cells pay for the movement of one glucose molecule to maintain concentration gradients.
• This is why people do these types of analysis to understand the energetics of what they call secondary active transport.

Equilibrium and Membrane Potential

• Without membrane potential, equilibrium is reached when concentrations inside and outside are identical.
• Living cells must have a membrane potential.

Free Energy and Transport

• Movement of molecules driven by changes in free energy.
• For uncharged molecules:
  • $\Delta G = RT \ln \frac{[A]<em>{\text{destination}}}{[A]</em>{\text{origin}}}$
• For charged molecules and membranes:
  • $\Delta G = RT \ln \frac{[A]<em>{\text{destination}}}{[A]</em>{\text{origin}}} + zF\Delta \Psi$
  • z = ionic charge
  • $\Delta \Psi$ = potential at destination - potential at origin
• Volts = Joules/Coulomb
• F is in Coulombs/mol, $\Delta G$ is in Joules/mol

Mediated Transport Types

• Facilitated diffusion: from high to low electrochemical potential (negative $\Delta G$), no energy input.
• Active transport: from low to high electrochemical potential (positive $\Delta G$), requires energy.
  • Energy from ATP hydrolysis or downhill transport of another molecule.

Types of Transporters

• Carriers: small molecules that move back and forth.
• Porins: holes in the membrane that are always open.
• Channels: specific, usually closed, open in response to stimuli.
• Transport proteins: change structure like a revolving door.

Valinomycin

• Valinomycin: composed of 12 building blocks (dodecadepsipeptide).
  • Valine (L and D isomers)
  • Lactic acid
  • Hydroxyisovaleric acid
• Held together by peptide and ester bonds.
• Cyclic molecule with nonpolar side chains.
• Six carbonyls make hydrogen bonds with amide nitrogens, canceling polarity.
• Other six carbonyls coordinate with the ion in the center.

Specificity for Potassium

• Valinomycin is 10,000 times more specific for potassium than sodium.