Lecture Notes: Protein Localization, Ion Channels and Membrane Potential

Protein Transport and Localization

• Recap of protein design:
  • Signal sequence + stop transfer sequence = membrane protein.
  • Signal sequence without stop transfer sequence = secreted protein.
• Membrane protein example:
  • Receptor and signaling domain.
  • Transport: ER membrane -> vesicles -> Golgi -> circulatory vesicles -> plasma membrane.

Secreted Ligands

• Signal sequence recognized by SRP, brought to ER, transferred to translocation channel.
• Protein threaded into ER lumen without stopping.
• Signal peptidase cleaves the signal sequence, releasing the protein into the ER lumen.
• Secreted protein transport:
  • ER lumen -> vesicle lumen -> Golgi lumen -> secretory vesicle -> secreted out of the cell.
• Secretory pathway involves passing the plasma membrane through translocation channel into the ER lumen, then through vesicles and Golgi.
• Discussion sections and homework will cover tracking membrane and secreted proteins through the cell.
• Problem-solving focuses:
  • Effects of mutations on protein localization (e.g., if COP is broken).
  • Determining protein location (lumen or membrane of ER).
  • Predicting protein orientation in the plasma membrane based on signal and stop-transfer sequences.

Transmembrane Proteins

• Single-pass vs. multi-pass transmembrane proteins.
• Multi-pass protein design example: two stop transfer sequences with intervening sequence and an additional N-terminal sequence.
• Start transfer sequence not at the N-terminus is not cleaved by signal peptidase.
• Effect of N-terminal signal sequence on protein processing:
  • If present and at the tip, it gets cleaved by signal peptidase.
  • Absence of the leading sequence leads to the signal sequence being cleaved.
  • A 4-pass protein becomes a 3-pass protein when the N-terminal start-transfer sequence is cleaved.

Midterm Information

• Upcoming midterm next Wednesday.
• Content covered: protein localization and ion channels/membrane potential.
• Homework due this week is the last material covered on the midterm.
• SRP binds to start transfer sequences, bringing them to the ER membrane.
• Signal peptidase cleavage depends on the proximity of the signal sequence to the N-terminus.
• Cleavage is essential for secreted proteins; otherwise, all proteins would be membrane-bound. The cell would not be able to secrete proteins into the lumen of the ER.

Membrane Potential

• Cell membrane separates extracellular space from the cytosol.
• Hydrophobic membrane core prevents charged ions from passing through.
• Ions: e.g., sodium (Na^+), potassium (K^+).
• Ion channels: membrane proteins forming tunnels for specific ions to pass through.
• Selective ion channels: allow only certain ions (e.g., potassium) to pass.
• Potassium ion channel: very common in cells.

Sodium-Potassium Pump

• Creates ion concentration gradients.
• Pumps sodium out of the cell and potassium into the cell, using ATP.
• Results: high sodium concentration outside the cell, high potassium concentration inside the cell.
• Concentration gradients: approximately 10x more sodium outside, 10x more potassium inside. (See textbook table for exact concentrations.)
• Asymmetry: more sodium outside, more potassium inside.

Diffusion

• Ions trapped inside and outside generate no voltage difference
• Diffusion: Movement of molecules from high to low concentration.
• Convection: Bulk movement of fluid (e.g., water flowing).
• Diffusion demonstrated with food coloring in water: dye spreads even without water flowing.
• Brownian motion: random movement of molecules due to thermal energy.
• Diffusion at the molecular level: random motion, but net movement from high to low concentration due to probability.
• No preferential direction for individual atoms.
• Higher probability of movement from high to low concentration areas.
• Fick's Law: mathematically describes the rate of diffusion (covered in BME 150).

Potassium Leak Channels and Equilibrium Potential

• Potassium channels open allowing potassium diffusion out of the cell, following its concentration gradient.
• Potassium efflux leads to:
  • Negative charge accumulation inside the cell.
  • Positive charge accumulation outside the cell.
• Electric field creation: from positive to negative charges across the membrane.
• Electric field direction: positive to negative.
• Potassium ions are pushed into the cell by the electric field.
• Membrane potential development: potassium leakage causes a voltage change.
• Equilibrium potential: the point where the electric field counteracts the concentration gradient, reaching a stable voltage (e.g., -70 mV).

Voltage-Gated Sodium Channels and Action Potential

• Resting membrane potential: -70 mV.
• A bump in voltage causes the voltage-gated sodium channel (Na^+) to open, which allows Na^+ to enter the cell following both the electric field and the concentration gradient.
• Threshold voltage: the voltage required to trigger the opening of the voltage-gated sodium channel.
• Na^+ entry into the cell causes positive charge to enter the cell and leaving behind negative charge.
• Depolarization: sodium influx causes the membrane to become more positive, and this process happens very quickly.
• Polarity switch: electric field reverses direction as the cell becomes more positive until an electric field balances the diffusion of Na^+, which occurs roughly at +60mV.

Voltage-Gated Potassium Channels and Repolarization

• Green K^+ channel opens when voltage is more positive.
• Potassium channel opens when the voltage gets positive.
• Potassium efflux: potassium rushes out of the cell due to the electric field and concentration gradient.
• The voltage drops quickly as the positive charge leaves the cell.
• Repolarization: potassium efflux returns the membrane potential to the equilibrium potential.
• Action potential: rapid voltage spike and return to resting potential (about 2 ms).

Action Potential Significance

• Neurons use action potentials to send information. This is learned in BME 120.
• Equilibrium potential depends on:
  • Ion concentration gradient (inside vs. outside).
  • Ion charge (e.g., Cl^- is negative, Ca^{2+} is +2).
• Discussion section will cover equilibrium potential calculations in more detail.
• Membrane potential and electrical changes are crucial for:
  • Governing essential cell functions.
  • Muscle contraction.
  • Insulin secretion.