CBIO3400 Lecture on Transport Across Biological Membranes

Passive and Active Transport

• Introduction to Transport Across Biological Membranes
  • Understanding the mechanisms of nutrient transport in cells, critical for health and function.

In-Class Activity

FRAP - Technical Overview

• Photobleaching experiment to study membrane dynamics.

  • Using a Laser Beam: A specific region of the membrane is treated with a laser beam resulting in the bleaching of fluorescently labeled lipids in that area.

• Recovery Phase: Following bleaching, the recovery of fluorescence in the bleached area is monitored over time, indicating the movement of unbleached lipids into the area.

  • Diagram Fig. 10-33 in MBOC describes the experimental setup.

Atorvastatin's Impact on Cell Membrane

• Experiment conducted to observe atorvastatin's effect on membrane fluorescence.

  • Experimental Setup: Cells treated with atorvastatin (cholesterol-synthesis inhibitor) and fluorescent dye incorporated into lipids.

  • Question Focus:

  • How does atorvastatin affect dye fluorescence?

  • What implications does this have for overall cell membrane properties due to cholesterol reduction?

Predicting Transmembrane Structures

In-Class Activity #2

Key Considerations

1. Overall length of the polypeptide chain (number of amino acids).
2. Hydropathy Plot Analysis:
   • X-axis = Amino Acids in Polypeptide Chain.
   • Y-axis = Hydrophobicity/Hydrophilicity scale.
3. Identification of candidate transmembrane (TM) helices.
   • If a hydrophobic stretch ≥20 amino acids, it might be a transmembrane domain.

• Example: Human Growth Factor Receptor examined using hydropathy plots.

Types of Membrane Transport

Learning Objectives

1. Need for transporters in cells.
2. Biological and medical importance of transporters.
3. Structure of transporters.
4. Energy sources for transporters.
5. Mechanistic understanding of transporter function.

Transport Mechanisms

1. Passive Transport: Movement along a concentration gradient without energy expenditure.

   • Types:
     • Diffusion: Movement of solute from high to low concentration.
     • Facilitated Diffusion: Requires transmembrane proteins for solute transport.

2. Active Transport: Movement against a concentration gradient, which requires energy (usually from ATP).

   • Utilizes various types of pumps including ATPase and co-transport mechanisms.

Membrane Permeability

• Biological membranes act as barriers for molecules.
• Permeability is affected by size, concentration, and specific membrane properties.

Transport Testing

• Methodology to determine the diffusion or transport characteristics of molecules across membranes.

Symport and Antiport

• Symport: When one solute moves down its gradient, it provides energy for another solute to move against its gradient.
• Antiport: Two solutes moving in opposite directions across the membrane, may involve one moving against and the other down its gradient.

Example of Sugar Transport

Steps of Glucose Transport into the Bloodstream

1. From Intestinal Lumen to Gut Cell:
   • Mechanism: Symporter involving Na+/Glucose.
2. From Gut Cell to Bloodstream:
   • Mechanism: Uniporter facilitating glucose diffusion.
3. Sodium Transport to Blood:
   • Mechanism: Na+/K+ ATPase, an active transporter.
4. Potassium Uniporter:
   • Restores ionic balance in gut cells.

Energy Sources for Transporters

• Transporters can use gradients formed by ion concentration differences to facilitate their actions.
• Ion Gradient Example:
  • 1 Na+: Enables enhanced glucose transport by a factor of 170.
  • 2 Na+: Enables a 30,000-fold enrichment of glucose transport against its gradient.
• Calculated Energy for Ion Movement:
  • For 1 Na+, $ext{ΔG} ext{ ~ -1.5 kcal/mol}$, for 2 Na+, $ext{ΔG} ext{ ~ -3.0 kcal/mol}$.

ATP-Dependent Active Transporters

Classes of Pumps

1. P-type Pump: Phosphorylates itself during transport.
2. ABC Transporter: ATP-binding cassette transporters.
3. V-type Pump: Acidifies organelles (e.g. vacuoles).
4. F-type Pump: Produces ATP.

Na+/K+ ATPase Function

• Maintains ionic gradients: high K+ and low Na+ within cells.
  • Concentrations: 5 mM Na+ outside, 140 mM Na+ inside; Vice versa for K+ (145 mM outside, 5-15 mM inside).

Structural Features of Na+/K+ Pump

• Key Domains:
  • ATP-binding domain
  • Phosphorylation domain
  • Na+ and K+ binding pockets.

Ion Channels

Types and Functions

1. Voltage-gated Ion Channels: Respond to changes in membrane potential.
2. Ligand-gated Ion Channels: Open upon binding of specific ligands.
3. Mechanosensitive Ion Channels: Open in response to mechanical stimuli.

Key Characteristics of Ion Channels

• Allow transport via hydrophilic pores at speeds of up to 100 million ions per second.
• Highly selective for specific ions.

Bacterial Potassium (K+) Channel

Structure and Function

• Composed of tetramer subunits: 2 transmembrane helices and a P-segment containing a selectivity filter.
• Mechanism:
  • Carbonyl oxygen in the selectivity filter attracts K+ while repelling Na+.

Regulation of Ion Channels

• Channels can exist in open or closed states modulated by various factors (voltage changes, ligands, etc.).

Behavioral Implications of Light-Activated Ion Channels

• Use of optogenetic techniques to manipulate aggressive behavior in mice by activating specific ion channels in the brain.

Nicotinic Acetylcholine Receptor

Role in Signal Transmission

• Functions at the neuromuscular junction, facilitating the transmission of signals necessary for muscle contraction via ion movement.

Comparative Characteristics of Transporters and Channels

Similarities

• Both form hydrophilic channels through membranes.
• Specificity for their solutes.

Differences

• Transporters: Change conformation for solute transport and require energy each time.
• Channels: Enable rapid ion transfer and respond dynamically to external stimuli (gating factors).

Suggested Reading

• MBOC Chapter 11 and Lodish Chapter 11 for deeper understanding of transport mechanisms.
• Lecture videos and additional questions posted on eLC for supplemental study.