2c Membrane Transport

Highly conserved family of transport proteins found across all phyla.
Minimum conserved structure:
- Two ATP binding domains (bottom).
- Two transmembrane domains.
Additional domains may be fused to these core elements.
Mechanism:
- Small solute enters the site, triggering a conformational change.
- ATP binds to ATPase domains and is hydrolysed (phosphorylation reaction).
- This opens the protein channel, allowing the molecule to leave the cell.
- Two ATP domains filled leading to two ADPs and a reset to the original conformation.

ABC transporters are highly expressed in many human cancer cells.
They pump drugs out of the cell, making cells resistant to cytotoxic drugs used in chemotherapy.
Transmembrane drug efflux pumps reduce intracellular drug concentration, generating resistance.
Certain drug efflux pumps extrude multiple types of drug molecules, leading to multidrug resistance (MDR).
P-glycoprotein (PGP) is the most prevalent MDR transporter.
- Expressed at elevated levels in many kinds of cancer cells, especially those that have survived chemotherapeutic treatment.
- A drug molecule binds to the pump's drug-binding pocket from the cytosol.
- ATP is required to pump drugs out of the cell.
- ATP must bind to the interior nucleotide-binding domains.
ATP binding causes a dramatic conformational change in the pump.
- Drugs or other toxins enter the pump and attach to the drug-binding pocket, resulting in an ATP-dependent shift in the conformation of the entire pump.
- The drug is then extruded into the extracellular space.

Transport against the electrochemical gradient (uphill).
Electrochemical gradient: more positive outside/top, more negative inside/bottom.
Types:
- Coupled Transporter: One transported molecule moves down its concentration gradient, driving the transport of another solute against its gradient.
- ATP-driven pump: Hydrolysis of ATP drives transport against the electrochemical gradient.
- Light-driven pump: Energy from light (e.g., bacterial rhodopsin in bacteria and archaea) drives transport of a solute (usually a proton) against its gradient.

Different parts of the cell interact with different environments, enabled by asymmetric transporter distribution.
Example: Intestinal epithelial cell:
- Gut lumen (orange) at the top, extracellular fluid (blue) at the bottom.

Non-uniform distribution allows transport of glucose from the intestinal lumen to the extracellular fluid and eventually to the blood.
Apical domain: Glucose enters via a symporter (both sodium and glucose enter together) against its concentration gradient.
- Sodium is higher in concentration outside the cell, so it moves down its concentration gradient.
Basal domain: Glucose exits through a glucose transporter via facilitated diffusion (high to low concentration, no energy needed).
Sodium is pumped back outside of the cell via the sodium-potassium pump (against its concentration gradient).
- Potassium is pumped from a low concentration outside the cell to a high concentration inside the cell.
- Energy-driven pump maintains the sodium gradient.

Two sodium ions to one glucose molecule enter the cell via the symporter.
Sodium enters with its electrochemical gradient; glucose is pumped against its concentration gradient.
Protein goes through alternate states: occluded, empty, outward opening.
Binding of one ligand increases the affinity for the second ligand (e.g., sodium binding first increases affinity for glucose).
When both molecules are bound, the protein closes and reopens towards the cytosolic side, releasing sodium and glucose into the cell.

Facilitated diffusion alone would only allow glucose to equilibrate across the membrane, leading to loss of glucose.
Active transport is required.
Apical side of the cell membrane allows facilitated diffusion when glucose levels are high in the intestines.
When glucose levels are high in the cell and low in the capillaries, GLUT2 allows facilitated diffusion from the cell.

Sodium-potassium ATPase maintains a low sodium concentration and a high cytosolic potassium concentration.
- ATP-dependent; pumps sodium out of the cell (against its gradient) and potassium into the cell (against its gradient).
SGLT1 (on the apical side) allows coupled transport of glucose into the cell against its concentration gradient, driven by the sodium gradient.
- Transport of glucose occurs with the transport of two sodium molecules.
GLUT2 (at the basal membrane) allows glucose to leave the cell into the capillary by facilitated diffusion.
This system allows glucose to flow from high to low levels, or to be pumped against the concentration gradient to maintain storage and reduce waste.

$K_m$ is a marker of the affinity of a protein for its substrate (in this case, glucose).
GLUT2: $K_m$ = 7-20 millimolar (lower affinity).
SGLT1: $K_m$ = ~2 millimolar (higher affinity).
SGLT1 has a higher affinity for glucose because it works when glucose levels are low and needs to move glucose against its gradient.
Net glucose uptake: the coupled reaction between SGLT1 and the sodium-potassium ATPase allows for three glucose molecules to be taken into the cell for every two ATP used.

Key points: