Lecture 5 - Carrier Mediated Transport Flashcards
Describe the structure of valinomycin
Valinomycin is a carrier protein with a ring like stricture composed from a trimeric repeating cycle of L-lactate, L-valine, D-hydroxy valeric acid and D-valine.
The outer ring of the protein is lipophilic meaning it can diffuse freely in the membrane.
It is selective to K+ over Na+ as K+ has a lower free energy of dehydration.
Describe valinomycins mechanism of transport.
- An empty pocket moves to the outer membrane due to the electrochemical gradient
- The K+ is dehydrated so it can bind to the pocket
- The pocket moves to the inner side of the membrane
- K+ is rehydrated and released
How good is valinomycin as a model carrier?
Valinomycin diffuses across the membrane and as a result exposes the ion binding sit alternately to each side of the membrane as evidenced in chilling experiments
The kinetic properties of valinomycin-mediated transport provides a useful kinetic model for carrier transport mechanisms
However valinomycin does not provide the basis for a molecular mechanistic model.
What are the kinetic properties of carriers?
- Binding of substrate from outside and formation of carrier substrate complex
- Re-orientation of carrier substrate complex to align binding site to inside
- Release of substrate
- Empty carrier reorientates
Carrier mediated transport can be described with Michaelis-Menten kinetics
Vmax = Transport rate independent of [Substrate] - reflects rate of carrier reorientation
Km = Transport rate dependent on substrate availability - reflects of binding and release of substrate
Kinetic constants help identify the physiological role of carrier
However valinomycin is a small peptide which diffuses across the membrane Gene cloning has revealed that most carriers are large proteins.
Describe uniport transport with GLUT2 as a model
- The glucose binding site is facing toward the outside and poised to bind glucose
- Glucose binds
- GLUT2 undergoes a conformational change to orientate the binding site to face inward
- Glucose is released
GLUT2 re-orientates to expose the biding site to the outside
Describe the structure function relationships for the carriers
E.g. The neurotransmitter sodium symporters (NSS)
* Major route for neurotransmitter clearance
* Require Na+ to drive uphill transport of neurotransmitters
* X-ray crystal structure reveals two hairpin loops which form gates which restrict the access to substrate binding sites.
* Alternating access model - alternate between different conformations to facilitate the movement of substances across a biological membrane, like the plasma membrane of a cell.
Give examples of some physiologically important carriers.
Facilitators
Sugar transporting GLUT proteins in mammalian cells - Major pathway for uptake of glucose into cells
* GLUT2 - Transporter of glucose between liver and blood, “glucose sensor” in pancreatic β cells, Insulin recruits more GLUT2 protein
* GLUT1 - Transporter of glucose to brain
Experiments demonstrating transport of glucose into red blood cells established the existence of membrane carriers. Use of D and L glucose. D-glucose the biologically active form demonstrated the existence of something that facilitated diffusion. The L isomer reflected simple diffusion across the membrane.
Yeast (Saccharomyces cerevisiae) glucose transport. The yeast evolved in a glucose rich environment so the uptake of glucose is not facilitated but other fungi uptake is coupled to H+ gradient to energise glucose uptake.
Secondary active transporters
* Na+ coupled sugar transport in mammalian cells
* 3Na+ - Ca2+ antiporter in cardiac muscle cell plasma membrane - maintains a Na+ gradient enabling Ca2+ efflux via the antiporter. Oubain is a treatment for heart failure that inhibits the carrier leading to elevated cytosolic Ca2+ increasing the force of heart muscle contractions
* Nutrient transport in plant roots - coupled to H+ gradient
