Lecture 15: Glucose Transport Flashcards
Name the three modes of transport for Na+, which is the symporter and which is the antiporter? What drives the Na/glucose symporter?
Na/k ATPase, Na/glucose symporter and glucose transporter (GLUT) antiporter. The na/k atpase creating high conc of extracellular Na+
Describe the properties of GLUTs.
- faster transport than diffusion as don’t need to move through the hydrophobic bilayer
- are specific to a single type of molecule
- reversible transport dependent on conc. gradient
What is the KM for glucose transport and what does this mean?
1.5mM so at this conc half the glucose transporters have bound glucose and transport is 50% of the max. rate.
How many TM domains do human GLUTs have? What are all family members? Where are the N and C terminal? how many classes, which TM are important for substrate binding?
12, glycosylated at different sites, cytosol, 3, TM7, 11, 12.
Which is the best understood and where is it found?
GLUT1, at the PM of most mammalian cells especially on the erythrocyte PM.
Why have GLUTs been studied in erythrocyte cells lots?
because they have no nucleus or organelles and easy to purify.
How are GLUT1 and 4 alike?
Sugar moiety found in both, similar substrate binding residues, similarity in intracellular loops and domains.
What is XylE?
A d-xylose transporter found in E.coli
What is the architecture of XylE?
12 TM formed of 2 x 6 helical domains with cytoplasmic N and C terminals.
Where is the sugar binding site of XylE? What is its substrate?
toward the C terminal and the substrate, D-cylose can access it from either side of the protein.
What residues form the proposed binding site of XylE?
Q282, Q283, N288 from TM7 and N317 from TM8 and N415 from TM11.
What is the N domain of XylE responsible for?
regulating the conf changes required for transport
What does the intracellular domain of XylE do?
Block the xylose from being released.
Compare the structures of XylE and GLUT1-4.
intracellular helices are conserved in GLUT1-4 and the residues that mediate inter domain interactions are conserved between XylE and GLUT1-4. There is a lot of conservation in the transmembrane domains.
How does D-xylose coordinate to XylE?
it is hydrogen bonded to the C domain by polar and aromatic residues. The aromatic residue, Tyr298 may prevent prevent D-xylose leaving to the extracellular side.
Which mutation in XylE has no effect?
N325
What can inhibit the transport of D-xylose and why?
D-glucose because they have similar chemical structures
What has more prominent function in binding D-glucose? How?
Gln168 as it forms 3 H bonds with it
What is similar between D-glucose recognition and GLUT1-4?
all residues apart from 2 that mediate D-glucose recognition are invariant in GLUT1-4
What does the latest GLUT1 model have?
4 alpha helices on the intracellular side
What should be very similar and why?
Recognition of D-glucose by GLUT1-4 as should be nearly identical to that observed in D-glucose bound to XylE as the vast majority of AAs involved in substrate binding are invariant between Cyle and GLUT1-4.
Mutations at which amino acids are predicted to impair conformational flexibility?
Gly73 and Gly130
When was the structure of GLUT1 predicted and from what?
2012-13 from bacterial
What happens in the mammalian brain? In what tissues is this the opposite?
They have constitutively high glucose requirement so GLUTs1-3 and constitutively targeted to the cell surface. Muscle and adipose tissue
How to erythrocyte cells ensure more glucose will keep entering the cell?
Glucose is phosphorylated to glucose -6-phosphate which cannot leave the cell back through a GLUT and means intracellular glucose levels are low so glucose keeps coming in down the conc gradient.
Where is GLUT4 predominantly expressed??
Adoposytes and muscle cells.
Where inside the cell is GLUT4 mainly distributed?
endosomes, trans-golgi network and endosomal sorting intermediate structures and GLUT4 storage vesicles (GSVs)
What happens to GFP-Glut4 when trigger cells with insulin?
get aggregation at the PM from
What is the ICH domain of GLUT1?
The intracellular helix bundle
What does GLUT4 translocate to the PM in response to?
Inslulin
What proportion of the insulin stimulated glucose uptake occurs in skeletal muscle and adipocytes?
90 and 10%
What are GSVs and what are they formed from?
GLUT specialised vesicles. Endosomes or TGN.
What do GSVs consistantly do but what limits their binding?
Sample the PM but are limited by lack of docking sites or tethering proteins.
How does ARF6 drive vesicle formation?
it is on the donor membrane and it rectuite adapter proteins which interact with clathrin and GSV resident proteins
What does the insulin receptor initiate and What protein creates targetting sites for the GLUT4 GSVs?
TC10 GTP bound
What does the insulin receptor initiate the pathway of? What is the consequence?
PI3K signalling cascade which recruits PISK which converts PIP2 to PIP3 which recruite PDK1 and AKT. Activation of AKT by PDK1 promotes GSV exocytosis
What is defective in diabetes 2?
Translocation of GLUT4 to PM
What is glucose stored as in adiposytes?
Fat
