Transporters Flashcards
symporter
Driving against the conc. gradient so need to couple transport to gradients of other molecules
antipoerter
Utilises energy gradient to allow passage
What is a solute carrier protein (SLC)
> 50 structurally related sub-classes
- 300 individual proteins
Involved in numerous process:
- Nutrient uptake
- Elimination of drugs
- Vesicle loading at synapse
- Neurotransmitter uptake
Targets for:
- Neurological conditions
- Diuretics
- Diabetic
Compare the SLC structure - Major facilitator superfamily and LeuT family
Major facilitator superfamily:
12 TMD
Two inverted pseudo-repeats (2 independently folded domains)
GLUT(SLC2), H+/peptide(SLC15)
leuT Family:
10 TMD
Two 5 TMD inverted repeats
Na+/Glucose (SLC5)transport, Na+/Cl- neurotransmitter uptake (SLC6)
Explain SLC in the CNS
Involved in glutamate reuptake (SLC1)
Transport of glutamate into vesicles (SLC17)
reuptake of monoamines (SLC6)
Explain vesicular transporters
- Transport driven by electrochemical H+ gradient created by vATPase
- Cations (e.g. ACh) – utilize ΔpH (the pH gradient) (exchange 2H+ for cation)
- Zwitterions (e.g. GABA, Gly) – utilize ΔpH and Δψ (electrical potential across the bilayer)
- Anions (e.g. Glu) – utilize primarily Δψ
Describe the transport process for a glutamate transporter
Glu binding site contains 2 Arganine residues - R88 and R322
Allosteric regulation by H+ (H199 and H434)
Glutamate exchanged with Cl- to offset charge
At low pH (5.6)(when internalised) transporter active, Cl- transported in exchange for Glu
At neutral pH (7.4) (when vesicle fuses with PM) transport blocked
Structure of neurotranmitter transporters
First structure/function from bacterial homologue Leucine transporter (LeuT)
12 TMD : twofold symmetry (11 and 12 - additional, not part of duplication event)
Broken TMD form binding site for ligand and Na+ ions
Structure solved in outward facing conformation
Pharmacophores involved in binding clearly not the same though!
Explain the serotonin transporter (SERT)
Transports Serotonin/5-HT, attenuating signalling through uptake from synapse
Utilizes Na+/Cl- gradient to drive transport
Target for antidepressants and physchostimulants: Inhibits uptake of serotonin so it acts on receptor for longer
Challenges of studying transporter structure
Integral membrane proteins
Unstable in detergent micelles
Mobile – by their very nature they are flexible
Solutions to challenges of studying transporter structure
Identify thermostable mutations :
- I291A, T439S (ts2) Km=4.5 μM, Vmax=21 pmol min-1 (WT: 1.9 μM, 23 pmol min-1)
- Y110A (ts3) No transport activity
Co-crystallize with Fab fragments (antibody fragments) to stabilize the structure
Architecture of SERT
Outward facing conformation
Location of mutations associated with disease.
Not dimeric, but have arisen through duplication with an inverted topological repeat.
Contain 10 TMD’s
Binding site halfway across the lipid bilayer
Ion binding sites halfway across the lipid bilayer
Coupling Na+/Cl- transport to seretonin
Na+/Cl- form part of the substrate binding site - in doing is help form it
Essentially coupling the transport of ions with the transport of serotonin
Located close to breaks in TMD I and VI
Transport tightly coupled
Drugs for SERT
Ibogaine
- Inhibitor – used to combat addiction.
Anti-depressants
- sertraline
- fluvoxamine
- paroxetine
(all relatively high binding affinity (Coleman 2018))
Anti-depressants act by binding to a central substrate binding cavity and locking the transporter in an open outward facing conformation.
Explain the binding of Paroxetine to SLC6
3 binding subsites that seem to are important
A Ring structure binds (from aromatic residue)
B Tryptophan side chain binds
Phenol group binding to subside C
Important that drugs possess all three of these properties for effective binding and therefore inhibition
Explain binding of Sertraline to SLC6
Again requires subsite A B and C binding for inhibition
Interactions that stabilise ligand binding site (SERT)
H-bonds from Ser/Thr to halide sites (near chloride binding site)
Aromatic interaction PheTyr and aromatic rings (pi-pi interactions between drug and binding sites)
Explain the techniques for discovering the structure of the transports at different steps of the catalytic transport cycle
SERT/Ibogaine/Fab (take SERT bound Ibogaine with an antibody fragment):
- Outward: High NaCl
- Occluded - medium salt conc
- Inward : Low NaCl/KCl
by modifying different buffer contributions they were able to drive SERT into different confirmational states
Fab fragments used to aid refinement – provides orientation and features
single particle analaysis using Cryo-EM to investigate the different steps of the transport process (~4.1Å) and their structure
Topology of open outward state
6TMD tilted outwards, substrate binding site accessable to outside the cell
1st TMD tucked in, blocking passage to inside of cell
Topology of Occluded state
6TMD -swung in, blocked access to outside of cell
1TMD still in also
Topology of open-inward state
first TMD swinging out to provide access to the inside of the cell from the binding site
Transport cycle: Gating
Outward facing vestibule
Ibogaine binds
helix 1b moves up and blocks vestibule from outside - now have occluded state with substrate trapped in centre
Movement of 6b and 1a - allows the inward facing vestibule conformation, due to binding site now being a low affinity site, molecule will break off into the cell
Challanges of cryo-EM for analysis of integral membrane proteins
Resolution 4.1Å
Substrates/ligands 10’s Å
Need higher resolution data to accurately fix the position of the sidechains/substrates
Allosteric regulation of transporters
Allosteric site between:
TM1s, TM6b, TM10, TM11, EL4 and EL6
Allosteric site malleable
Potential for a spectrum of inhibitors
Occludes the substrate binding site
Limits disassociation
