week 11: molecular mechanisms 22 Flashcards
(16 cards)
- Which of the following is a key difference between GlyT1 and GlyT2?
A) GlyT2 uses 2 Na⁺, GlyT1 uses 3 Na⁺
B) GlyT1 is found at inhibitory synapses
C) GlyT2 has a stronger glycine concentrating capacity
D) GlyT1 is found in presynaptic inhibitory neurons
Answer: C
- What is the likely mechanism of action for bioactive lipid inhibitors of GlyT2?
A) Directly competing with glycine at S1
B) Fully blocking transport by binding to S2
C) Binding to a separate extracellular allosteric site and slowing conformational changes
D) Blocking chloride ion co-transport
Answer: C
- Which statement about SERT inhibitors like SSRIs is TRUE?
A) They bind at the glycine binding site
B) They functionally act as competitive inhibitors by halting the transport cycle
C) They are non-competitive and irreversible
D) They enhance glycine availability for NMDA receptors
Answer: B
- What causes the severe side effects of early GlyT2 inhibitors?
A) They increased glycine release too much
B) They acted non-competitively and enhanced inhibition
C) They caused serotonin depletion
D) They irreversibly blocked glycine reuptake, halting glycine neurotransmission
Answer: D
- What best explains why NKCC1 and NSS transporters can be studied together?
A) They transport glycine and serotonin
B) They share the same amino acid sequence
C) They share the LeuT fold and similar alternating access mechanisms
D) They are both trimeric
Answer: C
- Compare and contrast the roles and ion coupling stoichiometry of GlyT1 and GlyT2.
Model Answer:
GlyT1 and GlyT2 are both glycine transporters in the NSS (SLC6) family, but they differ in their physiological roles and ion coupling:
GlyT1 is found mainly in glial cells at excitatory synapses and helps regulate extracellular glycine levels for NMDA receptor activation.
Stoichiometry: 1 glycine : 2 Na⁺ : 1 Cl⁻
GlyT2 is located in presynaptic inhibitory neurons at inhibitory synapses, where it recycles glycine for reuse in neurotransmission.
Stoichiometry: 1 glycine : 3 Na⁺ : 1 Cl⁻
This gives GlyT2 a stronger driving force and more efficient glycine reuptake.
- Explain why irreversible GlyT2 inhibition can lead to respiratory failure in rodents, while SERT inhibition does not cause similar toxicity.
Model Answer:
Irreversible GlyT2 inhibition blocks glycine reuptake at inhibitory synapses, depleting presynaptic glycine stores over time. Since glycine is crucial for inhibitory neurotransmission in the spinal cord and brainstem, its absence disrupts essential functions like motor control and breathing, potentially causing respiratory failure.
In contrast, SERT inhibitors (e.g., SSRIs) increase extracellular serotonin but do not deplete presynaptic stores or block all reuptake permanently. This modulates, rather than shuts down, serotonin signaling, making it safer in clinical use.
- Describe how bioactive lipid inhibitors affect GlyT2 activity and why they are considered non-competitive.
Model Answer:
Bioactive lipid inhibitors of GlyT2 bind to an extracellular allosteric site, not the glycine (S1) binding site. They do not prevent glycine binding or completely halt transport. Instead, they likely slow down conformational changes in the transport cycle, reducing the transport rate without full inhibition.
Because they bind outside the substrate site and their effects cannot be reversed by increasing glycine concentration, they are considered non-competitive inhibitors.
- Why are bioactive lipid GlyT2 inhibitors potentially more clinically viable than earlier GlyT2 inhibitors?
Model Answer:
Earlier GlyT2 inhibitors were irreversible and competitive, fully blocking glycine reuptake. This led to glycine depletion in presynaptic terminals and severe side effects, including respiratory failure in animal models.
In contrast, bioactive lipid inhibitors are non-competitive, reversible, and partial inhibitors. They allow some glycine transport, preserving inhibitory neurotransmission and reducing the risk of toxicity. This makes them more physiologically balanced and potentially safer for clinical use, especially in pain treatment.
- What structural feature allows EL4 to regulate substrate access in NSS family transporters?
Model Answer:
EL4 (Extracellular Loop 4) acts as a gate or lid in NSS transporters. After the substrate and ions bind in the extracellular vestibule, EL4 closes over the binding site, sealing the transporter and allowing the conformational change that shifts it to the inward-facing state.
This gating action of EL4 is crucial for alternating access and is also a target for inhibitors like SSRIs, which can prevent EL4 from closing, thereby arresting the transport cycle.
A drug binds in the extracellular vestibule of SERT and prevents EL4 from closing. What effect would this have on substrate transport, and why?
Model Answer:
If a drug binds in the extracellular vestibule and prevents EL4 from closing, the transporter will remain in the outward-open conformation. This halts the transport cycle because the closure of EL4 is required for the conformational shift to the inward-facing state that releases the substrate into the cytoplasm.
As a result, serotonin cannot be transported, leading to its accumulation in the synaptic cleft. This mechanism underlies the action of SSRIs, which functionally inhibit SERT and elevate serotonin signaling.
- Discuss how binding site location and functional outcome can differ for inhibitors—use SSRIs and bioactive GlyT2 inhibitors as examples.
Model Answer:
SSRIs bind to the S2 allosteric site in the extracellular vestibule of SERT. Although they do not occupy the main substrate site (S1), they functionally block serotonin transport by preventing conformational changes required for EL4 to close and the transport cycle to proceed. Therefore, SSRIs are functionally competitive, even though they bind allosterically.
Bioactive GlyT2 inhibitors also bind allosterically, but at a different extracellular allosteric site near the lipid membrane. They do not prevent glycine binding or EL4 movement, but likely slow the conformational transitions. Thus, they do not fully block transport, making them non-competitive and safer in physiological systems requiring continuous glycine recycling.
- Explain how the rocking bundle mechanism differs from the elevator mechanism, and identify which is used by NSS transporters like GlyT2 and SERT.
Model Answer:
The rocking bundle mechanism involves two domains: one relatively rigid scaffold and one mobile domain that “rocks” back and forth to alternate access to the substrate binding site from one side of the membrane to the other.
In contrast, the elevator mechanism involves a large vertical movement of a transport domain that physically moves the bound substrate across the membrane, like an elevator.
NSS transporters (e.g., GlyT2, SERT, DAT) use the rocking bundle mechanism, as shown by structural studies of LeuT and dDAT.
- How can site-directed mutagenesis and molecular dynamics (MD) simulations help identify allosteric binding sites on transporters?
Model Answer:
Site-directed mutagenesis allows researchers to mutate specific amino acids in a transporter to test whether they affect the binding or function of a drug or inhibitor. If mutating certain residues abolishes inhibition, those residues likely form part of the binding site.
Molecular dynamics (MD) simulations model how molecules move and interact over time. They can simulate how an inhibitor docks or “snorkels” into a transporter and interacts with specific residues, helping to visualize binding in the absence of a crystal structure.
Together, these techniques were used to show that bioactive GlyT2 inhibitors bind a novel extracellular allosteric site distinct from the substrate binding pocket or vestibule site.
- Why is the glycine clearance system particularly sensitive to transporter inhibition compared to dopamine or serotonin systems?
Model Answer:
The glycine clearance system is tightly coupled to inhibitory neurotransmission in the spinal cord and brainstem. GlyT2 is essential for recycling glycine into presynaptic terminals for reuse. If GlyT2 is fully inhibited, glycine cannot be recycled, leading to depletion of vesicular glycine, impaired synaptic inhibition, and severe physiological consequences (e.g., respiratory failure).
In contrast, dopamine and serotonin systems tolerate some degree of inhibition (e.g., by SSRIs or DAT inhibitors) because:
Neurotransmitters can persist in the cleft for longer without immediate failure
Their transporters are not the sole route of neurotransmitter clearance
These systems modulate mood and behavior, not essential reflexes like breathing
NKCC1 and KCC2 transporters.
epilepsy
NKCC1 brings Na⁺, K⁺, and 2 Cl⁻ into the neuron
→ This increases Cl⁻ concentration inside the cell
when GABA binds, accidentaly excitaotry as Cl- flows out, down conc gradient and out of the cell, causing positive charge == depolarisation, excitation of the neuron
KCC2 exports Cl⁻ out of the neuron, along with K⁺
→ This lowers internal Cl⁻ levels
when GABA binds, inhibitory as Cl- flows in, down conc gradient and in of the cell, causing negative charge == hyperepolarisation, inhibtiation of the neuron
🔁 What Happens in Epilepsy or Trauma?
KCC2 is downregulated (less active)
NKCC1 becomes dominant again
Cl⁻ builds up inside the neuron
GABA becomes depolarizing again → leads to excess firing → contributes to seizures
