week 10 Molecular mechanisms of pumps Flashcards
(17 cards)
Q3. Explain the catalytic cycle of Na⁺/K⁺-ATPase and its stoichiometry.
Model Answer:
Cycle Steps:
3 Na⁺ bind intracellularly.
ATP is hydrolyzed; aspartate is phosphorylated (D369).
Conformational change; Na⁺ is released extracellularly.
2 K⁺ bind from extracellular space.
Dephosphorylation of aspartate.
Conformational change returns pump to inward-facing state.
K⁺ is released intracellularly.
Stoichiometry: 3 Na⁺ out, 2 K⁺ in per ATP hydrolyzed. This generates an electrogenic gradient.
Q1. Describe the five classes of transporters in the Transporter Classification (TC) system. Which classes are covered in this course?
Model Answer:
The TC system categorizes membrane transporters into five broad classes:
Class 1: Channels and pores (e.g. α-helical channels, β-barrel porins)
Class 2: Electrochemical potential-driven transporters (secondary active transporters using symport or antiport)
Class 3: Primary active transporters (e.g. ATPases, ABC transporters)
Class 4: Group translocators (e.g. PTS system in bacteria)
Class 5: Transmembrane electron carriers (e.g. oxidoreductases)
Q2. Compare and contrast P-type, F-type, V-type, and ABC transporters.
Model Answer:
P-type ATPases: Use ATP to form a phosphorylated intermediate (e.g. Na⁺/K⁺-ATPase). Essential for ion homeostasis.
F-type ATPases: Function in ATP synthesis (e.g. mitochondrial ATP synthase), driven by proton gradients.
V-type ATPases: Pump protons to acidify compartments like lysosomes.
ABC transporters: Use two nucleotide-binding domains; transport diverse substrates; can mediate influx or efflux.
Q4. What structural features enable the Na⁺/K⁺-ATPase to function effectively?
Model Answer:
α-subunit: Contains the catalytic domains (P, A, N) and transmembrane helices.
β-subunit: Assists with membrane trafficking and stabilizes K⁺ affinity.
γ-subunit (FXYD): Modulates pump activity by altering Na⁺/K⁺ affinity.
Q5. List and explain two physiological roles of Na⁺/K⁺-ATPase in cells.
Model Answer:
Maintains resting membrane potential: Drives membrane potential to ~-70 mV, crucial for neuronal excitability.
Regulates osmotic balance: By moving Na⁺ out and K⁺ in, it prevents cellular swelling.
Q6. What are some diseases associated with mutations in Na⁺/K⁺-ATPase?
Model Answer:
Hypertension: Mutations lead to excess aldosterone production.
Neurological disorders: Familial hemiplegic migraine, rapid-onset dystonia Parkinsonism, alternating hemiplegia of childhood, CAPOS syndrome.
Cancer: Upregulation of FXYD3/5 in some tumors.
Q7. How do cardiotonic steroids like ouabain affect the Na⁺/K⁺-ATPase? Why is this medically relevant?
Model Answer:
Ouabain inhibits Na⁺/K⁺-ATPase by binding to extracellular TM domains.
This reduces Na⁺ gradient → reduces Na⁺/Ca²⁺ exchanger activity → increases intracellular Ca²⁺.
Used in heart failure treatment to enhance cardiac contractility.
Q1. Distinguish between uniporters, symporters, and antiporters. Give examples of each.
Model Answer:
Uniporters: Passive transporters that move a single substance down its concentration gradient. Example: GLUT1 (glucose transporter).
Symporters: Secondary active transporters that move two or more substances in the same direction. Example: EAATs (glutamate and Na⁺ co-transport).
Antiporters: Move substances in opposite directions. Example: Na⁺/K⁺/Cl⁻ cotransporter NKCC1.
Q2. Describe the ion coupling stoichiometry of EAATs and explain how this drives glutamate uptake.
Model Answer:
For each glutamate transported:
3 Na⁺ and 1 H⁺ are co-transported into the cell.
1 K⁺ is counter-transported out of the cell.
This results in a net positive charge (2+) into the cell, driving glutamate uptake against its concentration gradient.
Q3. Explain the clinical relevance of EAAT malfunction.
Model Answer:
EAAT failure, especially due to Na⁺/K⁺-ATPase dysfunction (e.g., during stroke), leads to impaired glutamate clearance.
This causes excitotoxicity due to excessive glutamate and Ca²⁺ influx via NMDA receptors, potentially resulting in cell death.
Q4. What is GltPh and why is it used as a model for EAATs?
Model Answer:
GltPh is a Na⁺-dependent aspartate transporter from Pyrococcus horikoshii.
It shares 37% identity with human EAAT2 and forms a trimeric structure.
It has been used to understand the structural and mechanistic basis of glutamate transport in mammals.
Q5. What transport mechanism does GltPh utilize?
Model Answer:
GltPh operates via the elevator mechanism:
The transport domain picks up substrate near the extracellular side.
It moves vertically within the membrane to release substrate intracellularly.
This is distinct from the rocker-switch mechanism in MFS transporters.
Q6. EAATs are both transporters and channels. Explain.
Model Answer:
EAATs also function as Cl⁻ channels, independent of their glutamate transport role.
These processes are thermodynamically uncoupled.
The Cl⁻ channel may assist in charge neutralization or osmotic balance during glutamate transport.
Q7. What is L-TBOA and how does it affect EAAT function?
Model Answer:
L-TBOA is a competitive inhibitor of EAATs.
It mimics aspartate but includes a benzyl ring that prevents hairpin closure needed for substrate occlusion.
This freezes the transporter in a non-functional conformation.
Q8. Describe how cysteine proximity assays were used to study transporter conformations.
Model Answer:
Two residues are mutated to cysteine and treated with oxidizing agents (CuPh).
Disulfide bond formation locks conformational states.
Functional changes are assessed by electrophysiology or SDS-PAGE mobility shifts, revealing proximity and movement of specific domains.
Q9. Compare the transport mechanisms of GLUT1 and EAATs.
Model Answer:
GLUT1 (MFS family) uses the alternating access model, with two 6-helix bundles rotating to expose the substrate site alternately.
EAATs use the elevator mechanism where the substrate-binding domain moves vertically.
GLUT1 is a uniporter (passive), while EAATs are symporters (secondary active).
Q10. How is Na⁺/K⁺-ATPase linked to EAAT function and what happens during ischemia?
Model Answer:
Na⁺/K⁺-ATPase maintains the Na⁺ gradient necessary for EAAT function.
During ischemia, ATP depletion leads to pump failure.
Result: Na⁺ gradient collapses, EAATs can’t clear glutamate → excitotoxicity → neuronal death.
