TY - BOOK
T1 - Studies on the C-terminal domain of the NMDA receptor subunit GluN1: allosteric modulation and channel properties
AU - Sevillano Quispe, Oscar Gabriel
PY - 2023/1
Y1 - 2023/1
N2 - The NMDA receptor (NMDAR) is a tetraheteromeric, ligand-gated ion channel involved in synaptic plasticity, memory, and learning. Although the receptor has been subject of several structural and functional studies, especially of its agonist-binding domain, the role of its long, disordered C-terminal domain (CTD) in channel function remains poorly understood. To study the role of the CTD, GluN1-1a mutants were designed, with particular focus on positive residues on cassettes C0 and C1, close to its transmembrane domain. We used the CTD-dependent NMDAR positive allosteric modulators pregnenolone sulfate (PS) and UBP684 to screen for relevant residues, and the effect they had on NMDAR function was measured using two-electrode voltage clamp on Xenopus oocytes. We found that a double mutant 840HK to AA, completely inhibits UBP684 potentiation, similar to a full CTD truncation, while decreasing PS potentiation unlike the truncation. We also observed that mutating the four basic-residue cluster 838KRHK into alanines increases the glutamate-evoked response. We determined that this effect is, at least in part, due to an increase in the open probability, by measuring the time constant of the decay when applying the open channel blocker MK-801, and by calculating the open ratio when applying the thiol-modifying agent MTSEA on A651C-GluN2B receptors. These studies provide a novel means of understanding the mechanisms that govern CTD-dependent NMDAR activity and advance our understanding of the molecular mechanisms that underlie synaptic plasticity.
AB - The NMDA receptor (NMDAR) is a tetraheteromeric, ligand-gated ion channel involved in synaptic plasticity, memory, and learning. Although the receptor has been subject of several structural and functional studies, especially of its agonist-binding domain, the role of its long, disordered C-terminal domain (CTD) in channel function remains poorly understood. To study the role of the CTD, GluN1-1a mutants were designed, with particular focus on positive residues on cassettes C0 and C1, close to its transmembrane domain. We used the CTD-dependent NMDAR positive allosteric modulators pregnenolone sulfate (PS) and UBP684 to screen for relevant residues, and the effect they had on NMDAR function was measured using two-electrode voltage clamp on Xenopus oocytes. We found that a double mutant 840HK to AA, completely inhibits UBP684 potentiation, similar to a full CTD truncation, while decreasing PS potentiation unlike the truncation. We also observed that mutating the four basic-residue cluster 838KRHK into alanines increases the glutamate-evoked response. We determined that this effect is, at least in part, due to an increase in the open probability, by measuring the time constant of the decay when applying the open channel blocker MK-801, and by calculating the open ratio when applying the thiol-modifying agent MTSEA on A651C-GluN2B receptors. These studies provide a novel means of understanding the mechanisms that govern CTD-dependent NMDAR activity and advance our understanding of the molecular mechanisms that underlie synaptic plasticity.
M3 - Ph.D. thesis
BT - Studies on the C-terminal domain of the NMDA receptor subunit GluN1: allosteric modulation and channel properties
PB - Århus Universitet
ER -