Analysis of M4 transmembrane domains in NMDA receptor function: a negative allosteric modulator site at the GluN1-M4 is determining the efficiency of neurosteroid modulation

• Main Author: Langer, Kai
• Format: Others
• Language: en
• Published: 2021
• Online Access: https://tuprints.ulb.tu-darmstadt.de/18595/1/Dissertation_Kai_Langer_05_2021.pdf; Langer, Kai <http://tuprints.ulb.tu-darmstadt.de/view/person/Langer=3AKai=3A=3A.html> (2021): Analysis of M4 transmembrane domains in NMDA receptor function: a negative allosteric modulator site at the GluN1-M4 is determining the efficiency of neurosteroid modulation. (Publisher's Version)Darmstadt, Technische Universität, DOI: 10.26083/tuprints-00018595 <https://doi.org/10.26083/tuprints-00018595>, [Ph.D. Thesis]

Ionotropic glutamate receptors (iGluRs), like the NMDA receptor, are ligand-gated ion channels. These receptors make up an important part of the mammalian central nervous system (CNS) as they allow rapid excitatory signal transduction. NMDA receptors are built of four structurally identical subunits. Of the structurally identical subunits, three main types exist (GluN1, GluN2, GluN3), which are freely combined, with GluN1 always being incorporated twice. The transmembrane regions of these subunits consist of four alpha-helices named M1 – M4 domains. M1, M2 and M3 of all GluN subunits were identified to form the ion channel pore. Ligand binding at NMDA receptors leads to an opening of this pore which allows an ion flow across a membrane. The role of the M4 domain within the NMDA receptor is not yet clearly defined and was therefore investigated in this thesis. Herein approaches of M4-truncations, M4 segment coexpression and introduction of point mutations into the M4 domain were used to get an insight into the role of the M4 domain in functionality, assembly, and steroid modulation of NMDA receptors. It could be shown that the M4 domains of GluN1/GluN2A and GluN1/GluN3A NMDA receptors are not involved in receptor assembly nor in surface trafficking. However, M4-truncation lead to an impeded functionality of the analyzed ion channels that could be rescued by coexpression of the respective M4 segment indicating its importance in NMDA receptor function. Introduction of point mutations identified two residues in the upper part of the GluN1-M4 domain to be necessary for this rescue effect. These residues form interaction points of the GluN1-M4 domain with M1 or M3 domains of neighboring GluN2 subunits. The upper part of the M4 was thought to affect the modulation of NMDA receptors by neurosteroids like Pregnenolone sulfate (PS) and an involvement of this M4 part in neurosteroid modulation could be verified in this work. Furthermore, two further residues in GluN1-M4 were found to influence the affinity of PS to and the effect of PS on GluN1/GluN2 and GluN1/GluN3 receptors indicating these residues to build a negative allosteric modulation site for PS. All in all, this thesis gives new insights into the function of the M4 domain within GluN1/GluN2 and GluN1/GluN3 receptors highlighting the role in regulating receptor function. Furthermore, the herein obtained data allow a better understanding of NMDA receptor modulation by compounds with PS-like properties and therefore form a basis for further research investigating therapeutic strategies for selective NMDA receptor modulation.