Abstract:
The NMDA receptor is a ligand-gated ion channel that mediates a fundamental component of excitatory synaptic transmission in the mammalian central nervous system. Aberrant NMDA receptor function causes and/or exacerbates multiple neurological and psychiatric disorders, thus creating the need for pharmacological modulation of its activity. The defining feature of ligand- gated ion channels is gating--the energetic coupling of ligand binding into opening of the associated ion channel pore. In an NMDA receptor subunit, gating is initiated in the extracellular ligand-binding domain (LBD) and is propagated via three linkers--S1-M1, M3-S2, and S2- M4--to the transmembrane domain (TMD) forming the ion channel. The M3-S2 linkers directly couples ligand binding to gating movements of the pore-lining M3 transmembrane segment, but how it does so, as well as the structural and functional contributions of the S1-M1 and S2-M4 linkers to the gating process, are unknown. My research focused on these LBD-TMD linkers to dissect out the mechanisms of gating in NMDA receptors. In an initial project, I identified key residues/positions within these linkers that are important to gating. These extracellularly accessible positions can modulate gating through a hypothesized disruption of transient contact interfaces dependent on the receptor gating state, thus qualifying for potential sites and mechanisms of drug action. I further studied the functional consequences of stabilizing such contacts amongst the linkers by engineered disulfide crosslinks. When M3-S2 was crosslinked to S2-M4 within an individual NMDA receptor subunit, I found symmetrical effects on the late gating steps comprising pore opening. Thus the NMDA receptor undergoes a pore opening mechanism through equivalent contributions of tightly-coupled subunits. When S1-M1 was crosslinked to S2-M4 either within or across subunits, I found strong impairments in pore opening. Therefore, while structurally at the periphery of the M3-S2/M3 central gating element, the conformational freedom of S1-M1 and S2-M4 is integral in mechanical opening of the channel pore. In conclusion, my work has provided new insights into mechanisms coupling ligand binding to ion channel gating in NMDA receptors, while also laying the groundwork for future targeting of the linkers to correct dysfunctional receptor activity.
