A new perspective on betaine: Targeting the glycine binding site of the NMDA receptor

Betaine, a methylated glycine derivative, shows promise in treating neuropsychiatric and neurological disorders, but its mechanisms remain unclear. This study investigates betaine's concentration-dependent effects on NMDA receptor-mediated excitatory field potentials (EFPs) in mouse medial prefrontal cortex slices and calcium influx in cultured cortical neurons. We further assessed subtype specificity using HEK293 cells stably expressing GluN1/GluN2A, GluN1/GluN2B, or GluN1/GluN2C NMDA receptor subtypes, measuring calcium flux and single-channel activity. In cortical slices, betaine alone did not alter EFPs but, when co-applied with glutamate, significantly increased EFP frequency and amplitude. Conversely, co-application with glutamate and glycine markedly reduced EFPs. Similarly, in cultured cortical neurons, betaine enhanced NMDA receptor-dependent calcium influx with glutamate alone but attenuated it when both glutamate and glycine were present. In HEK293 cells, betaine exhibited dual modulatory effects on NMDA receptor-mediated calcium influx, varying with GluN2 subtype and glutamate/glycine concentrations. Cell-attached patch-clamp recordings confirmed that betaine with glutamate induced NMDA receptor currents, which were reduced by glycine co-application. Molecular docking and dynamics simulations proposed that betaine binds effectively to the glycine-binding site on the GluN1 subunit, with stable interactions. These findings identify betaine as a context-dependent modulator of NMDA receptors via its interaction with the glycine-binding site, offering a novel mechanism that may underlie its therapeutic potential in disorders involving NMDA receptor dysfunction.