Glycine Gating of NR1/NR2A NMDA Receptors

Abstract

N-Methyl-D-Aspartate receptors (NMDARs) are ligand-gated ion channels that mediate excitatory neurotransmission in the mammalian central nervous system. They are required for normal neuronal function and are a factor in several neuropathies including Alzheimer's disease and schizophrenia. Classical NMDARs require both glycine (NR1) and glutamate (NR2) bound for receptor activation. Reaction mechanisms for NMDA receptors have been developed for several isoforms, however these models assume saturation of glycine sites and a quantitative understanding of glycine association and dissociation kinetics is currently inadequate. We measured current responses with fast application of glycine (in 1 mM glutamate) onto outside-out patches containing several NR1/NR2A receptors and observed relatively slow kinetics, with a rise time of 4.9 ± 0.6ms and deactivation of 105 ± 8ms. Anticipating a slow dissociation rate for glycine, we first developed models for two low-affinity glycine-site full-agonists: L-serine (95% efficacy; EC50=0.21mM) and 3,3,3-trifluoro-DL-alanine (132% efficacy; EC50=2.2mM). We used these data to select best fitting multi-state kinetic models of several arrangements and further validated these with cycles of simulations and experimental measurements which included macroscopic responses to several stimulation patterns. These models will be used to estimate glycine association and dissociation rate constants. Work is in progress to validate these results with macroscopic measurements. Knowledge about how glycine gates NMDA receptors will offer insight to glycine-dependent NR1/NR2A kinetic mechanisms and contribute to a more comprehensive understanding of the activation of these physiologically important receptors.