N-methyl-D-aspartate-selective glutamate receptors (NMDA receptors) are ligand-gated ion channels that mediate excitatory synaptic transmission in the brain and spinal cord. NMDA receptors are heteromultimers comprised of two glycine-binding NR1 subunits and two glutamate-binding NR2 subunits, of which there are four family members (NR2A,B,C,D). NMDA receptors are involved in many normal brain functions such as neuronal development, learning, memory, in addition to their roles in neuropathological conditions such as stroke, epilepsy, and neuropsychiatric disorders. Since its first description, the NR2B-selective NMDA receptor antagonist ifenprodil and its many analogues have been used in a multitude of studies exploring the role of this subunit in virtually every aspect of brain function including behavior, cognition, synaptic plasticity, neuronal development, and neuropathology. However, no highly selective antagonists have yet been described for NR2A-, NR2C-, or NR2D-containing receptors. We therefore developed an assay to identify non-competitive allosteric modulators of NR2C- and NR2D-containing NMDA receptors, and subsequently screened ~60,000 compounds for new subunit-selective antagonists and potentiators. The result was the identification of two structurally unique classes of compounds that are 100-500 fold selective inhibitors of NR1/NR2C and NR1/NR2D compared to NR2A- or NR2B-containing receptors or AMPA/kainate receptors. We also identified two structurally distinct classes of potentiators that are selective for NR2C/D-containing receptors. These new non-competitive allosteric modulators represent a breakthrough opportunity to study the role of the NR2C/D subunits in normal brain function and in neurological diseases. This proposal addresses 3 questions using these subunit-selective modulators. 1. What are the structural determinants of NR2C/D-selective inhibitors and potentiators? We will utilize site-directed mutagenesis, exploiting sequence differences between NR2A/B and NR2C/D, to identify key residues that mediate the actions of three classes of NR2C/D-selective potentiators and inhibitors. 2. What is the mechanism of action of NR2C/D selective inhibitors and potentiators? We will analyze macroscopic and single channel currents recorded under voltage clamp to define the mechanism of action of the non-competitive NR2C/NR2D inhibitors as well as NR2C/NR2D potentiators. 3. How do NR2C/D modulators alter synaptic signaling and neuronal excitability? We will evaluate the effect of inhibition and potentiation of NR2D-containing NMDA receptors at the cortical-subthalamic neuron synapse and NR2C/D-containing NMDA receptors at afferent excitatory synapses onto hippocampal interneurons in brain slices. Experiments will test the synaptic response to stimulus trains in the presence of NR2C/D modulators. We will also use these new pharmacological tools to investigate whether interneuron and subthalamic neuron excitability and spiking frequency can be altered through inhibition or potentiation of NR2C/D-containing receptors. PUBLIC HEALTH RELEVANCE: NMDA receptors mediate communication between neurons in the central nervous system, and thus play an important role in virtually all brain functions as well as numerous neurological diseases. The NMDA receptors are tetrameric assemblies comprised of two glycine-binding NR1 subunits and two glutamate-binding NR2 subunits, of which there are four subtypes (NR2A-D). Because different NR2 subunits are differentially expressed and have different functional properties, they serve a host of divergent roles in both normal brain function and disease. Although subunit-selective modulators have been proposed to be useful therapeutic agents in a number of neurological diseases, only one truly selective NR2 subunit-selective antagonist has been discovered. Ifenprodil and its analogues inhibit NR2B-containing receptors over 500-fold more potently than NR2A,C,D-containing NMDA receptors. Since the first description of the NR2B-selectivity of ifenprodil in 1993, no new highly selective subunit- specific compounds have been described. This lack of pharmacological reagents has led us to search for new ligands that act at NR2C- and NR2D-containing receptors. We have found at least three classes of antagonists of NMDA receptor function that are 100-500 fold more potent at NR2C- and NR2D-containing receptors when compared to NR2A- or NR2B-containing receptors. The current grant proposes experiments that define where on the NR2D subunit these novel compounds act, how they control NMDA receptor function, and what effect they have on synaptic transmission and neuronal excitability. This information will provide insight into receptor function, and define for the first time the role of the NR2C and NR2D subunits in synaptic transmission.