Summary The glutamate receptor gene family encodes AMPA, kainate, and NMDA receptors, which mediate excitatory synaptic transmission in the central nervous system. Two additional gene family members, GRID1 and GRID2, encode the enigmatic delta receptor GluD1 and GluD2 subunits, which can bind D-serine but do not appear to activate conventional signaling systems. There are four functional effects known for delta receptors: (1) Both delta receptors (GluD1 and GluD2) are activated when certain mutations occur in the transmembrane helices, which convert a non-gating receptor into a channel that is constitutively open, producing a tonic inward current. In addition, chimeric receptors in which the glutamate binding domain from a kainate or AMPA receptor replaces the analogous D-serine binding domain for GluD1 and GluD2 can be activated by glutamate, suggesting that the highly specialized machinery needed to convert agonist binding into pore opening is conserved in delta GluD1 receptors. (2) The binding of D-serine to GluD1 receptors harboring a TM3 mutation that renders them constitutively active can close the channel, raising that possibility that D-serine binding produces meaningful conformational changes with unknown physiological roles in WT receptors. (3) Ca2+ binding to a site at the dimer interface between two adjacent D-serine binding domains potentiates constitutive current in mutant receptors, suggesting Ca2+ could regulate GluD1 conformation and function. (4) The distal extracellular domain serves as a ligand for presynaptic Cbln2 and neurexin, which can alter synapse formation. Whereas Cbln2 and neurexin binding to GluD1 appears to be critical, it remains unclear whether channel gating, D-serine binding, or Ca2+ regulation of GluD1 play important roles in brain. A unique power of population genetics is that it can identify key functions of a protein in an unbiased manner. GRID1 is one of the least tolerant genes in the body, falling in the top 2 percentile for lacking variation, suggesting it plays essential roles. Consistent with this idea, patients with neurological conditions have been identified with missense variants in GRID1 that are absent in the general population. We will evaluate the effects of disease-associated variants in addition to well-tolerated variants commonly observed in the healthy population on three modalities associated with GRID1 function—constitutive activation, D-serine binding, Ca2+ binding. If any of these functional attributes are important, then we expect to find disease- associated variants that perturb them, while variants present in the standing population should be without effect. Three electrophysiological experiments will answer the following questions: Aim 1: Can missense variants produce active ion channels that are involved in neuropathology? Aim 2: Do missense variants alter the actions of D-serine and Ca2+ on constitutively active channels? Aim 3: Do missense variants alter the actions of glutamate on GluD1-GluK2 chimeric receptors?

Narrative Glutamate receptors mediate communication between neurons and thus play important roles in normal brain functions and a wide range of neurological diseases. Glutamate receptors include three well-known classes of synaptic receptors (NMDA, kainate, and AMPA), as well as an enigmatic class (delta receptors encoded by GRID1 and GRID2) that is poorly understood in that it is unclear whether it signals as an ion channel, a metabotropic receptor, or serves as a binding scaffold for other proteins. We will use population genetics and functional analysis of variants to explore the GRID1 gene to obtain new insight into its physiological role(s) in the brain. 1