LAUSR

Significance N-methyl-D-aspartate receptors (NMDARs), which are critical in the brain, are increasingly being shown to signal without ion flux (i.e., “metabotropically”). What controls the metabotropic function of NMDARs is unknown. We discovered that a form of metabotropic signaling—glycine priming—is controlled by alternative splicing of the mRNA encoding one NMDAR subunit, GluN1. Our discovery was surprising because the spliced exon encodes a peptide cassette in the extracellular region of GluN1 far from the plasma membrane, and yet, metabotropic function requires signaling across the neuronal membrane. Moreover, we found that this metabotropic function of NMDARs is neuron cell–type specific: excitatory neurons show glycine priming, whereas inhibitory neurons do not. These findings have widespread implications for NMDARs in health and disease. N-methyl-D-aspartate (NMDA) receptors (NMDARs), a principal subtype of excitatory neurotransmitter receptor, are composed as tetrameric assemblies of two glycine-binding GluN1 subunits and two glutamate-binding GluN2 subunits. NMDARs can signal nonionotropically through binding of glycine alone to its cognate site on GluN1. A consequence of this signaling by glycine is that NMDARs are primed such that subsequent gating, produced by glycine and glutamate, drives receptor internalization. The GluN1 subunit contains eight alternatively spliced isoforms produced by including or excluding the N1 and the C1, C2, or C2’ polypeptide cassettes. Whether GluN1 alternative splicing affects nonionotropic signaling by NMDARs is a major outstanding question. Here, we discovered that glycine priming of recombinant NMDARs critically depends on GluN1 isoforms lacking the N1 cassette; glycine priming is blocked in splice variants containing N1. On the other hand, the C-terminal cassettes—C1, C2, or C2’—each permit glycine signaling. In wild-type mice, we found glycine-induced nonionotropic signaling at synaptic NMDARs in CA1 hippocampal pyramidal neurons. This nonionotropic signaling by glycine to synaptic NMDARs was prevented in mice we engineered, such that GluN1 obligatorily contained N1. We discovered in wild-type mice that, in contrast to pyramidal neurons, synaptic NMDARs in CA1 inhibitory interneurons were resistant to glycine priming. But we recapitulated glycine priming in inhibitory interneurons in mice engineered such that GluN1 obligatorily lacked the N1 cassette. Our findings reveal a previously unsuspected molecular function for alternative splicing of GluN1 in controlling nonionotropic signaling of NMDARs by activating the glycine site.