Glutamate mediates the majority of fast excitatory neurotransmission in the vertebrate brain. Glutamate receptors (GluRs) transduce signals in two ways: metabotropic GluRs signal via intracellular G proteins, whereas ionotropic GluRs (iGluRs) open intrinsic ion channels in response to agonist binding. NMDA receptors (NMDARs) are glutamate- and glycine-gated iGluRs that play critical roles in spatial learning, contextual fear memory acquisition, synapse elimination, and chronic pain. Their particularly high calcium (Ca2+) permeability and strongly voltage-dependent channel block by external magnesium (Mg2+) distinguish NMDARs from other iGluRs. Mg2+ channel block of NMDARs inhibits current influx through the majority of agonist-bound, open NMDARs at resting membrane potentials (Vms), but this block is relieved by depolarization. Thus, significant current flow through NMDARs requires presynaptic activity (glutamate release) and postsynaptic activity (depolarization to relieve Mg2+ channel block), conferring on NMDARs a coincidence-detection capability that is central to their physiological importance. To mediate this and other important functions, NMDARs require tight regulation of the voltage-dependent Mg2+ block that provides crucial control of NMDAR-mediated current flow and Ca2+ influx. NMDARs are typically composed of NR1 and NR2 subunits. The four NR2 subunits (NR2A-D) contribute to four diheteromeric NMDAR subtypes (NR1/2A-NR1/2D), which differ in many respects, including the magnitudes of channel block by Mg2+, Ca2+ permeability, and single-channel conductance. Previously-gathered data from our lab demonstrates that the subtype specificity of Mg2+ block is principally conferred by a single amino acid site in the third transmembrane region (M3) of NR2 subunits. This "NR2 S/L site" contains a serine in NR2A and NR2B subunits and a leucine in NR2C and NR2D subunits. Surprisingly, the NR2 S/L site does not line the pore. I created several structural homology models of NMDARs to generate hypotheses regarding how the NR2 S/L site conveys its effects to the pore. I tested these hypotheses experimentally and found that the NR2 S/L site interacts with an NR1 subunit tryptophan in the pore-loop to regulate Mg2+ block properties. I further determined that the NR2 S/L site greatly contributes to the subtype variation in single-channel conductance, and likely plays a role in the subtype variation in Ca2+ permeability
