Abstract
Objective
N-methyl-D-aspartate (NMDA) receptors are expressed at synaptic sites where they mediate fast excitatory neurotransmission. NMDA receptors are critical to brain development and cognitive function. Natural variants to the GRIN1 gene, which encodes the obligatory GluN1 subunit of the NMDA receptor are associated with severe neurological disorders that include epilepsy, intellectual disability and developmental delay. Here we investigated the pathogenicity of three missense variants to the GRIN1 gene, p. Ile148Val [GluN1-3b(I481V)], p.Ala666Ser [GluN1-3b(A666S)] and p.Tyr668His [GluN1-3b(Y668H)].
Methods
Wild-type and variant-containing NMDA receptors were expressed in HEK293 cells and primary hippocampal neurons. Patch-clamp electrophysiology and pharmacology was used to profile the functional properties of the receptors. Receptor surface expression was evaluated using fluorescently tagged receptors and microscopy.
Results
Our data demonstrate that the GluN1(I481V) variant is inhibited by the open pore blockers, ketamine and memantine with reduced potency but otherwise has little effect on receptor function. By contrast, the other two variants exhibit gain-of-function molecular phenotypes. Glycine sensitivity was enhanced in receptors containing the GluN1(A666S) variant and the potency of pore block by memantine and ketamine was reduced, whereas that for MK-801 was increased. The most pronounced functional deficits, however, were found in receptors containing the GluN1(Y668H) variant. GluN1(Y668H)/2A receptors showed impaired surface expression, were more sensitive to glycine and glutamate by an order of magnitude and exhibited impaired block by extracellular magnesium ions, memantine, ketamine and MK-801. These variant receptors were also activated by either glutamate or glycine alone. Single receptor recordings revealed that this receptor variant opened to several conductance levels and activated more frequently than wild-type GluN1/2A receptors.
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